ABSTRACT Title of dissertation: THE C PROTEINS OF HUMAN PARAINFLUENZA VIRUS TYPE 1 EXERT BROAD CONTROL OVER THE HOST INNATE IMMUNE RESPONSE Jim Boonyaratanakornkit, Doctor of Philosophy, 2010 Dissertation directed by: Professor Peter Collins National Institutes of Health and Professor Siba Samal Veterinary Medical Sciences Human parainfluenza virus type 1 (HPIV1) is an important pediatric respiratory pathogen, and its virulence in vivo can be attenuated by introducing mutations into the C gene, a strategy that has been used to design live attenuated candidate vaccines. By tracking gene expression over time, we found that a HPIV1 mutant with a single point mutation in the C gene, referred to as CF170S, and a HPIV1 mutant with complete deletion of the C gene, referred to as P(C-), altered the expression of over 1000 genes, in sharp contrast to wild-type (WT) HPIV1. Using functional bioinformatics, we found that binding sites for the IRF and NF-?B family of transcription factors were over-represented in many of the C protein targeted pathways. By examining the activation of the major components of the type I interferon (IFN) enhanceosome, we found that the C mutant viruses, but not WT HPIV1, activated IRF3 phosphorylation and I?B? degradation, steps integral to the formation of the interferon enhanceosome. To investigate the basis for the observed antagonism of the host response by the WT C proteins, which are expressed from the C open reading frame as a nested set of carboxy-coterminal proteins, we searched for but were unable to identify C-interacting proteins among members of the RIG-I/MDA5 pathway. Furthermore, we also found that the WT C proteins supplied in trans could block IFN? induced by P(C-) HPIV1 infection but not by heterologous inducers of RIG-I and MDA5, namely RSV and poly I:C, respectively. These two lines of evidence suggested that the HPIV1 C proteins do not directly block type I IFN production, such as by interacting with a host factor of the RIG-I/MDA5 pathway. Using knockout mouse embryonic fibroblasts, we found that HPIV1-induced IFN? production relied mainly on MDA5. Consistent with this observation, a striking amount of intracellular dsRNA was detected during infection with the C mutant HPIV1 viruses but not with WT HPIV1. A marked increase in viral genome, antigenome, and mRNA as well as a decrease in viral protein accumulation provided compelling evidence for dysregulated viral RNA synthesis and an inhibition of viral protein synthesis in the absence of WT C proteins. We suggest that this resulted in an imbalance in the N protein-to-genomic RNA ratio, leading to incomplete encapsidation and an intracellular environment permissive for the generation of dsRNA. PKR activation followed the kinetics of dsRNA accumulation and contributed to both IFN? induction and the reduction in viral protein levels. This study establishes the profound effects that the C proteins of wild-type HPIV1 play in evading the host response to HPIV1 infection, and it extends our current understanding of the innate immune response to HPIV1 infection. THE C PROTEINS OF HUMAN PARAINFLUENZA VIRUS TYPE 1 EXERT BROAD CONTROL OVER THE HOST INNATE IMMUNE RESPONSE By Jim Boonyaratanakornkit Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2010 Advisory Committee: Professor Siba Samal, Chair Professor Peter Collins Professor Xiaoping Zhu Professor Nathaniel Tablante Professor Jeffrey DeStefano ? Copyright by Jim Boonyaratanakornkit 2010 ii Acknowledgements I would like to thank, first and foremost, Peter Collins, Brian Murphy, Siba Samal, Alexander Schmidt, Emmalene Bartlett, and Henrick Schomacker for their guidance. I am grateful to Sonja Surman and Emerito Amaro-Carambot for their technical assistance. I would also like to thank Shirin Munir and Christine Winter for providing the sucrose- purified GFP-tagged ?NS1/NS2 RSV. I am grateful to Owen Schwartz, Juraj Kabat, Lily Koo, and Steven Becker (NIAID Biological Imaging Core) for assistance with confocal microscopy. John Hiscott, McGill University, provided the VSV-GFP and Yasuhiko Ito, Mie University School of Medicine, provided the mouse anti-HPIV1 HN (8.2.2A and 4.5) antibodies. J. B. B. was supported in part by funding from the Howard Hughes Medical Institute Research Scholars Program. This research was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases. This research was also supported in part by a CRADA between NIH and MedImmune, LLC, for the development of PIV and RSV vaccines. iii Table of Contents Acknowledgements............................................................................................................. ii Table of Contents............................................................................................................... iii List of Abbreviations ......................................................................................................... iv Chapter 1: General Introduction ......................................................................................... 1 Background and Rationale............................................................................................... 1 Viral Tropism, Animal Models, and Cell Culture ........................................................... 2 Viral Attachment and Entry............................................................................................. 3 Viral Replication and Transcription ................................................................................ 3 The Innate Immune Response and Type I IFN................................................................ 6 Apoptosis ....................................................................................................................... 11 The HPIV1 C Proteins................................................................................................... 12 Reverse Genetics ........................................................................................................... 13 Study Aims and Outline ................................................................................................ 14 Chapter 2: Human parainfluenza virus type 1 C proteins are nonessential proteins that inhibit the host interferon and apoptotic responses........................................................... 16 Abstract.......................................................................................................................... 17 Introduction ................................................................................................................... 18 Materials and Methods .................................................................................................. 21 Results ........................................................................................................................... 32 Discussion...................................................................................................................... 43 Chapter 3: The C proteins of human parainfluenza virus type I (HPIV1) control the transcription of a broad array of cellular genes that would otherwise respond to HPIV1 infection ............................................................................................................................ 51 Abstract.......................................................................................................................... 52 Introduction ................................................................................................................... 53 Materials and Methods .................................................................................................. 58 Results ........................................................................................................................... 63 Discussion...................................................................................................................... 94 Chapter 4. The C proteins of human parainfluenza virus type 1 limit double stranded RNA accumulation that would otherwise trigger MDA5 and PKR activation............... 105 Abstract........................................................................................................................ 106 Introduction ................................................................................................................. 107 Materials and Methods ................................................................................................ 111 Results ......................................................................................................................... 116 Discussion.................................................................................................................... 137 General Conclusion......................................................................................................... 145 Appendices...................................................................................................................... 152 Supplementary Information for Chapter 3................................................................... 152 Supplementary Information for Chapter 4................................................................... 205 References....................................................................................................................... 209 iv List of Abbreviations 293: human embryonic kidney cell line A20: tumor necrosis factor alpha-induced protein 3 A549: human respiratory epithelial cell line aa: amino acid ADAR: adenosine deaminase, RNA-specific AGM: African green monkey AGT: angiotensinogen AKT: v-akt murine thymoma viral oncogene homolog ANOVA: analysis of variance AP-1: activator protein 1 ATF: activating transcription factor BAX: BCL2-associated X protein BCL: B-cell leukemia/lymphoma BHK: baby hamster kidney cell line BIRC: Baculoviral inhibitor of apoptosis repeat-containing protein bp: base pair BSA: bovine serum albumin C: C proteins, including C?, C, Y1, and Y2. cAMP: cyclic adenosine monophosphate CARD: caspase recruitment domain CASP: caspase CCL: C-C motif chemokine v cpe: cytopathic effect CTD: c-terminal domain CXCL: C-X-C motif chemokine DAPI: 4',6-diamidino-2-phenylindole DIABLO: Direct inhibitor of apoptosis-binding protein with low pI dsRNA: double stranded RNA DTT: dithiothreitol DUBA: deubiquitinating enzyme A EDTA: ethylenediaminetetraacetic acid EIF: eukaryotic translation initiation factor ELISA: enzyme-linked immunosorbent assay F: fusion protein F170S: a recombinant HPIV1 with the CF170S phenylalanine to serine substitution at amino acid 170 of the C proteins FACS: fluorescence-activated cell sorting FADD: Fas-associated via death domain FBS: fetal bovine serum FITC: fluorescein isothiocyanate FLC: full-length antigenomic cDNA clone FOX: forkhead box GAPDH: glyceraldehyde-3-phosphate dehydrogenase GFP: green fluorescent protein h: hours vi HA: hemagluttinin HAE: Human airway epithelial HEL: helicase domain HEp-2: Human epithelial cell line (HeLa contaminant) HLF: hepatic leukemia factor HN: hemagluttinin/neuraminidase protein HPIV: Human parainfluenza virus IFI: interferon-induced protein IFIT: IFN-induced protein with tetraicopeptide repeats IFN: interferon IFNAR: type 1 interferon receptor Ig: immunoglobulin IKK: I?B kinase IL: interleukin IP: immunoprecipitation IPS1 (or IPS-1): interferon beta promoter stimulator protein 1 IRF: interferon regulatory factor ISG: IFN-stimulated gene ISGF: interferon-stimulated gene factor ISRE: interferon-stimulated response elements IU: international unit I?B: inhibitor of of kappa light polypeptide gene enhancer in B-cells JAK: Janus kinase vii JNK: c-Jun N-terminal kinase JUN: v-jun sarcoma virus 17 oncogene homolog L: large polymerase protein LD50: 50% lethal dose M: matrix protein MAPK: mitogen-activated protein kinase MAVS: mitochondrial antiviral signaling protein MCL: myeloid cell leukemia MDA5 (or MDA-5): melanoma differentiation-associated protein 5 MEF: mouse embryonic fibroblast MEKK1: mitogen-activated protein kinase kinase kinase 1 MEM: minimum essential medium MIAME: minimum information about a microarray experiment min: minutes MK2: Rhesus monkey kidney cell line MOI: multiplicity of infection MRC-5: normal human fetal lung cell line MTC: multiple testing correction MVA: Modified Vaccinia Ankara MX: myxovirus (influenza virus) resistance MYB: v-myb myeloblastosis viral oncogene homolog n.s.: non-specific N: nucleoprotein viii nAb: neutralizing antibody NEMO: NF-?B essential modulator NF-?B: nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 NS: nonstructural protein NSm: medium segment nonstructural protein NSs: small segment nonstructural protein NU: neutralizing unit OAS: oligoadenylate synthetase ORF: open reading frame P(C-): a recombinant HPIV1 that does not express any of the C proteins p.i.: post-infection P: phopho or phosphate or also HPIV1 phosphoprotein P38: mitogen-activated protein kinase 14 PBS: phosphate buffered saline PFA: paraformaldehyde PFU: plaque forming unit PI3K: phosphatidylinositol 3-kinase PKR: double stranded RNA-dependent protein kinase Poly I:C: polyinosinic:polycytidylic acid PRD: positive-regulatory domain PVDF: polyvinylidene fluoride Rel: v-rel reticuloendotheliosis viral oncogene homolog rHPIV: recombinant HPIV ix RIG-I: retinoic acid-inducible gene-I RLR: RIG-I-like receptors RSV: respiratory syncytial virus RT: room temperature or reverse transcription RT-qPCR: reverse transcription-quantitative polymerase chain reaction SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis SE (or S.E.): standard error SeV: Sendai virus sh: small hairpin shCON: control small hairpin RNA shPKR: PKR small hairpin RNA SOCS: suppressor of cytokine signaling SOX: sex determining region Y-box SPIB: Spi-B transcription factor ssRNA: single stranded RNA STAT: signal transducer and activator of transcription TAK1: TGF?-activated kinase 1 TANK: TRAF family member-associated NF-?B activator TBK1: TANK-binding kinase 1 TCF1: transcription factor 1, also known as hepatocyte nuclear factor 1? or IFN production regulator factor TCID50: 50% tissue culture infectious dose TFBS: transcription factor-binding site x TFIIH: transcription factor II H TIR: Toll-interleukin-1 receptor domain TLR: Toll-like receptor TM: transmembrane TNF: tumor necrosis factor TNFRSF: tumor necrosis factor receptor superfamily member TNFSF: tumor necrosis factor ligand superfamily member TRADD: TNFRSF1A-associated via death domain TRAF: TNF receptor-associated factor TRAIL: TNF-related apoptosis-inducing ligand TRAIL-R: TRAIL receptor TRIF: TIR domain-containing adapter protein inducing interferon beta TRIM: tripartite motif-containing protein Ub: ubiquitin UDP: uridine diphosphosphate Vero: African green monkey kidney cell line VISA: virus-induced-signaling adapter VP: viral protein vRNA: viral RNA VSV: vesicular stomatitis virus WB: Western blot WT (also wt): wild-type ZNF: zinc finger 1 Chapter 1: General Introduction Background and Rationale Human parainfluenza virus type 1 (HPIV1) is an important and uncontrolled pediatric respiratory pathogen that causes a significant burden of disease, mainly in young children, the immunocompromised, and the elderly (30, 42, 58, 79, 134, 154). HPIV1 is the single most common cause of laryngotracheobronchitis or croup. Other clinical manifestations of HPIV1 range from mild disease, including rhinitis, pharyngitis, and otitis media, to severe lower respiratory disease, including bronchitis, bronchiolitis, and pneumonia (Figure 1) (30). In all, 4 major serotypes of human parainfluenza virus have been described, including HPIV1, 2, and 3 which, collectively, are the second most common cause of pediatric respiratory hospitalizations, only surpassed by respiratory syncytial virus (RSV) (42, 134). Disease caused by HPIV4 is less frequently diagnosed and typically less severe (13). Licensed vaccines are currently not available against any of the HPIVs but ongoing vaccine development efforts that employ cDNA derived live attenuated viruses have progressed to phase 1/2 clinical evaluation (89, 91, 93). 2 Figure 1. The clinical manifestations of HPIV1 infection. Viral Tropism, Animal Models, and Cell Culture HPIV1 was first isolated from infants and children with lower respiratory tract disease (23). The virus infects and replicates in the epithelial cells that line the respiratory tract, including the mucus membranes of the nose and throat, larynx, trachea, bronchi, and lower airways (143). In vivo, HPIV1 is generally considered a pneumotropic virus since it buds from the apical surface instead of the basolateral surface (10, 136). The virus replicates well in the respiratory tract of hamsters, guinea pigs, cotton rats, ferrets, and non-human primates but infection is typically self-limited and asymptomatic with minimal pulmonary pathology (26, 37). In cell culture, HPIV1 replicates well in both simian and human kidney cells and human respiratory cells but trypsin must be supplied in the medium. 3 Viral Attachment and Entry The hemagglutinin-neuraminidase (HN) transmembrane surface glycoprotein mediates attachment by binding sialic acid on cell surfaces. The HN protein also triggers fusion by interacting with the F protein (130, 151). The F protein promotes viral entry by mediating fusion between the viral envelope and the cell membrane. The inactive precursor F0 protein is converted to the fusogenic F1 and F2 polypeptides after cleavage by a secretory protease, and a hydrophobic fusion peptide in F1 initiates fusion (211). Together, the two surface glycoproteins HN and F constitute the major protective and the sole neutralizing antigens against infection (94, 153, 175). Viral Replication and Transcription HPIV1 is an enveloped, single stranded, negative sense, nonsegmented RNA virus in the Paramyxoviridae family (Figure 2). Upon attachment and fusion, the viral genome enters, replicates in the cytoplasm, and buds as pleomorphic, spherical virions through the plasma membrane (191). The viral genome, 15.6 kb in length, consists of 6 genes (3? ? N-P/C-M-F-HN-L ? 5?) that encode the nucleoprotein (N), phosphoprotein (P), C proteins (C), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and the large polymerase protein (L). Replication of the parainfluenza viral genome proceeds through an intermediate positive sense antigenome. Viral replication occurs in a nucleocapsid that consists of viral RNA-dependent RNA polymerase and P protein in association with N-bound genome and antigenomes. The L polymerase 4 catalyzes both RNA replication and transcription. The P protein binds to both the N and L proteins and serves as a polymerase cofactor that links the L protein to the N-RNA template (74). The N protein has traditionally been thought to function as a highly stable and ribonuclease resistant coat that protects the viral genome and antigenome from nuclease digestion (100). N encapsidation also leads to the formation of a helical structure that may align the major promoter at the 3? end of the viral genome and that may provide interaction sites for the assembly of nucleocapsids during viral budding (112, 186). The nucleotide length of the viral genome and antigenome must be an even multiple of six for efficient replication to occur. This ?rule of six? is thought to arise from the need to fully encapsidate the entire length of the genome with a chain of N protein monomers that span exactly six nucleotides so that the phase or position of the nucleotides of the major promoter relative to each N monomer, i.e. positions 1 through 6, is conserved (102, 170, 194). A single promoter at the 3? end of the genome promotes replication and leads to sequential transcription. Each gene is flanked by an upstream gene start region that directs viral polymerase initiation and mRNA capping and a downstream gene end that directs transcriptional termination and polyadenylation (138). Since polymerase reinitiation is imperfect, polymerase drop-off leads to a gradient of mRNA transcription that decreases with increasing distance from the 3? major promoter, i.e. N mRNA is more abundant than L mRNA (27). 5 Each gene of the HPIV1 genome encodes a single major protein with the exception of the P/C gene, which encodes the P protein in one open reading frame and a nested set of four carboxy-coterminal C proteins (C', C, Y1, and Y2) expressed from individual start sites in a second open reading frame. An unconventional GUG start codon for C' reduces translation initiation efficiency and thereby allows leaky scanning by the ribosome to initiate at the next downstream start codon for C. In contrast, the ribosome accesses the Y1 and Y2 start codons by discontinuous scanning-independent shunting from the 5? cap (32, 34). Recent studies, including the findings from this dissertation and from others in the lab, have shown that the C proteins play a critical role in HPIV1 virulence by inhibiting apoptosis, regulating type I interferon (IFN) production and signaling, and controlling the transcription of a large number of host genes (9, 17, 18, 192). 6 Figure 2. Diagrammatic representation of the HPIV1 virion (top) and its genome organization (bottom). The N gene and the N proteins encapsidating the viral genome are colored in green. The P gene and the P proteins associated with the viral polymerase are colored in yellow. The C ORF is colored in orange. The M gene and the M proteins located on the inner face of the viral envelope are colored in blue. The F gene and the F surface glycoproteins are colored in aqua. The HN gene and HN suface glycoproteins are colored in red. The L gene and the L polymerase are colored in purple. The Innate Immune Response and Type I IFN Innate immunity, considered the front-line of host defense, comprises the early phases of the host response to infection and generally discriminates between groups of related 7 pathogens. The innate immune response also contributes, by antigen presentation and the secretion of cytokines, to the induction of effectors in the adaptive immune response, including cytotoxic T lymphocytes and secretory and serum neutralizing antibodies (77, 172, 184). An important component of the innate immune response to viral infection is the production of type I IFN, signaling through its receptor, and the subsequent establishment of an antiviral state. The induction of IFN synthesis following virus infection depends on a number of pattern-recognition receptors that recognize conserved pathogen-associated molecular patterns and initiate downstream signaling cascades (Figure 3). The presence of dsRNA, an intermediate of RNA viral replication, is recognized as a pathogen-associated molecular pattern by Toll-like receptor 3 (TLR3) and two caspase recruitment domain (CARD)-containing RNA helicases, retinoic acid- inducible gene I (RIG-I) and melanoma associated-differentiation gene 5 (MDA5), which act as intracytoplasmic sensors of dsRNA (2, 76, 147, 180, 212, 214). Whereas TLR3 mainly senses extracellular dsRNA on the surface and in the endosomal compartment of antigen-presenting cells, RIG-I and MDA5 are constitutively present and detect intracellular dsRNA (2, 214). RIG-I activation by dsRNA requires ubiquitination of its CARD domain by the ubiquitin ligase TRIM25 (45). The exact nature of the dsRNA ligands recognized by RIG-I and MDA5 is still controversial (203). Currently, it is thought that RIG-I binds to 5? triphosphorylated dsRNA, whereas MDA5 binds higher- order webs of dsRNA and ssRNA (148, 165). TLR3 signals through an adaptor called TIR domain-containing adaptor inducing IFN? (TRIF), while RIG-I and MDA5 recruit another CARD-containing adaptor called mitochondrial antiviral signaling protein (MAVS, also referred to as IPS-1, Cardif, or VISA). MAVS contains a transmembrane 8 domain that targets the protein to the mitochondria, implicating a role for the mitochondria as a signaling platform for the innate immune response (167). MAVS relays the signal to two ubiquitin ligases TRAF3 and TRAF6. The deubiquitinases DUBA and A20 negatively regulate IFN? induction by removing ubiquitin from TRAF3 and TRAF6, respectively. TRAF3 activates the kinases TBK1 and IKK?, which phosphorylate IRF3 (interferon regulatory factor 3) (41, 99, 140, 167, 207). Once activated, IRF3 translocates into the nucleus and binds the positive regulatory domain III (PRDIII) of the IFN? promoter. TRAF6 activates IKK? and induces the proteasome- mediated degradation of I?B which allows NF-?B to translocate into the nucleus and bind PRDII (167, 207). After the host cell recognizes and signals the presence of foreign RNA, IRF-3 and NF-?B, together with ATF-2/c-Jun (AP-1), form an enhanceosome that binds to the IFN? promoter, inducing transcription of the IFN? gene. Although crystal structures of ATF-2 and c-Jun bound to PRDIV of the IFN? promoter are known, the viral triggers that activate the MAPK pathway leading to ATF-2 and c-Jun phosphorylation and dimerization are less well understood (142). Once type I IFN is produced and secreted, it binds to the type I IFN receptor on the surface of the infected and neighboring cells to induce a robust antiviral response (121, 183). 9 Figure 3. Diagrammatic representation of the cellular signal transduction pathways that can be activated by RNA virus infection and lead to IFN? production. IFN? expression relies on the activation of three transcription factors IRF3, NF-?B, and AP-1 that bind the IFN? enhanceosome. Dotted lines represent protein-protein interactions. The dotted line with an arrowhead signifies binding of the AP-1 transcription factor to PRDIV. The dashed lines signify that the viral triggers that activate MEKK1 and the MAPK signal transduction pathway leading to ATF2 and JUN phopshorylation are not yet fully characterized. The transcriptional activation of type I IFN is biphasic. One gene encodes the prototypical IFN?, while more than a dozen IFN species comprise the IFN? family (183). In humans, at least 23 gene loci on chromosome 9 correspond to the IFN? family, and at 10 least 14 are known to encode functional proteins (144). Recognition of viral infection results in phosphorylation of constitutively expressed IRF3 to induce IFN? and IFN?1 expression in human cells as part of the early response to virus infection. Once early type I IFN is produced and secreted, it binds to the type I IFN receptor on the surface of the infected and neighboring cells and activates the JAK-STAT signal transduction cascade. The JAK1 and TYK2 kinases are activated by autophosphorylation and phosphorylate STAT1 and STAT2, inducing the formation of IFN-stimulated gene factor 3 (ISGF3) which consists of a heterotrimeric complex of STAT1, STAT2, and IRF9 (Figure 4). ISGF3 binds to the IFN-stimulated response elements (ISRE) within the promoter regions of several hundred IFN-inducible genes, including 2?-5?-oligoadenylate synthetase (OAS), dsRNA dependent protein kinase (PKR), and IRF7. The de novo produced IRF7, which is highly homologous to IRF3, can also undergo phosphorylation by TBK1, dimerize, translocate into the nucleus, and then activate production of additional type I IFNs. Unlike IRF3 though, IRF7 can form a homodimer or heterodimerize with IRF3 to efficienty activate the expression of both the IFN? gene and the full set of IFN? genes (73, 163). Thus, immediate-early expression of IFN? and IFN?1 occurs soon after viral infection is detected, while delayed expression of the entire type I IFN family, induced by IRF7, occurs only after IFN? and IFN?1 secretion, receptor binding, and signaling to amplify a robust feed-forward antiviral response (121, 183). 11 Figure 4. Diagrammatic representation of the type 1 IFN signaling pathway from IFN receptor activation to the formation of the STAT1, STAT2, and IRF9 transcription factor complex (ISFG3). Apoptosis Many viruses have evolved strategies to regulate host cell apoptotic responses to virus infection (reviewed in (127)). Apoptosis, a process of programmed cell death mediated by the activation of a group of caspases, results in systematic cellular self-destruction in response to a variety of stimuli (reviewed in (40)). There are two major apoptotic pathways, the extrinsic and intrinsic pathways, which converge at a step involving the activation of the effector caspase 3. The activation of effector caspases, nuclear 12 condensation and fragmentation, and cell death are the final steps in the apoptosis pathway. Viral proteins that are able to modulate the host apoptotic response include pro-apoptotic viral proteins such as West Nile virus capsid protein (208) and bunyavirus NSs proteins (28), and anti-apoptotic viral proteins such as RSV NS proteins (14), Bunyamwera virus NSs (101) and Rift Valley fever NSm protein (204). However, these studies are complex and some viral proteins such as influenza A virus NS1 have been reported to have both pro- and anti-apoptotic functions (166, 220). SeV C proteins have been implicated in the regulation of apoptosis but their role in this process remains incompletely defined (15, 71, 83, 105, 145). A role for HPIV1 proteins in apoptosis had not been investigated to date (9). The HPIV1 C Proteins The C proteins of SeV have been shown to have multiple functions that include inhibition of host innate immunity through antagonism of interferon (IFN) induction and/or signaling (51, 56, 104), regulation of viral mRNA synthesis by binding to the L polymerase protein (21, 33, 75, 110, 185), participation in virion assembly and budding via an interaction with AIP1/Alix, a cellular protein involved in apoptosis and endosomal membrane trafficking (66, 82, 156), and regulation of apoptosis. SeV mutants containing deletions of all four C proteins are viable, but are highly attenuated in vitro and in mice (55, 109, 110). To date, the HPIV1 C proteins have not been as extensively studied as those of SeV. However, the HPIV1 C proteins, like the SeV C proteins, are nonessential, play a role in evasion of host innate immunity through inhibition of type I IFN production and signaling, and regulate the host apoptotic response (9, 18, 192). 13 Reverse Genetics Our laboratory has established a reverse genetics system for HPIV1 that allows the generation of infectious virus from cDNA (85, 137). In the reverse genetics system for HPIV1, a full length HPIV1 antigenomic cDNA, under the control of a T7 promoter, is transfected with support plasmids encoding the N, P, and L proteins into cells that constitutively express the T7 RNA polymerase, i.e. BHK-T7 cells, or into cells infected with the replication-deficient vaccinia virus MVA-T7 that encodes the T7 polymerase (206). HPIV1 can then be recovered from the supernatant. Using this system, we generated a number of HPIV1 mutants to investigate C protein function as well as to generate novel live virus vaccines that would be attenuated by disturbing the IFN antagonistic function of the C proteins, a strategy that has been shown to result in attenuation of replication in vivo (5, 6, 192). Previously, a phenylalanine to serine substitution of amino acid 170 of Sendai Virus (SeV, also referred to as murine PIV1) was shown to significantly attenuate a highly virulent SeV strain, increasing the lethal dose 50% (LD50) 20,000-fold in mice (50). This C F170S mutation was introduced into the P/C gene of recombinant HPIV1 by reverse genetics (85, 137). Whereas WT HPIV1 infection prevented IRF3 dimerization, its nuclear translocation, and IFN? production, infection with HPIV1 containing the CF170S mutation (F170S virus) led to IRF3 activation and IFN? production (192). WT HPIV1, but not the F170S mutant virus, also inhibited the antiviral state induced by type I IFN, most likely due to inhibition of STAT1 nuclear translocation in human lung cells (18, 192). Not unexpectedly, the F170S mutant was attenuated for replication in the respiratory tract of hamsters and African green monkeys 14 (AGMs) (5, 192). We also constructed a HPIV1 mutant referred to as P(C-) that does not express any of the four C proteins due to point mutations that silence the C ORF, and this mutant was even more attenuated than F170S HPIV1 in vivo (9). Current strategies to develop live attenuated vaccine candidates against this important pediatric pathogen include the introduction of attenuating mutations within the viral genome so that the vaccine strain causes little or no disease while, at the same time, confers protection from subsequent infections. The induction of IFN production, IFN signaling, and apoptosis that results from mutating or silencing the C gene may augment the establisment of an antiviral state, enhance antigen presentation and the adaptive immune response, and limit the spread of virus from infected cells. Interferon has been detected in the nasal washes of children infected with HPIV1 and likely contributes to host defense (63). The attenuation achieved by mutating or silencing the C gene underscores the importance of the HPIV1 C proteins as virulence factors during infection and as targets for developing candidate live attenuated virus vaccines. Study Aims and Outline This study addresses seven major aims: first, to create by reverse genetics a virus named P(C-) HPIV1 in which expression of the C proteins is silenced (this mutant virus was recovered by E. Bartlett); second, to compare the level of replication and of type I IFN induced in vitro during infection with P(C-) and WT HPIV1; third, to characterize the transcriptional response of human respiratory epithelial cells to infection with WT HPIV1 or HPIV1 in which the C gene is mutated or ablated; fourth, to examine the contribution 15 of IFN? to this response; fifth, to determine the components of the type I interferon (IFN) response that are activated upon infection with C mutant HPIV1 viruses; sixth, to identify the stimuli that trigger host IFN? production in response to these HPIV1 C mutants; and seventh, to examine the mechanisms by which the wild-type C proteins hold in check the induction of a potent host innate immune response. Aims one and two are described in chapter 2, aims three and four are described in chapter 3, and aims five through seven are described in chapter 4. Supplementary tables and figures are provided in the appendix. 16 Chapter 2: Human parainfluenza virus type 1 C proteins are nonessential proteins that inhibit the host interferon and apoptotic responses Adapted from Journal of Virology 2008 Sep; 82(18): 8965?8977 Emmalene J. Bartlett,1 Ann-Marie Cruz,1 Janice Esker,1 Adam Casta?o,1 Henrick Schomacker,1 Sonja R. Surman,1 Margaret Hennessey,2,3 Jim Boonyaratanakornkit,1 Raymond J. Pickles,2,3 Peter L. Collins,1 Brian R. Murphy,1 and Alexander C. Schmidt1 1Laboratory of Infectious Diseases, RNA Viruses Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services. Bethesda, MD, 20892-2007, USA. 2Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill. Chapel Hill, NC, 27599- 7248, USA. 3Department of Microbiology and Immunology, University of North Carolina at Chapel Hill. Chapel Hill, NC, 27599-7248, USA. 17 Abstract Recombinant human parainfluenza virus type 1 (rHPIV1) was modified to create rHPIV1-P(C-), a virus in which expression of the C proteins (C? , C, Y1, and Y2) was silenced without affecting the amino acid sequence of the P protein. Infectious rHPIV1- P(C-) was readily recovered from cDNA, indicating that the four C proteins were not essential for virus replication. Early during infection in vitro, rHPIV1-P(C-) replicated as efficiently as wild-type (wt) HPIV1, but its titer subsequently decreased coincident with the onset of an extensive cytopathic effect not observed with wt rHPIV1. rHPIV1-P(C-) infection, but not wt rHPIV1 infection, induced caspase 3 activation and nuclear fragmentation in LLC-MK2 cells, identifying the HPIV1 C proteins as inhibitors of apoptosis. In contrast to wt rHPIV1, rHPIV1-P(C-) and rHPIV1-CF170S, a mutant encoding an F170S substitution in C, induced interferon (IFN) and did not inhibit IFN signaling in vitro. However, only rHPIV1-P(C-) induced apoptosis. Thus, the anti-IFN and antiapoptosis activities of HPIV1 were separable: both activities are disabled in rHPIV1-P(C-), whereas only the anti-IFN activity is disabled in rHPIV1-CF170S. In primary human airway epithelial cell cultures, rHPIV1-P(C-) was considerably attenuated, suggesting that disabling the anti-IFN and antiapoptotic activities of HPIV1 had additive effects on attenuation. Its highly restricted replication in primary human airway epithelial cell cultures suggests that it might be overattenuated for use as a vaccine. Thus, the C proteins of HPIV1 are nonessential but have anti-IFN and antiapoptosis activities required for virulence. 18 Introduction Human parainfluenza virus type 1 (HPIV1) is a member of the Paramyxoviridae family, which includes a number of other medically important human pathogens such as HPIV2 and 3, respiratory syncytial virus (RSV), measles virus, mumps virus, and human metapneumovirus (90). The HPIVs are enveloped, non-segmented, single-stranded, negative-sense RNA viruses that are classified in the genera Respirovirus (HPIV1 and HPIV3) and Rubulavirus (HPIV2). HPIV1, 2 and 3 are significant respiratory pathogens for infants and young children, with clinical manifestations ranging from mild disease, including rhinitis and pharyngitis, to more severe disease, including croup, bronchiolitis, and pneumonia (31, 68, 69, 90, 122, 154). The contribution of HPIV infections to pediatric respiratory hospitalizations varies between studies and ranges from 7-21% overall for HPIV1, 2 and 3. The HPIVs collectively are the second leading cause of pediatric hospitalizations for viral respiratory disease behind RSV and ahead of influenza (86, 90, 134). A licensed vaccine is currently not available for the prevention of HPIV disease, but experimental live attenuated candidate vaccines are under development for HPIV1, 2 and 3, with those for HPIV3 in clinical trials (8, 12, 61, 89, 92, 139). The HPIV1 genome is 15,600 nucleotides in length and contains six genes in the order 3?-N-P/C-M-F-HN-L-5? (138). Each gene encodes a single protein with the exception of the P/C gene that encodes the phosphoprotein P in one open reading frame (ORF) and up to four accessory C proteins, C', C, Y1 and Y2, in a second, overlapping ORF. The synthesis of the C proteins initiates at four separate translational start codons in the C ORF in the order C', C, Y1, and Y2, and the four proteins are carboxy co-terminal (90). 19 However, it is unclear whether the Y2 protein is actually expressed during HPIV1 infection (152). C proteins are expressed by members of the Respirovirus, Morbillivirus and Henipavirus genera but not by viruses that belong to the Rubulavirus and Avulavirus genera. The paramyxovirus C proteins studied to date are non-essential accessory proteins that contribute significantly to virus replication and virulence in vivo (5, 84, 109, 110). The C proteins of Sendai virus (SeV), a member of the Respirovirus genus and the closest homolog of HPIV1, are the most extensively characterized. The C proteins of SeV have been shown to have multiple functions that include inhibition of host innate immunity through antagonism of interferon (IFN) induction and/or signaling (51, 56, 104), regulation of viral mRNA synthesis by binding to the L polymerase protein (21, 33, 75, 110, 185), participation in virion assembly and budding via an interaction with AIP1/Alix, a cellular protein involved in apoptosis and endosomal membrane trafficking (66, 82, 156), and regulation of apoptosis (see below). SeV mutants containing deletions of all four C proteins are viable, but are highly attenuated in vitro and in mice (55, 109, 110). To date, the HPIV1 C proteins have not been as extensively studied as those of SeV. However, the HPIV1 C proteins, like the SeV C proteins, play a role in evasion of host innate immunity through inhibition of type I IFN production and signaling (18, 192). Type I IFN was not detected during infection with HPIV1 wild type (wt) in A549 cells, a human epithelial lung carcinoma cell line, but was induced during infection with a recombinant HPIV1 (rHPIV1) mutant bearing a F170S amino acid substitution in C, designated rHPIV1-CF170S (192). HPIV1 wt virus, but not the rHPIV1-CF170S mutant virus, inhibited the antiviral state induced by type I IFN, most 20 likely due to inhibition of STAT1 nuclear translocation in human lung cells (18, 192). Many viruses have evolved strategies to regulate host cell apoptotic responses to virus infection (reviewed in (127)). Apoptosis, a process of programmed cell death mediated by the activation of a group of caspases, results in systematic cellular self-destruction in response to a variety of stimuli (reviewed in (40)). There are two major apoptotic pathways, the extrinsic and intrinsic pathways, which converge at a step involving the activation of the effector caspase 3. The activation of effector caspases, nuclear condensation and fragmentation, and cell death are the final steps in the apoptosis pathway. Viral proteins that are able to modulate the host apoptotic response include pro-apoptotic viral proteins such as West Nile virus capsid protein (208) and bunyavirus NSs proteins (28), and anti-apoptotic viral proteins such as RSV NS proteins (14), Bunyamwera virus NSs (101) and Rift Valley fever NSm protein (204). However, these studies are complex and some viral proteins such as influenza A virus NS1 have been reported to have both pro- and anti-apoptotic functions (166, 220). SeV C proteins have been implicated in the regulation of apoptosis but their role in this process remains incompletely defined (15, 71, 83, 105, 145). A role for HPIV1 proteins in apoptosis has not been investigated to date. In the present study, a rHPIV1 mutant was generated in which the C protein ORF was modified using reverse genetics to preclude expression of any of the four C proteins while maintaining expression of a wt P protein. This mutant, designated rHPIV1-P(C-), was evaluated for replication in vitro and in vivo in order to determine: i) whether C was 21 essential for HPIV1 replication; ii) what effect the deletion of C protein expression had on the host response to virus infection; and iii) whether a C protein deletion mutant had properties that warranted further development as a live attenuated HPIV1 candidate vaccine. In contrast to HPIV1 wt (but similar to rHPIV1-CF170S), rHPIV1-P(C-) was found to induce a robust IFN response. Additionally, in contrast to HPIV1 wt (and also in contrast to rHPIV1-CF170S), rHPIV1-P(C-) induced a potent apoptotic response. Both phenotypes appeared to contribute to attenuation in cultures of human ciliated airway epithelium. Materials and Methods Cells and Viruses LLC?MK2 cells (ATCC CCL 7.1) and HEp?2 cells (ATCC CCL 23) were maintained in Opti?MEM I (Gibco?Invitrogen, Inc. Grand Island, NY) supplemented with 5% FBS and gentamicin sulfate (50 ?g/ml). A549 cells (ATCC CCL-185) were maintained in F-12 nutrient mixture (HAM) (Gibco-Invitrogen, Inc.) supplemented with 10% FBS, gentamicin sulfate (50 ?g/ml) and L-glutamine (4 mM). Vero cells (ATCC CCL-81) were maintained in MEM (Gibco-Invitrogen, Inc.) supplemented with 10% FBS, gentamicin sulfate (50 ?g/ml) and L-glutamine (4 mM). BHK-T7 cells, which constitutively express T7 RNA polymerase (20), were kindly provided by Dr. Ursula Buchholz, NIAID, and were maintained in GMEM (Gibco-Invitrogen, Inc.) supplemented with 10% FBS, geneticin (1 mg/ml), MEM amino acids, and L-glutamine (2 mM). Human airway tracheobronchial epithelial (HAE) cells were isolated from airway specimens of patients without underlying lung disease provided by the National 22 Disease Research Interchange (NDRI, Philadelphia, PA) or from excess tissue obtained during lung transplantation, provided by the UNC Cystic Fibrosis Center Tissue Culture Core under protocols approved by the University of North Carolina at Chapel Hill (UNC) Institutional Review Board. Growth and differentiation of these cells on semi-permeable Transwell inserts at the air-liquid interface generated ciliated human airway epithelium, as previously described (149). All infections were incubated at 32?C except where indicated otherwise. Biologically-derived wt HPIV1 Washington/20993/1964, the parent of rHPIV1, was isolated from a clinical sample in primary AGM kidney (AGMK) cells, passaged 2 more times in primary AGMK cells (135) and once in LLC-MK2 cells (6). This preparation has a wt phenotype in AGMs, and will be referred to here as HPIV1 wt, but it was previously referred to as HPIV1LLC1 (6). The rHPIV1 wt referred to in this study also contains a mutation in the HN gene, HNT553A, that has previously been shown not to have an effect on virus replication (5) and is therefore considered the equivalent of wt virus. rHPIV1 wt was generated as previously described (6, 138, 192). The rHPIV1 wt was used in all experiments, with the exception that the biological HPIV1 wt was used for the hamster challenge and in the AGM studies, as indicated. The generation and characterization of the rHPIV1-CF170S mutant also was described previously (6); this virus contains a single nucleotide substitution in the P/C gene that creates a phenylalanine-to- serine substitution at amino acid 170 (numbered relative to the C' protein) that affects all four C proteins and is silent in the P protein. Media used for propagation and infection of HPIV1 wt and rHPIV1 mutants in LLC?MK2 cells did not contain FBS but contained 23 1.2% TrypLE Select, a recombinant trypsin (Gibco-Invitrogen, Inc.), in order to cleave and activate the HPIV1 fusion (F) protein, as described previously (138). Purified virus stocks were obtained by infecting LLC-MK2 cells, followed by centrifugation and banding of virus containing supernatant in a discontinuous 30/60% (w/v) sucrose gradient, steps designed to minimize contamination with cellular factors, especially IFN. Recombinant vesicular stomatitis virus expressing the green fluorescent protein (VSV- GFP) was originally obtained from John Hiscott (176). Stocks of VSV were propagated in Vero cells and sucrose purified as indicated above. Virus titers in samples were determined by 10-fold serial dilution of virus in 96-well LLC-MK2 monolayer cultures, using two or four wells per dilution. After 7 days of incubation, infected cultures were detected by hemadsorption with guinea pig erythrocytes, as described previously (169). Virus titers are expressed as log10 50% tissue culture infectious dose per ml (log10 TCID50/ml). VSV stock titers were determined by plaque assay on Vero cells under a 0.8% methyl cellulose overlay. Antibodies Polyclonal antisera directed against the HPIV1 C or P proteins were generated by repeated immunization of rabbits with the following KLH-conjugated peptides: (i) QMREDIRDQYLRMKTERW (amino acid (aa) residues 153-170 of HPIV1 C'; directed against the carboxyl terminal region of C', C, Y1 and Y2), for Skia-31; and (ii) RDPEAEGEAPRKQES (aa 10-24 of P), for Skia-2. Antisera were generated at Spring Valley Labs (Woodbine, MD). Two murine monoclonal antibodies directed against the 24 HPIV1 HN protein, designated 8.2.2.A and 4.5, were kindly provided by Dr. Yasuhiko Ito (103). Construction of mutant rHPIV1-P(C-) cDNA Nucleotide insertions, deletions and substitutions were introduced into the P/C gene of rHPIV1 wt (Figure 1A) (137) in order to silence the expression of the C', C, Y1, and Y2 proteins without affecting the P protein (Figure 1B and C). The 93 nucleotides between the P/C gene start signal and the P start codon, including the C' start codon, were deleted (Figure 1B and C, mutation 2) and replaced with a 6 nucleotide insertion to act as a ?linker? (Figure 1B and C, mutation 1). The sequence immediately upstream of the P start codon was modified by the addition of the ?linker?: CGA(ATG) to AAC(ATG), making the P start site more efficient by Kozak?s rules (106) and reducing translational initiation at the downstream start codons (Figure 1B and C, mutation 1). The C start codon was modified (ATG to ACG) (Figure 1B and C, mutation 3), and three codons were converted to stop codons, including one immediately downstream of the Y1 start codon (TCA to TGA), which will affect all of the C proteins except Y2, and two downstream of the Y2 start codon (TCG to TAG; TTG to TAG), which will affect all of the C proteins (Figure 1B and C, mutations 4, 5, and 6). All of the introduced changes are silent in the P protein. These changes were achieved using a modified PCR mutagenesis protocol described elsewhere (129) and the Advantage?HF PCR Kit (Clontech Laboratories, Palo Alto, CA). The entire PCR amplified gene product was sequenced using a Perkin?Elmer ABI 3100 sequencer with the Big Dye sequencing kit (Perkin?Elmer Applied Biosystems, Warrington, UK) to confirm amplification of the 25 desired sequence containing the introduced changes. Full?length antigenomic cDNA clones (FLCs) of HPIV1 containing the desired mutations were assembled in T7 polymerase-driven plasmids using standard molecular cloning techniques (138), and the region containing the introduced mutation in each FLC was sequenced as described above to confirm the presence of the introduced mutation and absence of adventitious changes. Each virus was designed to conform to the rule of six, i.e., the nucleotide length of each genome was designed to be an even multiple of six (102), a requirement for efficient replication of HPIV1. Figure 1. Designing the HPIV1-P(C-) viral cDNA. (A) The HPIV1 wt genome, shown 3? to 5?, includes the P/C gene that encodes the phosphoprotein P from one ORF and the four carboxy-coterminal C proteins, C', C, Y1 and Y2, from a second, overlapping ORF. The coding regions for these proteins are shown, with the initiation and termination codons numbered according to the P/C gene sequence. (B) Various mutations were introduced into the HPIV1 P/C gene to silence expression of the four C proteins without affecting the amino acid sequence of the P protein. Panel B shows the sequence of the upstream end of the P/C gene, with the transcription gene-start signal and the translational start signal for each protein boxed. Nucleotide (nt) substitutions and an insertion in the rHPIV1-P(C-) sequence are indicated in boldface, and a deletion is indicated with a dotted line. These mutations are identified with circled numbers that correspond with a description in panel (C) of the effect of each mutation. Briefly, 93 nt were deleted between the gene-start signal and P start codon and replaced with a 6-nt spacer CCCAAC (mutations 1 and 2), thus eliminating the first 11 codons of C' including 26 its start codon. The sequence immediately upstream of the P start codon was modified: CGA(ATG) to AAC(ATG), which will also optimize the Kozak sequence and reduce translational initiation at the downstream start codons (mutation 1). The methionine start codon of the C protein was converted to threonine (mutation 3), and one stop codon was introduced downstream of the Y1 start codon (mutation 4) and two stop codons were introduced downstream of the Y2 start codon (mutations 5 and 6). This cDNA was used to recover infectious rHPIV1-P(C-). 27 Recovery of infectious rHPIV1-P(C-) rHPIV1-P(C-) was recovered using a reverse genetics system, similar to previously described methods (138), in BHK-T7 cells constitutively expressing T7 polymerase (20) that were grown to 90 to 95% confluence in six-well plates. Cells were transfected with 5 ?g of the FLC, 0.8 ?g each of the N and P, and 0.1 ?g of the L support plasmids in a volume of 100 ?l of Opti-MEM per well. Transfection was carried out with Lipofectamine 2000 (Invitrogen, Inc., Carlsbad, CA), according to the manufacturer's directions. The transfection mixture was removed after a 24 h incubation period at 37?C. Cells were then washed and maintained in GMEM supplemented with amino acids and 1.2% TrypLE Select and transferred to 32?C. On day 2 following transfection, the supernatant was harvested. Virus was amplified by passage on LLC-MK2 cells and cloned biologically by two successive rounds of terminal dilution using LLC?MK2 monolayers on 96?well plates (Corning Costar Inc., Acton, MA). To confirm that rHPIV1-P(C-) contained the appropriate mutations and lacked adventitious mutations, viral RNA (vRNA) was isolated from infected cell supernatants using the QIAamp viral RNA mini kit (Qiagen Inc., Valencia, CA), reverse transcribed using the SuperScript First?Strand Synthesis System (Invitrogen, Inc., Carlsbad, CA), and amplified using the Advantage HF cDNA PCR Kit (Clontech Laboratories). The viral genome was sequenced in its entirety, confirming its sequence. Western Blot 28 LLC-MK2 monolayers grown in 6 well plates (Costar) were mock-infected or infected at an input multiplicity of infection (MOI) of 5 TCID50/ml with sucrose-purified rHPIV1 wt or rHPIV1-P(C-). Cell lysates were harvested 48 h post infection (p.i.) with 200 ?l of 1X Loading Dye Solution sample buffer (Qiagen, Inc) and purified on QIAshredder (Qiagen, Inc.) spin columns. Ten ?l (for Skia-31 probing) or 6 ?l (for Skia-2 probing) of each sample was reduced, denatured and loaded onto 10-well 10% Bis-Tris gels (Invitrogen, Inc.). Gels were run in MOPS buffer (Invitrogen, Inc.), and protein was transferred onto PVDF membranes (Invitrogen, Inc.) and blocked overnight at 4?C in PBS/Tween (0.1%) containing 3% BSA. PDVF membranes were incubated with 15 ml of a 1:1000 dilution of primary antibody in PBS/Tween with 1% BSA at room temperature (RT) for 2 h and then were washed 3 times for 10 min with PBS/Tween. Membranes were incubated for 1 h at RT with a 1:20,000 dilution of peroxidase labeled goat anti-rabbit IgG (KPL, Gaithersburg, MD) as the secondary antibody. After washing 3 times for 10 min with PBS/Tween, SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) was added for 10 min at RT. Membranes were developed on Kodak MR films (Kodak, Rochester, NY). Kinetics of replication of rHPIV1-P(C-) The rHPIV1 wt and rHPIV1-P(C-) viruses were compared in multi-cycle growth curves. Confluent monolayer cultures of LLC-MK2 cells in 6-well plates were infected in triplicate at a MOI of 0.01 TCID50/cell. Virus adsorption was performed for 1 h in media containing trypsin. The inoculum was then removed and cells were washed three times, after which fresh medium containing trypsin was added and then harvested as the day 0 29 sample and replaced with fresh media containing trypsin. On days 1-7 p.i., the entire supernatant was removed for virus quantitation and was replaced with fresh medium containing trypsin. Supernatants containing virus were frozen at -80?C, and virus titers (log10 TCID50/ml) were determined with endpoints identified by hemadsorption. Cytopathic effect (cpe) was visually monitored. The amount of cpe observed under the microscope was given a score ranging from 1-5 based on the percentage of cells in the monolayer showing cpe. Cpe of less than 20% of cells was scored as 1; 21-40% as 2; 41%-60% as 3; 61-80% as 4; 81-100% as 5. Immunostaining and confocal microscopy LLC-MK2 cells were seeded onto 24 well plates containing 12 mm glass cover slips, were mock-infected or infected with rHPIV1-P(C-) or rHPIV1 wt at a MOI of 10 TCID50/cell, and were incubated for 72 h. Media was removed and cover slips were washed twice with PBS. Cells were then fixed with 3% formaldehyde solution in PBS for 40 min at RT, washed once with PBS, permeabilized with 0.1% Triton X-100 in PBS for 4 min at RT, and washed twice with PBS prior to blocking with PBS containing 0.25% BSA and 0.25% gelatin for 1 h at RT. HPIV1 HN staining was performed using a 1:4000 dilution of a mixture of HPIV1-HN 8.2.2.A and HPIV1-HN 4.5, two murine antibodies directed against the HPIV1 HN protein, kindly provided by Yasuhiko Ito, Mie University School of Medicine, as primary antibody. After incubation at RT for 1 h, cells were washed twice with PBS and stained with a 1:1000 dilution of Texas Red conjugated donkey anti-mouse IgG (Jackson Immunochemicals, West Grove, PA), as secondary antibody, for 1 h at RT. Activated caspase 3 was detected using a 1:25 dilution of a 30 FITC-conjugated rabbit anti-human activated caspase 3 antibody (BD Pharmingen, San Jose, CA). Cells were washed twice with PBS and immediately mounted onto slides with the DAPI-containing antifade reagent, ProLong Gold (Invitrogen, Inc.). Slides were covered with foil and left to dry overnight at RT, then stored at -20?C until microscopy was performed on a Leica SP5 confocal microscope. FACS analysis LLC-MK2, Vero, or A549 cells in 6-well plates were mock-infected or infected with rHPIV1 wt or rHPIV1-P(C-) at a MOI of 5 TCID50/cell. Cells were harvested at 24, 48, and 72 h p.i. by scraping cells into 2 ml of fresh FACS buffer (PBS; 1% FBS) and pelleting at 1200 rpm for 10 min at 4?C. Cells were resuspended in 1 ml of 3% paraformaldehyde (PFA) and fixed for 15 min on ice, then rinsed twice in 3 ml FACS buffer. Cells were permeabilized and stained with the following antibodies diluted in FACS buffer containing 0.1% Triton-X-100: i) rabbit anti-human activated caspase 3 FITC (1:100; BD Pharmingen); and ii) mouse anti-PIV1 HN (1:2000 of a 1:1 mix of HPIV1-HN 8.2.2.A and HPIV1-HN 4.5). Staining was performed for 45 min at RT in a dark environment then cells were rinsed twice with 2 ml FACS buffer and stained with APC-conjugated goat anti-mouse IgG (1:1000; Jackson ImmunoResearch Laboratories, West Grove, PA), diluted in FACS buffer containing 0.1% Triton-X-100. Staining was carried out for 30 min at RT. Finally, cells were rinsed twice in FACS buffer and resuspended in 250 ?l FACS buffer for analysis. Sample analysis was carried out on a FACSCalibur (BD Biosciences, San Jose CA) using CellQuestPro software. Further analysis was performed using FlowJo software (TreeStar Inc., Ashland, OR). 31 Type I IFN bioassay The amount of type I IFN produced by HPIV1-infected A549 cell cultures was determined by an IFN bioassay, as previously described (192). Type I IFN concentrations were determined by measuring the ability of samples to restrict replication of VSV-GFP on HEp-2 cell monolayers in the samples in comparison to a known concentration of a human IFN-? standard (AVONEX; Biogen, Inc., Cambridge, MA). Briefly, samples were treated at pH 2.0 to inactivate virus and acid-labile type II IFN prior to being serially diluted 10-fold in duplicate in 96-well plates of HEp-2 cells along with the IFN-? standard (5000 pg/ml). After 24 h, the cells were infected with VSV-GFP at 6.5x104 PFU/well. After an additional 24 to 36 h, plates were read for GFP expression using a typhoon 8600 phosphorimager (Molecular Dynamics, Sunnyvale, CA). The dilution at which the level of GFP expression was approximately 50% of that in untreated cultures was determined as the end-point. The end-point of the AVONEX standard was compared to the end-point of the unknown samples, and IFN concentrations were determined and expressed as mean ? SE (pg/ml). Viral inoculation of HAE Apical surfaces of HAE were rinsed with PBS to remove apical surface secretions and fresh media was supplied to the basolateral compartments prior to inoculation. The apical surfaces of HAE were inoculated with HPIV1s at a low input MOI (0.01 TCID50/cell) in a 100 ?l inoculum, and the cultures were incubated at 37?C. The inoculum was removed 2 h p.i., and apical surfaces rinsed for 5 min with PBS and then 32 incubated at 37?C. At days 0-7 p.i., apical samples were collected by incubating the apical surface with 300 ?l of media for 30 min at 37?C, after which the media was recovered. Samples were stored at -80?C prior to determination of virus titer. Statistical analysis The Prism 5 (GraphPad Software Inc., San Diego, CA) one-way ANOVA test (Student- Newman-Keuls multiple comparison test) was used to assess statistically significant differences between data groups (P < 0.05). Results Construction and recovery of a rHPIV1 mutant not expressing any of the four C proteins The P/C gene of HPIV1 wt encodes the phosphoprotein, P, in one ORF and four carboxy co-terminal C proteins, C', C, Y1 and Y2, in a second, overlapping ORF (Figure 1A). We engineered rHPIV1 to silence expression of all four C proteins without affecting the P protein, creating the mutant virus rHPIV1-P(C-) (Figure 1B and C). The changes introduced to silence expression of the C proteins included the deletion of the 3? portion of the P/C gene containing the C' start, conversion of the C start to an ACG codon, and the introduction of three stop codons into the C ORF immediately downstream of the Y1 and Y2 start codons (Figure 1B and C). Importantly, all of the introduced changes were silent in the P protein (Figure 1B and C). The Y1 and Y2 start codons were not modified since any changes introduced at these sites would have altered P protein amino acid assignments. The AUG to ACG change at the start site of C would not necessarily silence its expression entirely, since ACG functions (inefficiently) as a start codon for the 33 C' protein of SeV, but other changes at this site could not be accommodated without affecting P coding, and in any event any residual expression of C would be ablated by the three stop codons that were introduced downstream. The recombinant virus was recovered from this mutant cDNA in cell culture and the virus replicated to 8.0 log10 TCID50/ml. Sequence analysis of the entire virus genome revealed that rHPIV1-P(C-) contained all the intended mutations and no unintended changes (data not shown). Western blot analysis of infected LLC-MK2 cell lysates using an antibody directed against the carboxy terminus of the C proteins demonstrated the expression of the C' and C proteins in cells infected with HPIV1 wt, but not rHPIV1-P(C-) (Figure 2A). C' was found to be the most abundant C protein, and Y1 and Y2 were not detected. An additional unidentified species, indicated with an asterisk in Figure 2A, was detected in cells infected with rHPIV1-P(C-), but not in cells infected with rHPIV1 wt (Figure 2A). This species was not detected using an antibody directed against the amino terminus of the C' and C proteins (data not shown). An ATG codon in the C ORF that is downstream of the last inserted stop codon in Y2 could potentially give rise to a truncated protein that would be carboxy-coterminal with the C proteins and would be 157 aa in length, compared to 204 aa for the C protein and 175 aa for Y2 (Figure 1A). This 47 aa difference in predicted size between C and the unknown protein in Figure 2A would correspond to an approximately 5 kDa difference in the proteins? apparent molecular weights, consistent with the mobility difference observed in our Western blot (Figure 2A). The P protein could be detected in both rHPIV1 wt- and rHPIV1-P(C-)-infected cells (Figure 2B). The ability to recover the rHPIV1-P(C-) mutant indicates that the four 34 wild type C proteins are not essential for replication in vitro, with the caveat that there was expression of a new species that may have been a truncated C protein. Figure 2. Identification of HPIV1 C and P proteins in lysates from infected LLC-MK2 cells. Lysates were prepared 48 h p.i. from LLC-MK2 cells that were mock-infected or infected with sucrose-purified rHPIV1 wt or rHPIV1-P(C-) at an input MOI of 5 TCID50/cell. Reduced, denatured cell lysates were resolved by SDS-PAGE electrophoresis and Western blots were prepared and analyzed using rabbit anti-peptide antisera against (A) the HPIV1 C proteins and (B) the HPIV1 P protein. The asterisk (*) in panel A indicates a new band of unknown identity, detected only in the rHPIV1-P(C-)- infected cell lysates. 35 The rHPIV1-P(C-) mutant replicates efficiently in vitro but causes increased cpe compared to rHPIV wt Multi-cycle replication of the rHPIV1-P(C-) mutant was assessed in LLC-MK2 cells infected at a MOI of 0.01 TCID50/cell (Figure 3A). LLC-MK2 cells were chosen since HPIV1 wt replicates efficiently in this cell line. rHPIV1-P(C-) and rHPIV1 wt replicated to similar titers until day 3 p.i., when rHPIV1 wt continued to increase in titer whereas rHPIV1-P(C-) decreased in titer. Concomitantly, rHPIV1-P(C-)-infected LLC-MK2 cells developed extensive cpe while rHPIV1 wt-infected cells did not (Figure 3B). This also is evident in photomicrographs of LLC-MK2 cells taken 72 h following infection at MOIs of 0.01 or 5 TCID50/cell (Figure 3A). In LLC-MK2 cells infected at a MOI of 5 TCID50/cell, increased cpe associated with rHPIV1-P(C-) but not rHPIV1 wt became evident at approximately 48 h p.i. (data not shown). Similarly, enhanced cpe associated with infection by the rHPIV1-P(C-) mutant was observed in A549 cells (data not shown), which were evaluated since they are a human respiratory tract-derived continuous cell line and in vivo HPIV1 replicates preferentially, if not exclusively, in respiratory epithelial cells. In summary, rHPIV1-P(C-) and rHPIV1 wt replicated with equal efficiency early in infection, but there was a subsequent decrease in rHPIV1-P(C-) titers that was temporally associated with development of extensive cpe, a phenomenon not seen in rHPIV1 wt-infected cells. 36 Figure 3. Comparison of the replication of rHPIV1 wt and rHPIV1-P(C-) viruses in vitro. (A) Multi-cycle replication in LLC-MK2 cells infected at a MOI of 0.01 TCID50/cell. On days 0-7 p.i., the overlying medium was harvested for virus titration, shown as the means of 3 replicate cultures. On days 1-7 p.i., the cell monolayers were monitored for cpe and assigned a score of 1-5 according to the extent of cpe (Materials and Methods), shown as the means of the 3 replicate cultures. (B) LLC-MK2 cells were mock-infected or infected with rHPIV1 wt or rHPIV1-P(C-) at a MOI of 0.01 or 5 TCID50/cell, as indicated in parentheses below the virus names. Photomicrographs taken at 72 h p.i. show increased cytopathic effect (cpe) in the rHPIV1-P(C-)-infected cultures (magnification, x10). 37 Infection with rHPIV1-P(C-) infection induces apoptosis To further explore the basis of the enhanced cpe associated with the rHPIV1-P(C-) mutant, we assayed virus-infected LLC-MK2 cells for activation of caspase 3, the major effector caspase in the apoptotic pathway. Activated caspase 3 was visualized by immunofluorescence (Figure 4A) and by FACS analysis (Figure 4B) using an antibody that specifically recognizes the cleaved, activated form of the enzyme. Replicate LLC- MK2 monolayers were infected at a MOI of 10 TCID50/cell, incubated for 24, 48 and 72 h, fixed, permeabilized, and stained with antibodies for HPIV1 HN (Texas-Red) and for activated caspase 3 (FITC). The HPIV1 HN antigen was detected in the vast majority of cells infected with either virus (Figure 4A). By 72 h p.i., activated caspase 3 was detected in the majority of the rHPIV1-P(C-)-infected cells but not rHPIV1 wt-infected cells (Figure 4A). In addition, cell rounding and nuclear condensation was seen in the majority of rHPIV1-P(C-)-infected cells but not in rHPIV1 wt-infected cells (Figure 4A, asterisk), consistent with the interpretation that the rHPIV1-P(C-)-induced cpe is the direct result of virus-induced apoptosis. We next determined the frequency of apoptosis in infected cells using flow cytometry. The rHPIV1-CF170S mutant, which encodes a F170S substitution in C, and which has previously been associated with type I IFN induction and effective type I IFN signaling, but not with cpe in vitro, was included here for comparison. Replicate cultures of LLC- MK2 cells were infected with rHPIV1 wt, rHPIV1-CF170S, or rHPIV1-P(C-) at a MOI of 5 TCID50/cell and, at 24, 48, and 72 h p.i., were fixed, permeabilized, and immunostained 38 for HPIV1 HN protein and activated caspase 3 (Figure 4B). More than 70% of cells in the rHPIV1-P(C-)-infected cultures were positive for activated caspase 3 by 72 h p.i., compared to approximately 5% and 7% in the rHPIV1 wt- and rHPIV1-CF170S-infected cell cultures respectively (Figure 4C). Similar studies in Vero and A549 cells confirmed that rHPIV1-P(C-) was a potent activator of caspase 3 activation while rHPIV1 wt was not, although the level of caspase 3 activation in these cells was lower than in LLC-MK2 cells (data not shown). Vero cells were also evaluated here since vaccine viruses to be used in clinical trials are prepared in this cell line and therefore it is important to determine the growth characteristics of potential vaccine viruses in this cell line. By 72 h p.i., approximately 12% and 18% of rHPIV1-P(C-)-infected Vero and A549 cell cultures, respectively, were positive for activated caspase 3, compared to approximately 4% of rHPIV1 wt-infected cell cultures for both cell types. Figure 4. Infection with rHPIV1-P(C-) induces activation of caspase 3, indicative of apoptosis. Caspase 3 activation was evaluated by immunostaining and FACS analysis. (A) Evaluation of caspase 3 activation by immunofluorescence. LLC-MK2 cells were mock-infected or infected with rHPIV1 wt or rHIV1-P(C-) at a MOI of 10 TCID50/cell. At 72 h p.i., cells were fixed, permeabilized, and stained for HPIV1 HN protein (red) and activated caspase 3 (green), and nuclei were stained with DAPI (blue). Cells were visualized by confocal microscopy and scale bars represent 10 ?m. (B) Evaluation of caspase 3 activation by FACS analysis. LLC-MK2 cells were mock-infected or infected with rHPIV1 wt, rHPIV1-CF170S, or rHPIV1-P(C-) at a MOI of 5 TCID50/cell in triplicate. Cells were harvested at 24, 48, and 72 h p.i., fixed, permeabilized and stained for HPIV1 39 HN and activated caspase 3 in FACS buffer prior to analysis. Sample analysis was carried out using a FACSCalibur flow cytometer and FlowJo software. Dot plots of representative data for samples from the 48 h time point are shown. (C) Percentage of cells positive for activated caspase 3 at 24, 48 and 72 h p.i., as determined by FACS analysis ? S.E. rHPIV1-P(C-), but not rHPIV1 wt, induces type I IFN production and signaling HPIV1 C proteins have been shown to inhibit production of and signaling by type I IFN (18, 192). We have previously demonstrated that type I IFN was not detected during infection of A549 cells with HPIV1 wt but was efficiently produced in response to rHPIV1-CF170S (192). To determine the relative effect of deleting all four C proteins on the ability of HPIV1 to inhibit the type I IFN response, we infected A549 cells at a MOI 40 of 5 TCID50/cell with rHPIV1-P(C-), rHPIV1 wt or rHPIV1-C F170S and subsequently quantified type I IFN in medium supernatants using a bioassay based on the inhibition of infection and GFP expression by VSV-GFP (Figure 5A). As shown previously, rHPIV1 wt inhibited the IFN response effectively (Figure 5A), with barely detectable levels of IFN-? appearing late in infection, at 72 h p.i. In contrast, both rHPIV1-P(C-) and rHPIV1-CF170S induced a robust type I IFN response, with IFN detectable in the supernatant as early as 24 h p.i. and until 72 h p.i., achieving a peak concentration of approximately 300 pg/ml at 48 h p.i. These data suggest that C proteins play a critical role in antagonism of type I IFN production but that IFN generation is unrelated to the apoptotic response seen with the C mutants since the F170S mutant generates similar levels of IFN with no observable cpe beyond wt HPIV1. We have previously demonstrated that type I IFN signaling leading to the establishment of an antiviral state is inhibited following infection with HPIV1 wt, but not with mutants that encode defective C proteins, e.g. rHPIV1-CF170S (192). To determine the relative effect of deleting all four C proteins on the ability of HPIV1 to inhibit type I IFN signaling, Vero cells were mock-infected or infected with rHPIV1 wt, rHPIV1-CF170S, or rHPIV1-P(C-) at a MOI of 5 TCID50/cell for 24 h, treated with 0, 100 or 1000 IU of IFN- ? for 24 h, and infected with 200 PFU/well of VSV-GFP. The number of VSV-GFP foci were counted 48 h later and the percent inhibition due to IFN-? treatment was calculated relative to cells that did not receive IFN-? (Figure 5B). In control cells that were not infected with rHPIV1, VSV-GFP replication was completely inhibited by IFN-? treatment. In contrast, infection with rHPIV1 wt ablated the ability of 100 IU/ml of IFN- 41 ? to inhibit VSV-GFP replication and blunted the inhibitory effect of 1000 IU/ml of IFN- ?, indicating that rHPIV1 wt can prevent IFN-? signaling and the induction of an antiviral state. Infection with rHPIV1-P(C-) did not inhibit the antiviral effect of IFN-? at either concentration, an effect similar to that for rHPIV1-CF170S (Figure 5B). In summary, unlike rHPIV1 wt, rHPIV1-P(C-) is unable to inhibit both the production of type I IFN and the induction of an antiviral state by IFN-?. Figure 5. rHPIV1 wt, but not rHPIV1-P(C-), inhibits type I IFN induction and signaling. (A) Induction of type I IFN. A549 cell monolayers were either mock-infected or infected with rHPIV1 wt, rHPIV1-CF170S, or rHPIV1-P(C-) at a MOI of 5 TCID50/cell. Aliquots of the overlying medium were taken at 0, 24, 48 and 72 h p.i. and assayed on fresh cells for the ability to inhibit infection and GFP expression by VSV-GFP as measured with a phosphorimager. IFN concentrations were determined by comparison with a standard curve prepared in parallel with an AVONEX? IFN-? standard and are expressed in pg/ml ? SE based on triplicate samples. The lower limit of detection was 39.1 pg/ml (dashed line). (B) Type I IFN signaling. Vero cells in 6-well plates were infected with the indicated rHPIV1s at a MOI of 5 TCID50/cell and incubated for 24 h. Cells were then left untreated or were treated with 100 or 1000 IU/ml IFN-? (1 well per treatment per virus) for 24 h. The cells were then infected with VSV-GFP and incubated for 48 h. The VSV-GFP foci were visualized using a phosphorimager and counted. The graph represents the percent inhibition of VSV-GFP replication in IFN-? treated versus untreated cells based on two independent experiments. 42 Replication of rHPIV1-P(C-) is restricted in human airway epithelial (HAE) cells in vitro HPIV1 wt has previously been shown to infect ciliated apical cells in an in vitro model of the human airway epithelium (11). Here, we characterized the ability of the rHPIV1- P(C-) to infect HAE in a multiple cycle growth curve. Following apical inoculation at low input MOI (0.01 TCID50/cell), rHPIV1 wt replicated efficiently in HAE, reaching a peak titer of 7.4 log10 TCID50/ml in the apical wash fluid. However, replication of the 43 rHPIV1-P(C-) was severely restricted in human ciliated cells, reaching a barely detectable peak of 1.8 log10 TCID50/ml (Figure 6). Figure 6. rHPIV1-P(C-) replicates very poorly in primary human airway epithelial (HAE) cells compared to rHPIV1 wt. HAE cultures were inoculated on the apical surface with either virus at a MOI of 0.01 TCID50/cell, and virus titers were determined in apical surface washes at days 0-7 p.i. These are shown as the means of triplicate cultures from two donors ? S.E., and the limit of detection is 1.2 log10TCID50/ml. Discussion A recombinant HPIV1 mutant, rHPIV1-P(C-), that does not express any of the four wild type C proteins but does express a wild type P protein was generated and characterized in 44 vitro and in vivo. rHPIV1-P(C-) was found to replicate efficiently in vitro, implying that the HPIV1 C proteins are non-essential accessory proteins. However, rHPIV1-P(C-) expressed a novel protein not seen with rHPIV1 that may have been a truncated form of C, and thus we cannot yet conclude that C-related proteins are completely dispensable. rHPIV1-P(C-) replicated with the same efficiency as HPIV1 wt early after infection of human- and monkey-derived cell lines, but its replication subsequently decreased coincident with the onset of extensive cpe that was not observed with rHPIV1 wt. The C proteins of SeV have been extensively characterized as non-essential gene products with multiple functions (105, 109, 110). However, SeV and HPIV1 differ with regard to the genetic organization of their accessory proteins and the phenotypes specified by accessory protein mutations. First, SeV encodes a V protein in addition to the C proteins, whereas HPIV1 does not (90). Second, deletion of all four C proteins in SeV significantly restricted its replication in vitro (66, 105, 109), whereas loss of the wild type forms of all four HPIV1 C proteins did not appear to reduce the replication efficiency of rHPIV1-P(C-) apart from the indirect effect of its enhanced cpe. Third, a six amino acid deletion in the N terminal region of the SeV C protein had a profound effect on replication in its natural host, i.e., rodents (47), whereas a similar mutation in HPIV1 C did not affect replication in non-human primates, the closest available animal model to its natural human host (5). Fourth, the F170S mutation in SeV C induced apoptosis in primary mouse pulmonary epithelial cells (83), whereas the same mutation in HPIV1 failed to specify this phenotype in the present study. Since the genetic organization of the accessory proteins of SeV and HPIV1 differ and since the phenotypes of C protein mutants differ significantly in vitro and in vivo, the functions of the HPIV1 C proteins 45 cannot be reliably inferred from findings obtained with SeV C protein mutants, and therefore they must be determined directly. An amino acid alignment of the HPIV1 and Sendai virus C proteins is provided in Figure 7. In the case of HPIV1, this information has added importance since live attenuated vaccine candidates that are presently being prepared for clinical trials include mutations in the C protein. Figure 7. CLUSTAL W alignment of the C protein amino acid sequences of Sendai virus and HPIV1 (Genbank accession numbers NP_056874 and NP_604436, respectively). The alignment was generated using the MacVector software program (MacVector Inc., Cary, NC) using default parameters. Conserved amino acids are indicated with asterisks. Replication of rHPIV1-P(C-) in cell culture peaked early and then decreased steadily coincident with the development of extensive cpe that was not observed with HPIV1 wt. This cpe was associated with caspase 3 activation, cell rounding, nuclear condensation and nuclear fragmentation, indicating that it was apoptotic in nature. In this report, we 46 identified a novel protein expressed by rHPIV1-P(C-), therefore there is also the alternative possibility - albeit unlikely - that the observed apoptosis during infection with rHPIV1-P(C-) was a gain-of-function due to the novel protein. However, in other work in progress, we have generated additional HPIV1 mutants that also express this novel protein but do not induce apoptosis and do not specify an attenuation phenotype (data not shown). This indicates that the absence of the four C proteins, and not the presence of the additional protein, is associated with the apoptosis-inducing (and also the attenuation) phenotypes of rHPIV1-P(C-). Taken together, our data indicate that one function of the HPIV1 C proteins is to delay or prevent the apoptotic response of the infected cell. Apoptosis has also previously been associated with Sendai virus C protein mutants. SeV C deletion mutants have been shown to induce apoptosis in vitro, whereas SeV wt did not (83, 105), suggesting that both SeV C proteins and HPIV1 C proteins act as inhibitors of apoptosis. In addition, the SeV mutant Ohita MVC11, containing the CF170S substitution, was observed to induce cell death in vitro while SeV wt did not (83). Similar to our observations of rHPIV1-P(C-), Ohita MVC11 titers peaked early and decreased concomitant with the induction of apoptosis (83). However, data we have presented suggests that despite the relatively high degree of amino acid conservation (70% identity) between HPIV1 and Sendai virus C proteins (Figure 7), the induction of apoptosis and/or virus-mediated inhibition of apoptosis may be mediated by different mechanisms for HPIV1 and SeV C proteins. For example, although SeV Ohita MVC11 induces apoptosis, the HPIV1 mutant containing the homologous CF170S substitution, rHPIV1- CF170S, does not induce apoptosis. Furthermore, IRF-3 activation was recently shown to be required for apoptosis during SeV infection in human cell lines (145), but we have 47 previously shown that rHPIV1-CF170S, which does not induce apoptosis, stimulates IRF-3 activation (192). Studies to define the mechanism of apoptosis inhibition by HPIV1 have been initiated. The most extensively characterized function of paramyxovirus C proteins is their type I IFN antagonist activity. HPIV1 C proteins have been shown to disrupt the host type I IFN response by (i) inhibiting IRF-3 activation and thereby inhibiting the production of type I IFN, and (ii) inhibiting STAT nuclear translocation (18) and thereby inhibiting the JAK-STAT signaling pathway (192). Our results support these previous findings by demonstrating that, in the absence of C proteins, type I IFN is produced in response to HPIV1 infection and is able to successfully establish an antiviral state in respiratory epithelial cells. In contrast, infection with HPIV1 wt inhibits type I IFN production as well as the establishment of an antiviral state that results from the activation of the JAK/STAT pathway (54, 60, 114, 158, 183, 198). This pathway controls transcription of a group of more than 300 genes termed the IFN-stimulated genes, which have antiviral, antiproliferative, immunomodulatory, and apoptosis modulating functions (114). Therefore, it is not surprising that many viral proteins that have been characterized as IFN antagonists have also been identified to play a role in the regulation of apoptosis. Examples of such proteins include the Bunyamwera virus NSs proteins (101, 197), the RSV NS1 and NS2 proteins (14, 173) and the influenza A virus NS1 protein (166, 182, 196, 220). Our data demonstrate that the HPIV1 C proteins also act both as type I IFN antagonists and as apoptosis antagonists. Interestingly, however, the anti-IFN and anti- apoptosis activities of the HPIV1 C proteins were separable: while the rHPIV1-P(C-) and 48 rHPIV1-CF170S mutants were indistinguishable with regard to the induction of IFN production and signaling in cell culture, only rHPIV1-P(C-) induced apoptosis. Since the type I IFN response and apoptosis are both components of the host?s innate antiviral response, viruses that have lost the ability to inhibit these responses are often attenuated. The attenuation phenotype of the rHPIV1-P(C-) virus was evaluated in an in vitro model of human ciliated airway epithelium (HAE). In order to obtain an assessment of attenuation prior to the initiation of clinical trials, replication of rHPIV1-P(C-) was characterized in a HAE model, which uses primary human airway epithelial cells grown at an air-liquid interface to generate a differentiated, pseudo-stratified, ciliated epithelium that bears close structural and functional similarity to human airway epithelium in vivo (149, 219). A similar model has been used previously to evaluate the attenuation of RSV vaccines (205). We have also recently characterized the replication of HPIV1 wt and several HPIV1 vaccine candidates in the in vitro HAE model and observed a strong correlation between attenuation in AGMs and attenuation in HAE cells, indicating the potential usefulness of the HAE model for evaluating potential respiratory virus vaccine candidates (11). In our current HPIV1 study, growth of rHPIV1-P(C-) in HAE cells was barely detectable, whereas rHPIV1 wt grew to high titer. This suggests that this virus might be over-attenuated in humans, potentially limiting its efficacy as a vaccine. Therefore, we are currently proceeding to clinical trials with a previously characterized virus, rHPIV1-CR84G/?170HNT553ALY942A (8), that is less attenuated than rHPIV1-P(C-). Only if rHPIV1-CR84G/?170HNT553ALY942A proves to be insufficiently attenuated will we advance rHPIV1-P(C-) into clinical trials. 49 Previous studies of rHPIV1 C mutants demonstrated that in vivo attenuation correlated with the ability to stimulate an effective type I IFN response, including IFN production and IFN signaling (5, 192). This was demonstrated in detail for rHPIV1-CF170S (192). The rHPIV1-P(C-) virus was indistinguishable from rHPIV1-CF170S with regard to the induction of type I IFN production and signaling to establish an antiviral state in vitro. However, rHPIV1-P(C-) was much more attenuated in AGMs than rHPIV1-CF170S. This increased level of attenuation might be due to the induction of apoptosis by rHPIV1-P(C- ), a property not shared by rHPIV1-CF170S. We did not determine whether apoptosis was induced in vivo following rHPIV1-P(C-) infection, however, a SeV mutant containing the CF170S mutation was shown to induce apoptosis both in vitro and in vivo, in the bronchial epithelium of mice, and this virus was also attenuated in its natural host (49, 83). It is possible that the in vitro apoptosis phenotype of rHPIV1-P(C-) can translate to in vivo effects. This would suggest that the greater level of attenuation of rHPIV1-P(C-) versus rHPIV1-CF170S is based on two additive effects: i) the ability (of both viruses) to activate the type I IFN response; and ii) the ability of rHPIV1-P(C-) (but not PIV1-CF170S) to induce apoptosis. However, it is also possible that a C protein function other than inhibition of the IFN response and apoptosis contributed to the high level of attenuation of rHPIV1-P(C-). In summary, we have demonstrated that the HPIV1 C proteins are non-essential viral proteins that play an important role in viral replication in vitro and in vivo and act as antagonists of the type I IFN response and of apoptosis. Our HPIV1 C protein deletion 50 mutant is highly attenuated in primary human airway epithelium. It remains to be determined whether such a highly attenuated virus would be useful as a vaccine candidate against PIV1 in humans. 51 Chapter 3: The C proteins of human parainfluenza virus type I (HPIV1) control the transcription of a broad array of cellular genes that would otherwise respond to HPIV1 infection From Journal of Virology 2009 Feb; 83(4): 1892-1910 Jim B. Boonyaratanakornkit, Emmalene J. Bartlett, Emerito Amaro-Carambot, Peter L. Collins, Brian R. Murphy and Alexander C. Schmidt Laboratory of Infectious Diseases, RNA Viruses Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892-8007, USA. 52 Abstract HPIV1 is an important respiratory pathogen in children and the most common cause of viral croup. We performed a microarray-based analysis of gene expression kinetics to examine how wild-type (wt) HPIV1 infection altered gene expression in human respiratory epithelial cells and what role IFN? played in this response. We similarly evaluated HPIV1-P(C-), a highly attenuated and apoptosis-inducing virus that does not express any of the four C proteins, and HPIV1-CF170S, a less attenuated mutant that contains a single point mutation in C and, like wt HPIV1, does not efficiently induce apoptosis, to examine the role of the C proteins in controlling host gene expression. We also used this data to investigate whether the phenotypic differences between the two C mutants could be explained at the transcriptional level. Mutation or deletion of the C proteins of HPIV1 permitted the activation of over 2000 cellular genes that otherwise would be repressed by HPIV1 infection. Thus, the C proteins profoundly suppress the response of human respiratory cells to HPIV1 infection. Cellular pathways targeted by the HPIV1 C proteins were identified and their transcriptional control was analyzed using bioinformatics. Transcription factor binding sites for IRF and NF-?B were over- represented in some of the C protein targeted pathways, but other pathways were dominated by less known factors such as forkhead transcription factor FOXD1. Surprisingly, the host response to the P(C-) and CF170S mutants was very similar, and only subtle differences in the expression kinetics of caspase 3 and the TRAIL receptor 2 were observed. Thus, changes in host cell transcription did not reflect the striking phenotypic differences observed between these two viruses. 53 Introduction Human parainfluenza virus type 1 (HPIV1) is the principal cause of laryngotracheobronchitis or croup, and HPIV1, 2, and 3 are collectively the second most common cause of pediatric respiratory hospitalizations, only surpassed by respiratory syncytial virus (42, 134). The clinical manifestations of HPIV1 range from mild disease, including rhinitis, pharyngitis, and otitis media, to severe disease, including croup, bronchiolitis, and pneumonia (30). Licensed vaccines are currently not available against any of the HPIVs but ongoing HPIV3 vaccine development efforts that employ cDNA derived live attenuated viruses have progressed to phase 1/2 clinical evaluation (89, 91, 93). HPIV1 is an enveloped, negative sense, nonsegmented RNA virus. The viral genome, 15.6 kb in length, consists of 6 genes (3? ? N-P/C-M-F-HN-L ? 5?) that encode the nucleoprotein (N), phosphoprotein (P), C proteins (C), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and the large polymerase protein (L). The C proteins comprise a nested set of four carboxy-coterminal proteins, designated C', C, Y1, and Y2, that are expressed from individual start sites in a second open reading frame (ORF) within the P/C gene and are thought to play an important role in HPIV1 virulence (138). The C proteins of Sendai virus (SeV), or murine PIV1, are the best studied of the paramyxovirus C proteins, and there is considerable sequence conservation between the SeV and HPIV1 C proteins. However, the P/C gene organization of SeV differs from that of HPIV1 in that SeV expresses a second accessory protein, the V protein, in addition to the C proteins. There are also phenotypic differences between SeV 54 and HPIV1 C protein mutants. SeV mutants that do not express any of the C proteins (SeV C knockouts) could be recovered using reverse genetics which identified the C proteins as non-essential viral proteins (108). However, SeV C knockouts are restricted in replication in vitro and in vivo, which reflects the observation that the C proteins are multifunctional (48, 83, 123, 152). SeV C proteins have been shown to regulate viral replication (33), promote viral assembly (66), confer virulence in mice (85), suppress apoptosis in HEp-2 cells (105), and interfere with antiviral host defense through interferon (IFN) antagonism (57, 96). Recent studies evaluating the HPIV1 C proteins have also identified them as non-essential (9) and demonstrated an important role for the HPIV1 C proteins as IFN antagonists following infection of A549 and MRC-5 human respiratory epithelial cells (18, 192). Specifically, the HPIV1 C proteins inhibit the induction and signaling of IFN in the host cell. The induction of IFN synthesis following virus infection depends on a number of pattern- recognition receptors that recognize conserved pathogen-associated molecular patterns and initiate downstream signaling cascades (87). The IFN antiviral pathway can be activated by double stranded RNA (dsRNA), 5'-triphosphate single stranded RNA or viral infection. The presence of dsRNA, an intermediate of viral replication, is recognized as a pathogen-associated molecular pattern by Toll-like receptor 3 (TLR3) and two caspase recruitment domain (CARD) containing RNA helicases, retinoic acid-inducible gene I (RIG-I) and melanoma associated-differentiation gene 5 (MDA-5), which are constitutively expressed and act as intracytoplasmic sensors of dsRNA (2, 180, 214). RIG-I also specifically binds single stranded RNA with 5?- triphosphate ends to 55 distinguish self from non-self RNA (76, 147, 180, 212). TLR3 uses the adaptor TIR domain-containing adaptor inducing IFN? (TRIF) while RIG-I and MDA-5 recruit another CARD-containing adaptor mitochondrial antiviral signaling protein (MAVS, also referred to as IPS-1, Cardif or VISA) to relay the signal to the kinases TBK1 and IKK-i, which phosphorylate IRF3 (interferon regulatory factor 3), and to IKK?, which activates the NF-?B pathway (41, 99, 140, 167, 207). Once activated, IRF3 translocates into the nucleus and binds the positive regulatory domain III (PRDIII) of the IFN? promoter. RIG-I and MDA-5 can distinguish between different RNA viruses (98). IFN production in response to SeV infection is specifically dependent on RIG-I expression, whereas IFN production in response to picornavirus infection is dependent on MDA-5 expression (98). IRF-3 and NF-?B, together with ATF-2/c-Jun, form an enhanceosome that binds to the IFN? promoter, inducing transcription of the IFNB gene. IFN?, in turn, controls the expression of a broad array of antiviral genes (210). Transcriptional activation of type I IFN is biphasic. One gene encodes the prototypical IFN?, while more than a dozen IFN species comprise the IFN? family (183). Recognition of viral infection results in phosphorylation of constitutively expressed IRF3 to induce IFN? and IFN?1 expression in human cells as part of the early response to virus infection. Once early type I IFN is produced and secreted, it binds to the type 1 IFN receptor on the surface of the infected and neighboring cells, activates the JAK-STAT signal transduction cascade, and induces formation of IFN-stimulated gene factor 3 (ISGF3) which consists of a heterotrimeric complex of STAT1, STAT2, and IRF9. ISGF3 binds to the IFN-stimulated response elements (ISRE) within the promoter regions of several hundred IFN-inducible genes, including 2?-5?-oligoadenylate synthetase (OAS), dsRNA dependent protein kinase 56 (PKR), and IRF7. The de novo produced IRF7 is phosphorylated and can then activate production of additional type I IFNs, including IFN? and the full set of IFN??s (163). Thus, immediate-early expression of IFN? and IFN?1, induced by IRF3, NF-?B, and ATF-2, occurs soon after viral infection is detected, while delayed expression of the entire type I IFN family, induced by IRF7, occurs only after IFN? and IFN?1 secretion, receptor binding, and signaling to amplify a robust feed-forward antiviral response (121, 183). Our laboratory has established a reverse genetics system for HPIV1 that allows the generation of infectious virus from cDNA (85, 137). Using this system, a number HPIV1 mutants containing mutations in the C gene have been generated to investigate C protein function and to generate novel live attenuated experimental vaccines that are attenuated by disturbing the IFN antagonistic function of the C gene (192). Previously, a phenylalanine to serine substitution of amino acid 170 of SeV was shown to significantly attenuate a highly virulent SeV strain, increasing the lethal dose 50% (LD50) 20,000-fold in mice (50). This CF170S mutation, which affects all four C proteins without affecting the P protein, was introduced into the P/C gene of recombinant (r) HPIV1 by reverse genetics (85, 137). This mutant, designated as rHPIV1-CF170S and also referred to here as CF170S, was previously found to be defective in inhibiting IFN? induction and signaling in vitro and to be attenuated for replication in the respiratory tract of hamsters and African green monkeys (AGMs) (5, 192). Whereas wild-type (wt) HPIV1 infection abrogated IRF3 dimerization and nuclear translocation, infection with rHPIV1- CF170S resulted in IRF3 activation and IFN? production (192). The wt HPIV1 C proteins were also shown to 57 inhibit IFN signaling by blocking STAT1 and STAT2 nuclear translocation, and wt HPIV1 could overcome a pre-existing IFN?-induced antiviral state in MRC-5 cells (18). In contrast to wt HPIV1, rHPIV1- CF170S did not inhibit the establishment of an antiviral state (192). More recently, we constructed a mutant HPIV1 that does not express any of the four C proteins, which is designated rHPIV1-P(C-) and also is referred to here as P(C-) (9). In this virus, the C' start codon was deleted, the C start codon was mutated, and several stop codons were inserted to ensure that the C', C, Y1, and Y2 proteins could not be expressed. All of the above mutations were designed to be silent in the P ORF. Interestingly, the P(C-) and CF170S mutant viruses differ in their in vitro and in vivo phenotypes. The P(C-) mutant is highly attenuated in vivo in AGMs whereas the CF170S mutant is only moderately attenuated. In addition, infection with the P(C-) mutant leads to early and significant induction of apoptosis while CF170S and wt HPIV1 are very weak inducers of apoptosis (9). Microarray-based analyses are increasingly being used in virology and have helped to elucidate virus-host interactions for a number of viruses (reviewed in (88)). In the present study, we used a microarray-based analysis of the kinetics of gene expression to examine: 1) how wt HPIV1 infection altered human respiratory epithelial cell gene expression; 2) what role IFN? played in this response; 3) how the response to infection with the C mutant viruses CF170S and P(C-) compared to the response to wt HPIV1; and 4) whether the phenotypic differences between the two C mutant viruses (level of 58 attenuation and apoptosis phenotype) could be explained on a transcriptional level. We compared the mRNA levels in A549 cells treated with IFN? or infected with wt HPIV1, CF170S, or P(C-) using a microarray that represented the full complement of known human genes. To examine the function of the C proteins in the infected cell, the expression patterns of cells infected with HPIV1 C mutant viruses were compared to that of cells infected with wt HPIV1. We show that the C proteins of HPIV1 profoundly suppress the innate response of human respiratory cells to infection with this important pediatric respiratory virus. Materials and Methods Cell culture and viruses A549 human respiratory epithelial cells (ATCC catalog number CCL-185, Manassas, VA) were maintained in F-12 medium supplemented with 0.1mg/mL gentamicin sulfate, 4 mM L-glutamine (Gibco-Invitrogen, Carlsbad, CA) and 5% fetal bovine serum (Hyclone, Logan, UT). LLC-MK2 cells (ATCC) were maintained in Opti-MEM I (Gibco-Invitrogen) supplemented with 0.1mg/mL gentamicin sulfate and 5% fetal bovine serum. Recombinant wt HPIV1 and mutant CF170S and P(C-) were each recovered from cDNA as previously described (9, 137, 138). All viral infections were carried out at 32?C in media containing 1.2% recombinant trypsin TrypLE Select (Gibco-Invitrogen) without fetal bovine serum. Virus stocks were generated by infecting LLC-MK2 cells at a multiplicity of infection (MOI) of 0.01 50% tissue culture infectious dose (TCID50) per cell and by 59 harvesting supernatant on day 7 post infection (p.i.). Virus particles in the supernatant were purified by centrifugation in a discontinuous 30%/60% sucrose gradient in 0.05M HEPES and 0.1M MgSO4 (Sigma-Aldrich, St. Louis, MO) at 120,000g for 90 minutes at 4?C. This purification was performed to minimize contamination of virus suspensions used to infect cells with cellular proteins, including IFN. Virus titers were determined by infecting LLC-MK2 cell monolayers with serial 10-fold dilutions of virus, and infected cultures were detected 7 days p.i. by hemadsorption with guinea pig erythrocytes (168). The titer of sucrose purified wt HPIV1, CF170S, and P(C-) was determined at 8.9, 9.1, and 8.5 log10TCID50 per mL, respectively. Viral genomic RNA was isolated from the sucrose purified stocks for sequence analysis using the QIAamp viral RNA Mini Kit (Qiagen, Valencia, CA), then reverse transcribed using the Superscript II First Strand Synthesis System (Invitrogen), amplified using the Advantage HF-PCR Kit (Clontech, Mountain View, CA), and purified using the High Pure PCR Purification Kit (Roche, Indianapolis, IN) per the manufacturers? protocols and as previously described (138). The identity of each virus was confirmed by sequencing the entire viral genome using BigDye Terminator v1.1 on a DNA Analyzer 3730 (Applied Biosystems, Foster City, CA) and analysis with Sequencher 4.7 (Gene Codes Corporation, Ann Arbor, MI). Microarray sample preparation and expression analysis A549 cells were mock infected or infected in triplicate at a MOI of 5 TCID50 per cell with wt HPIV1, CF170S, or P(C-) for 6, 12, 24, and 48 h. In an independent experiment, 60 additional A549 cell cultures were treated in triplicate with 300 pg/mL (60 IU/mL) of IFN? (Avonex, Biogen Inc, Cambridge, MA) for 6 or 24 h. We chose this concentration because we previously found that infection of A549 cells with CF170S and P(C-) led to IFN secretion that resulted in an IFN concentration of approximately 300 pg/mL in the cell supernatant (9, 192). Thus, four treatment groups, i.e. IFN? treatment, as well as infection with wt HPIV1, CF170S, or P(C-) were separately analyzed; IFN treatment at two time points and each of the three virus infections at 4 time points. Cellular RNA was extracted from infected/treated cells using the RNeasy Mini Kit and treated with the RNase-free DNase set (Qiagen). RNA from virus infected and IFN? treated A549 cells was labeled with Cy5 while RNA from mock infected samples was labeled with Cy3 using the 2-Color Low RNA Input Linear Amp Kit PLUS (Agilent Technologies, Foster City, CA) as per the manufacturer?s protocol. Labeled RNA was hybridized onto Whole Human Genome 44K Oligo Microarrays (catalog number G4112F) and was scanned with a DNA Microarray Scanner (Agilent Technologies) per the manufacturer?s protocol. This microarray detects the expression of over 41,000 genes and expressed sequence tags, representing all known genes in the human genome. Spot detection, signal quantitation, dye normalization by Lowess regression, and quality control assessment of control RNA spike-ins was performed using Feature Extraction Software v9.5 (Agilent Technologies). Data was then loaded into GeneSpring GX 7.3.1 (Agilent Technologies) for signal normalization, statistical tests, and hierarchical clustering. Relative gene expression ratios in virus infected and IFN? treated cells compared to mock were calculated by dividing the intensity of the Cy5 signal by the Cy3 61 signal intensity. Each array was normalized to the median signal ratio of all probes on the array. Genes were defined as being differentially expressed between two samples if they were flagged as present in at least one sample and exhibited at least a 4-fold difference that was statistically significant after multiple testing correction with a Benjamini- Hochberg false discovery rate of P < 0.01. The 4-fold cutoff was chosen to minimize false positives and increase confidence in our data interpretation. Wei, et al. give sample sizes required for detecting differential gene expression with 1.5-, 2-fold, and 4-fold change cutoffs. Lower fold-change cutoffs lead to a greater rate of false positives and a requirement for much larger sample sizes to detect significant changes by statistical tests (200). The phenotypic differences between the C mutant viruses were striking and were expected to be associated with large changes in expression that could be detected above 4-fold. 2,612 genes were identified as significantly differentially expressed compared to mock in at least one of the four infection/treatment groups. To identify differences in gene expression between wt HPIV1, CF170S, and P(C-), we selected 1,314 of the 2,612 genes that demonstrated significant differential expression in at least one pair-wise comparison between virus infections at the same time point and/or between IFN? treatment and mock. Hierarchical clustering was performed on individual samples based on the expression ratios at each time point using a Pearson correlation as a measure of similarity between expression profiles. These clusters are groups of genes that share similarities in the pattern with which their expression varies over all sampling time points (38). All of the microarray data was deposited according to the MIAME standards to the Gene Expression Omnibus with accession number GSE12664 at http://www.ncbi.nlm.nih.gov/geo/. 62 Functional genomic bioinformatics The clusters identified above were further examined for commonalities in functional characteristics including over-representation of biological pathways and over- representation of transcription factor binding sites (TFBSs) within the respective cluster. Over-represented functional pathways within each cluster of genes were searched against the Agilent Whole Human Genome Reference Set using Ingenuity Pathway Analysis 5.5.1 (Ingenuity Systems, Redwood City, CA). Statistically over-represented functional pathways (P < 0.05 using a right-tailed Fisher?s exact test) were retained for further analysis. A P value less than 0.05 in the Fisher exact test indicates that the proportion of probes in the cluster, compared to every other probe on the Agilent Whole Genome array, involved in a pathway is greater than would be expected by chance. TFBSs that were over-represented within each cluster were identified using oPOSSUM Single Site Analysis software to search the 2000 base pair (bp) upstream sequence of each gene in the cluster against the JASPAR database (72, 161, 193). JASPAR is an open-access, curated, non-redundant database of 123 sequence motif matrices of experimentally defined TFBSs. Genes with assigned Entrez Gene identifiers were loaded into oPOSSUM to examine upstream sequences in all of the genes within the respective cluster of genes. oPOSSUM retrieves upstream promoter sequences from Ensembl (www.ensembl.org), uses phylogenetic footprinting to identify conserved, functional, non-coding DNA, and aligns sequences to position specific scoring sequence matrices in JASPAR in order to identify high-quality, predicted, evolutionarily conserved TFBSs. We used an 85% matrix match threshold, a Z-score > 10, and a Fisher exact test P < 0.05 to select for 63 statistically over-represented TFBSs within each cluster compared to the background of all genes in oPOSSUM. These criteria have been shown by testing randomly generated gene lists to yield a specificity of 86% (72). Real-time quantitative PCR (RT-qPCR) expression analysis Total RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) and amplified using the TaqMan Universal PCR Master Mix (Applied Biosystems). TaqMan Gene Expression Assays (Applied Biosystems) for IFNB1 (Hs01077958_s1), IRF7 (Hs00242190_g1), MX1 (Hs00182073_m1), NFKB1 (Hs00231653_m1), TRAF1 (Hs01090170_m1) and GAPDH (Hs99999905_m1) were chosen to validate microarray expression profiles, and samples were prepared for analysis as per the manufacturer?s protocol. The microarray analysis verified that GAPDH was an appropriate endogenous control since GAPDH expression was constant throughout all conditions and time points. Samples were run on the 7900HT Fast Real-time PCR System (Applied Biosystems). Quantitative PCR analysis was performed using SDS 2.3 and RQ Manager 1.2 (Applied Biosystems). All expression values were normalized against the GAPDH endogenous control and then normalized against mock infected samples to obtain relative gene expression ratios using the Pfaffl method (146). Results Differential gene expression following wt HPIV1 infection goes beyond modulation of the type 1 IFN response 64 In order to better understand the host cell response to HPIV1 infection, A549 human respiratory epithelial cells were infected with wt HPIV1 at a MOI of 5 TCID50 per cell or were mock infected, and total cellular RNA was extracted at 6, 12, 24, and 48 h p.i. RNA was reverse transcribed and changes in gene expression were compared to mock infected cells using a two-color 60-mer cDNA microarray platform representing more than 41,000 human genes and expressed sequence tags. To directly identify IFN-inducible genes in A549 cells for comparison to the wt HPIV1 induced genes, additional A549 cells were treated with 300 pg/mL (60 IU/mL) of IFN? or left untreated, and analyzed in the same way as the wt HPIV1 infected cells. This dose of IFN? was chosen because it approximates the concentration previously shown to be induced in this cell line by infection with the CF170S and P(C-) viruses (9, 192). An overview of this data set is depicted in Figure 1, column I. Each treatment group (i.e. wt HPIV1 or IFN?) at each time point consists of three replicates. A considerable number of known IFN-inducible genes, e.g. IRF7 and MX1, were up-regulated late in the course of wt HPIV1 infection, i.e., 48 h p.i., even though wt HPIV1 is known to inhibit type 1 IFN production and signaling in A549 cells (192). A total of 343 genes were differentially expressed (i.e. a four-fold or greater change compared to mock and P < 0.01) at one or more time points following infection with wt HPIV1 and/or IFN? treatment; overall, 284 genes for HPIV1 and 191 genes for IFN? (Fig. 2). 65 Figure 1. Comparison of A549 cellular genes responsive to infection with wt HPIV1 (referred to here as WT) or treatment with IFN?. Column I) Hierarchical clustering of the 343 genes that were differentially expressed (4-fold or greater change relative to mock, P < 0.01 with multiple testing correction) following wt HPIV1 infection and/or IFN? treatment, with the fold-change indicated in color. Each colored row represents an individual transcript, with representative genes identified immediately to the right. Each colored column consists of three replicates, side by side, with the treatment condition/time point identified at the bottom. The genes were clustered into five groups (A through E, demarcated to the right) according to similarity of expression kinetics and magnitude of expression versus mock at each time point using a Pearson correlation as a measure of similarity. The number of genes in each cluster is given in parentheses. Three of the clusters (A, B, and D) contain genes that were predominantly responsive to IFN? and/or wt HPIV1; one cluster (C) contains genes that were predominantly responsive to IFN? but not wt HPIV1, and one cluster (E) contains genes were predominantly responsive to wt HPIV1 but not IFN? (see the text). Column II) The gene expression profiles for clusters A through E are shown in panels A through E, respectively, with the geometric mean gene expression (y-axis) plotted against each treatment condition at the indicated time-points in h p.i. (x-axis). Column III) Functional pathways that are over- represented in clusters A through E were determined using Ingenuity Pathway Analysis and are indicated on the left, next to the expression profile of the respective cluster. TFBSs that are over-represented among the genes within each cluster were identified using oPPOSUM to search the JASPAR core database, and are indicated to the right. The JASPAR database contains IRF1 and IRF2 but does not include an entry for other IRFs. 66 To identify potential binding sites for other IRFs and ISGF3, we also scanned all clusters for the shared core sequence motif for the IRFs and ISGF3, GAAANNGAAA. 67 Figure 2. Total number of genes differentially expressed during HPIV1 infection and IFN? treatment compared to mock over time in h p.i. Specifically, the number of genes that were significantly differentially expressed, in comparison to mock infected cells, in cells infected with WT, CF170S, or P(C-) viruses at 6, 12, 24, and 48 h p.i., or in IFN? treated A549 cells at 6 and 24 h is indicated. Total represents the cumulative number of genes differentially expressed taking all time points into consideration. Infection with CF170S or P(C-) viruses led to a significant alteration of gene expression in many more genes than did infection with WT or treatment with IFN?. The 343 genes that were differentially expressed in response to wt HPIV1 and/or IFN? treatment were grouped into clusters based on the kinetics and magnitude of gene expression. This was done using a hierarchical clustering analysis of the 343 genes across 68 both treatments and all of the time points (e.g. wt HPIV1 at 6, 12, 24 and 48 h p.i., and IFN? at 6 and 24 h). Five different gene expression profile patterns were identified, including 3 clusters containing a combined total of 209 genes that were predominantly responsive to both IFN and wt HPIV1 (Fig. 1, clusters A, B and D), one cluster of 17 genes that was predominantly responsive to IFN? but not wt HPIV1 (cluster C), and one cluster of 117 genes that was predominantly responsive to wt HPIV1 but not IFN? (cluster E). Although, in total, the 226 genes from clusters A, B, C, and D can be grouped together based on the predominant kinetics of gene expression after IFN? treatment, only 191 of the 226 genes satisfied the 4-fold change criteria for IFN-responsive genes (Fig. 2). The remaining 34 genes were up-regulated less than 4-fold with IFN? treatment but greater than 4-fold with wt HPIV1 infection. Some representative members of each cluster are indicated in Fig. 1, column I; a complete list of the genes and their expression levels is provided in supplementary Table S1 of the appendix. The graphs in Fig. 1, column II represent the geometric mean value for the fold change over time compared to mock for all genes within each cluster and indicate a signature profile for the genes in that cluster. Nearly all of the 170 genes in cluster A were responsive to IFN? treatment, and a majority also were responsive to wt HPIV1 infection, although the response to wt HPIV1 infection was delayed by approximately 24 h (Fig. 1, column II, cluster A). In both treatment groups, the fold change increased with time. We next analyzed cluster A (and each succeeding cluster) for over-representation of functional pathways within each 69 cluster of genes using a software tool that determines the likelihood of a given number of genes being represented within a defined pathway (Ingenuity Pathway Analysis). The P value (Fisher?s exact test) for that likelihood is indicated next to the pathways in Fig. 1, column III. Genes involved in the Toll-like receptor and NF-?B pathways were over- represented in cluster A, which is not surprising given the observed up-regulation in response to IFN? and wt HPIV1 (Fig. 1, column III and Table S1). Aiming to understand the regulation of gene expression within a given cluster, the JASPAR database of TFBSs was searched for over-representation of specific TFBSs within the genes contained in each cluster. In cluster A, IRF1 and IRF3/ISGF3 TFBSs, which all share the same conserved consensus sequence, were found to be significantly over-represented (Fig. 1, column III). Since ISGF3, a heterotrimeric complex of STAT1, STAT2, and IRF9, is induced by binding of IFN? to the IFN receptor, this finding was not unexpected. Translocation of ISGF3 into the nucleus activates the transcription of genes with ISREs. IRF1 similarly binds to and activates upstream cis-acting elements of the IFNA and IFNB genes. Cluster B contained 27 genes that were predominantly up-regulated by IFN? and wt HPIV1. The mRNAs in cluster B were rapidly induced at 6 h post IFN? treatment but, on average, failed to increase further at 24 h, whereas HPIV1 infection led to a steady increase in the expression of the cluster B genes (Fig. 1 column II, cluster B). Genes within this cluster seem to be functionally involved in IFN signaling and IL6 signaling. Analysis of upstream cis-acting elements reveals that the family of NF??B binding sites 70 was significantly over-represented amongst these genes. IFIT2 (IFN-induced protein with tetratricopeptide repeats-2/ISG-54) was one of the most strongly expressed genes in cluster B and was up-regulated 85-fold by IFN? treatment and more than 500-fold following wt HPIV1 infection (Table S1). The 17 genes in cluster C were up-regulated by IFN? but not by wt HPIV1. Up- regulation was rapid, occurring by 6 h with continued increase at 24 h (Fig. 1, column II, cluster C). The genes in cluster C formed a relatively heterogeneous group that contained no significantly over-represented functional pathways or TFBSs, and contained only a few genes with known antiviral function such as the tripartite motif protein 34 (TRIM34) (115). It is significant that infection with wt HPIV1 did not induce or activate expression of the subset of IFN?-responsive genes that are represented in cluster C, suggesting that HPIV1 might activate a selective negative feedback mechanism that inhibits transcription of a subset of IFN?-responsive genes. Cluster D is comprised of 12 genes that as a group were strongly induced by wt HPIV1 infection but weakly induced with IFN? treatment. Only 2 genes were significantly induced by IFN? beyond 4-fold. Genes involved in IFN? and chemokine signaling, such as CCL3, CCL5, CXCL10 and IL28A/IFN?, were over-represented in this cluster, and both IRF1 and c-REL binding sites dominated in the promoter regions of cluster D genes (Fig. 1, column III, Table S1). 71 Cluster E contained 117 genes that were predominantly induced by wt HPIV1 but not by IFN?. Induction of the genes in cluster E was significantly delayed, i.e., it was not observed until 48 h p.i. This cluster notably included the cytokines IL6, IL8, TNF (tumor necrosis factor ?), and CXCL3, and, as a result, pathways involved in apoptosis, NF-?B, and death receptor pathways as well as in IL6 signaling were significantly over- represented in cluster E. The family of NF-?B TFBSs, but not ISRE or IRF, were statistically over-represented in the promoters of these genes. The five species of NF-?B transcription factors, RELA (p65), RELB, c-REL, NF-?B1 (p105/p50), and NF-?B2 (p100/p52), are present in homo- and heterodimers that are bound by I?B proteins and localized in the cytoplasm until I?B is phosphorylated by the I?B kinase, leading to its ubiquitination and proteasome-mediated degradation. This releases the various NF-?B dimers for nuclear translocation and binding to NF-?? TFBS in target genes. RELA, RELB, and c-REL each contain a transactivator domain that strongly activates transcription whereas NF-?B1 and NF-?B2 lack this domain and, when binding as homodimers, can function as transcription repressors (188). Taken together, two key signaling pathways, the NF-?B and the IFN pathway, are central to regulating the cellular antiviral and inflammatory response to wt HPIV1 infection. The HPIV1 C proteins inhibit the induction of gene expression in A549 cells The HPIV1 C proteins play an important role in the inhibition of IFN? production and signaling (18, 192). The recently generated rHPIV1 mutant that does not express any of the C proteins, designated P(C-), is similar to CF170S with regard to its IFN phenotype in A549 cells but differs from CF170S in that P(C-) is a strong inducer of apoptosis in LLC- 72 MK2 and A549 cells whereas CF170S more closely resembles wt HPIV1 in that it is a weak inducer of apoptosis (9, 192). In addition, P(C-) is more attenuated than CF170S in the respiratory tracts of hamsters and AGMs (9). We first sought to compare the number of genes that were differentially expressed by infection with wt HPIV1 or HPIV1 C mutant viruses to assess the role of the C proteins in controlling the host response to viral infection. Therefore, the number of significantly differentially regulated genes (4-fold cutoff, P < 0.01) in A549 cells infected with either the CF170S or the P(C-) mutant was compared to that with wt HPIV1 infection or IFN? treatment. Surprisingly, infection with CF170S or P(C-) altered the expression of a substantially greater number of genes than wt HPIV1 infection or IFN? treatment. Specifically, taking all of the time points into consideration, the CF170S mutant significantly induced 1,734 genes and suppressed 690 genes, whereas P(C-) induced 1,430 genes and suppressed 170 genes by 48 h p.i. In contrast, wt HPIV1 significantly induced 281 genes and suppressed 3 genes (Fig. 2). This indicates the profound effect the C proteins have on suppression of the host cell response to HPIV1 infection. Treatment with IFN? differentially induced 190 genes and suppressed only 1 expressed sequence tag at 6 h and/or 24 h post-treatment, based on a four-fold or greater change compared to mock-treatment, for a total of 191 IFN?-responsive genes (Fig. 2). For comparison, previous microarray-based studies that surveyed only a fraction of the human genome identified 14, 56, and 268 genes whose expression changed 10-fold, 4- fold, and 2-fold, respectively, in IFN? treated HT1080 human fibrosarcoma cells by 6 h and 42 genes whose expression changed 5-fold in IFN? treated A549 cells by 24 h (36, 73 59, 160). Interestingly, to the best of our knowledge, 66 of the 190 induced genes whose expression changed 4-fold or greater in the present study have not been previously described as IFN-regulated (Table S1) (36, 59, 160). Pair-wise comparisons of the fold change in the global gene expression (i.e., the entire microarray) was conducted for CF170S versus wt HPIV1, P(C-) versus wt HPIV1, and CF170S versus P(C-) at 6, 12, 24, and 48 h p.i. (Fig. 3). In this comparison, each gene is represented by a single dot, and the accumulation of dots along the center diagonal line in each plot in Fig. 3 indicates a similar fold change between the viruses represented on the x- and y-axes, whereas the two outer lines indicate a four-fold difference. This comparison of the kinetics of the cellular response to infection showed that global gene expression in cells infected with CF170S or P(C-) at 6 and 12 h p.i. did not differ significantly from that of wt HPIV1 infected cells (Fig. 3, 6 h and 12 h panels). Points deviate from the central diagonal only at 24 h and/or 48 h p.i., especially when either of the C mutant viruses are compared to WT due to the potent effects of infection with mutant viruses that are unable to suppress the cellular response to viral infection. By 24 h p.i., a significant proportion of genes were either up- or down-regulated more than four- fold in both the CF170S and P(C-) groups, as indicated by the number of dots outside the two outer boundaries that run parallel to the diagonal, while wt HPIV1 virus did not have this effect (Fig. 3, 24 h). At 48 h p.i., this difference in gene expression between each of the C mutants versus wt HPIV1 was even more pronounced. It was also important to compare transcriptional patterns between the two C mutant viruses in order to investigate why complete deletion of the C gene conferred a greater level of attenuation and an 74 apoptotic phenotype, in contrast to CF170S. Surprisingly, there was no apparent difference in global gene expression between the CF170S and P(C-) infected cells with the exception of a slightly more pronounced gene induction in P(C-) infected cells at 24 h p.i. (Fig. 3, right column). However, none of the genes differed four-fold or more in expression between these two C mutant viruses. Because of the remarkable lack of obvious differential gene expression between CF170S and P(C-) infected cells, the sequence identity of CF170S and P(C-) was confirmed in the supernatant of the respective cell cultures used in the RNA extraction for gene expression analysis (data not shown). 75 Figure 3. Pair-wise comparisons of global gene expression in A549 cells indicates that the CF170S and P(C-) viruses induce a highly similar host cell response that differs greatly from the wt HPIV1 (WT) -induced response. Each plot depicts the geometric mean difference for individual transcripts (represented by dots) from triplicate samples in pair- wise comparisons between cells infected with CF170S versus WT (left panels), P(C-) versus WT (middle panels), and CF170S versus P(C-) (right panels) at 6, 12, 24, and 48 h p.i. (rows 1, 2, 3, and 4, respectively). The central diagonal line indicates equal fold change between the virus pair under comparison; the outer boundaries indicate a four- fold change. 76 The observation that there was not a single gene that differed with statistical significance by four-fold or more between the two C mutant viruses at any time point might seem inconsistent with the data in Fig. 2 where, for example, the overall number of genes that were differentially regulated for each C mutant virus versus the mock-treated control was 1,600 for the P(C-) virus and 2421 for the CF170S mutant, an apparent difference of 821 genes. This apparent discrepancy can be reconciled by constructing a Venn diagram and inspecting the genes that are differentially expressed only by P(C-) infection compared to mock or only by CF170S infection compared to mock (Fig. S1). The difference in the number of differentially regulated genes, i.e. 821, between the P(C-) and CF170S groups compared to mock does not accurately represent the total number of genes that were differentially expressed between the two C mutant viruses because not every one of the 1600 genes that was differentially expressed by P(C-) infection was also differentially expressed by CF170S infection. The number of genes that are differentially expressed compared to mock during P(C-) or CF170S infection can be grouped into three sets: 169 genes that were altered by P(C-) infection only, 990 genes that were altered by CF170S infection only, and 1,431 genes that were altered by both C mutant infections (Fig. S1). Thus, in total, there were 1159 genes that were identified as differentially expressed compared to mock during infection with one C mutant but not the other. Of these 1159 genes, none was significantly diffesrentially expressed by 4-fold or more between the two viruses, suggesting that the differences were small and generally in the same direction, as indicated in the examples given in Table 1. Thus, fold-change and statistical filters that compared the two mutant viruses directly were needed to identify genes that differed 77 significantly so that subsequent functional bioinformatics analyses could be performed (Table 2 and Fig. S3). Organization of genes that are differentially regulated in wt and mutant HPIV1and in IFN? treatment groups into hierarchical clusters We further analyzed those genes that were differentially regulated (four-fold or greater difference, P < 0.01 with multiple testing correction) in any of the four treatment groups (wt HPIV1, CF170S, P(C-), and IFN?) versus the mock-treated control at any time point. These genes, which numbered 2,612, were subjected to pair-wise comparisons among the three virus treatment groups, analyzing each time point separately, in order to identify virus-to-virus differences that might account for phenotypic differences. Of the 2,612 genes, 1,238 were differentially regulated in at least one pair-wise comparison between any two virus groups at the same time point. Consistent with the analysis in Fig. 3, we did not identify a single gene that was significantly differentially expressed between CF170S and P(C-) infection when using a four-fold cutoff and multiple testing correction. The 1,238 genes included all but 76 of the 191 IFN?-responsive genes detected in this study; therefore, these additional 76 genes were added to the comparison so that the full 78 complement of IFN?-responsive genes could serve as a benchmark for analysis of a possible transcriptional basis for the phenotypic differences seen between wt HPIV1, CF170S and P(C-) infection, and also to permit an analysis of the behavior of IFN- responsive genes during infection with the HPIV1 C protein mutants. The total of 1,314 genes were hierarchically clustered into groups A-H based on similarities in the gene expression kinetics and magnitude, as described for Fig. 1 (Fig. 4 columns I and II; Table S2). Figure 4. Infection of A549 cells with CF170S or P(C-) drastically alters the expression of a large number of genes involved in diverse cellular functions. A total of 1,238 genes that were differentially expressed at 6, 12, 24, and 48 h p.i. following infection with CF170S, P(C-), or WT viruses were combined with 76 genes that were differentially regulated at 6 or 24 h after IFN? treatment and subjected to hierarchical clustering based on similarity of expression kinetics and magnitude of expression as described in the legend to Fig. 1, yielding eight clusters (A through H). Column I) As in Fig. 1, the clusters are depicted by a hierarchical clustering matrix with the columns representing treatment and time point and the rows indicating individual transcripts, with representative transcripts identified to the right. The number of genes in each cluster is given in parentheses. Column II) The geometric mean gene expression profiles for clusters A through H are shown in panels A through H, respectively, with the geometric mean gene expression (y- axis) plotted against each treatment condition at the indicated time-points in h p.i. (x- axis). Column III) Functional pathways that are over-represented in clusters A through H were determined using Ingenuity Pathway Analysis and are indicated on the left, next 79 to the expression profile of the respective cluster. TFBSs that are over-represented among the genes within each cluster were identified using oPOSSUM to search the JASPAR core database. Cluster F represented a distinct group of 223 mRNAs that individually were responsive to most or all of the four treatments (IFN?, CF170S, P(C-), or wt HPIV1) (Fig. 4, column I; Table S2). Cluster F included most of the 191 IFN?-responsive genes detected in this study. Cluster F is similar, but not identical, to cluster A in Fig. 1. In addition, the 80 majority of genes in cluster C of Fig. 1, which were up-regulated predominantly by IFN? treatment and not at all by wt HPIV1 infection, are found in cluster F of Fig. 4. The expression of 16 of these 17 genes was significantly more highly induced by infection with the C mutant viruses beginning at 24 h p.i. (Table S1 vs Table S2). Paradoxically, the remaining gene, namely angiotensinogen (AGT), in this group of 17 was down- regulated by the C mutant viruses even though it was induced by IFN?. This is an indication that the effects of the C proteins are more complex than a general inhibition of IFN production and signaling. For the genes in cluster F as a whole, the mean fold change induced by IFN? was approximately 3-fold at 6 h and approximately 8-fold at 24 h (Fig. 4, column II, cluster F). Infection with wt HPIV1 induced a delayed response that, at 48 h p.i., was similar in magnitude to the IFN? induced response at 24 h, whereas CF170S and P(C-) induced a far earlier and greater response than wt HPIV1 that equaled or exceeded that of IFN?. By 24 h p.i., CF170S and P(C-) induced on average a greater than 10-fold up- regulation of cluster F gene expression, exceeding the response induced by IFN? treatment for 24 h (Fig. 4 column II, cluster F). Amongst the genes within cluster F, genes involved in IFN signaling, antigen presentation, ubiquitination, and Toll-like receptor signaling were over-represented. As expected, ISGF3, IRF1, and IRF3 TFBSs were over-represented within the upstream promoter sequences of cluster F genes (Fig. 4, column III, cluster F). Virtually every member of cluster F was induced much earlier and to a greater extent by the CF170S and P(C-) mutants compared to wt HPIV1, suggesting that functional C proteins are crucial in suppressing the transcription of antiviral genes within this cluster. 81 The remaining 7 gene clusters (Fig. 4, column ??) contained only a few IFN??responsive genes and consisted mainly of genes that were differentially expressed between virus groups. Genes in clusters A, B, and C shared three features: 1) they were not significantly up-regulated by IFN?; 2) they were up-regulated by all three viruses; and 3) the fold increase in up-regulation was much greater for the C mutant viruses than for wt HPIV1. Nonetheless, each cluster exhibited a clear difference in the kinetics or magnitude of the up-regulation of expression. The 337 genes in cluster A, on average, were induced strongly following infection with either C mutant within 12 h and to a much more limited and delayed degree following wt HPIV1 infection (Fig. 4 columns I and II, cluster A). The 102 genes in cluster B shared a similar expression profile. However, infection with P(C-) up-regulated cluster B gene expression at 24 h p.i. to an even higher level than in cluster A, and expression was maintained at that level through 48 h p.i. In cluster B, wt HPIV1 infection led to a slightly more pronounced average gene induction at 48 h than in cluster A (Fig. 4 column II, cluster B). Cluster A included genes involved in the p38 MAPK, NF-?B, p53, and death receptor pathways, while genes involved in death receptor, IL6, and apoptosis pathways were over-represented in cluster B (Fig. 4 column III, clusters A and B). Over-representation of TFBSs suggested a role for forkhead transcription factors FOXD1 and FOXD3 in the regulation of cluster A genes and for c- REL, RELA, and NF-?B1 in the regulation of cluster B genes (Fig. 4, column III, panels A and B). The specific function FOXD1 and FOXD3 have not yet been well defined, but the family of forkhead transcription factors are best known for their role in regulating cellular development and differentiation (150). The 334 genes in cluster C behaved similarly to those in clusters A and B, but the magnitude of expression was less than that 82 in clusters A and B, and over-representation of only one functional pathway (lipid metabolism) was found in this large cluster of genes (Fig. 4, column III, cluster C). Clearly, functional C proteins partially suppress the activation of genes in clusters A, B, and C. Cluster D stood out because of its small size (only 9 genes were assigned to this cluster) and because these genes were up-regulated rapidly and to a very high level following wt or mutant HPIV1 infection. These findings indicate that wt C proteins permit the activation of these 9 genes. However, since the level of expression of these genes is slightly lower in the wt versus mutant HPIV1 infected groups, this indicates that the C proteins can have a quantitative effect on modulating the overall level of gene induction in response to viral infection (Fig. 4, column II, cluster D). Cluster D included mRNAs encoding IL6, IFN? and IL28A/ IFN?2, and the chemokines CX3CL1, CCL2, and CCL5 (Fig. 4, columns I-III, cluster D). c-REL TFBSs were over-represented in this set of genes. Cluster D genes contrast with those in clusters E and G which fail to activate during wt HPIV1 infection. The 25 genes in cluster E and the 48 genes in cluster G were induced at a relatively low level and only late following infection with CF170S or P(C-) but not at all following either wt HPIV1 infection or IFN? treatment (Fig. 4, column II, clusters E and G). The two clusters differed in the magnitude of expression between 24 h and 48 h p.i. since, after infection with the C mutant viruses, average gene expression increased substantially for cluster E but remained flat or decreased for cluster G. The genes in cluster E were only 83 marginally over-represented in the lipid metabolism pathway, and none of the TFBSs were over-represented in this cluster (Fig. 4, column II, cluster E). The 48 genes in cluster G, however, including pro-inflammatory genes (e.g. IL1B) as well as inhibitors of inflammation (e.g. SOCS3), were over-represented in the acute phase response and the IL10 pathway, with complex transcriptional regulation (Fig. 4, column III, cluster G). The transcription factors over-represented in this cluster included FOXI1, zinc finger protein 42 (ZNF42), and FOXD1. ZNF42 and both forkhead transcription factors FOXI1 and FOXD1 are best known for regulating cellular development and differentiation (78, 150). Lastly, cluster H included all of the genes down-regulated by infection with CF170S or P(C-) (Fig. 4, column II, cluster H). Infection with either C mutant virus significantly suppressed the expression of the 234 genes in this cluster. Genes involved in xenobiotic, hydrocarbon, and cellular stress / protein metabolism pathways were over-represented in this cluster, including members of the cytochrome P450 superfamily, glutathione transferases, and UDP-glucuronosyltransferase. Wt HPIV1 infection and IFN? treatment had no effect on these genes. The only transcription factor statistically over-represented was TCF1, also known as IFN production regulator factor, a protein that initially became known to control important liver specific genes involved in metabolism but more recently was found to be expressed in lung tissue as well (164) (Fig. 4, column III, cluster H). The gene expression profile of cluster H suggests that the C proteins expressed by wt HPIV1 inhibit the suppression of cluster H gene expression. 84 Taken together, the analysis of global gene expression in A549 cells infected with wt HPIV1, CF170S, or P(C-) suggests that wt HPIV1 is able to control a large and diverse array of host genes involved in the recognition of non-self and the initiation of an antiviral response. Ablation of HPIV1 C protein function, by mutation or deletion, allows for an innate response of epithelial cells that encompasses a multitude of biological processes, including IFN signaling, cell death, inflammation, and metabolic regulation. We have observed that infection with P(C-) rapidly induces apoptosis, which is not seen during infection with CF170S or wt HPIV1. The similarity of the host transcriptional response to infection with CF170S and P(C-) was unexpected (Figs. 2, 3, and 4 column I) since the C proteins are multifunctional. We applied a principal component analysis, using all genes on the microarrays to examine the level of similarity between treatments, and found that overall gene expression during P(C-) and CF170S infection was highly similar (Fig. S2). Furthermore, both C mutant viruses induced gene expression patterns that were significantly different from WT infection. This identifies a high degree of similarity between the global gene expression profiles of the two mutants, but it does not rule out the possibility that small, specific differences exist between the two mutants, as addressed below. One might have expected that the host cell response to infection with CF170S, the virus containing only a single point mutation in each of the four C proteins, would not be as dramatic as the response to infection with P(C-), the virus not encoding any of the C proteins. Real-time quantitative PCR confirms microarray-based observations regarding differential gene expression 85 In order to validate the kinetics of gene expression observed in the microarray study, five differentially expressed genes that encompassed a range of expression levels, kinetic patterns, and pathways representing all four treatment groups were selected for RT-qPCR analysis (Fig. 5). For all five mRNAs, the gene expression profiles determined by microarray analysis were generally confirmed by the RT-qPCR analysis, indicating that this microarray platform yielded reliable semiquantitative results when using triplicate samples. However, the microarray-generated data underestimated the magnitude of expression compared to RT-qPCR by a factor of 10 to 1000. This probably reflected the more limited dynamic range in microarray analyses and probe saturation with high mRNA levels, as reported previously in a comparison of expression levels determined by Northern blot versus microarray (52). However, the expression profiles derived from microarray data and RT-qPCR data, in general, followed the same trend over time. This confirmed that IRF7 and MX1 mRNAs were strongly induced by IFN? treatment, whereas IFN? mRNA was only weakly induced, and NFKB1 (transcription factor NF-?B p100/p52) and TRAF1 (tumor necrosis factor receptor-associated factor 1, involved in signal transduction) mRNAs were not induced at all by this treatment (Fig. 5). In addition, IRF7, NFKB1, and TRAF1 mRNAs were barely induced by wt HPIV1 infection but strongly induced by infection with either C mutant (Fig. 5). IFN? and MX1 mRNAs were also induced by wt HPIV1 infection, but not as strongly as following CF170S or P(C-) infection, confirming the previous expression pattern observed for cluster D genes (Fig. 4, column II, cluster D). 86 Figure 5. Comparison of the kinetics and magnitude of expression of representative genes quantified by microarrays versus RT-qPCR. An aliquot of the total RNA that was used for microarray analysis was reverse transcribed and amplified using TaqMan probes for A) IFNB, B) IRF7, C) MX1, D) NFKB1, and E) TRAF1. These include both IFN- responsive (A, B, and C) and IFN-independent (D, E) genes. Values were normalized to the endogenous control gene GAPDH and expressed as fold-increase relative to mock infection. Microarray expression values are indicated with a continuous line, while the dotted line represents qPCR expression values. Note, both axes are in logarithmic scale, and the qPCR data (left-sided axes) spans a much larger dynamic range than the microarray data (right-sided axes). 87 A more detailed examination of the IFN pathway Since ablation of the function of IFN antagonist proteins results in restriction of replication of many viruses in vivo (46, 192, 216) and since CF170S and P(C-) are attenuated in vivo but differ in their level of replication, we took a closer look at individual genes in the IFN pathway to examine whether subtle differences in their expression level in CF170S and P(C-) infected cells could explain the greater level of attenuation of P(C-). We examined the kinetics and magnitude of expression of genes encoding type 1 IFNs and their receptors (Fig. 6A), as well as that of genes involved in IRF3 and IFN signaling (Fig. 6B). Since the number of genes in these pathways is large, only selected genes representative of most pathway members are presented. Interestingly, CF170S and P(C-) infection, but not wt HPIV1 infection or IFN? treatment, induced transcription of members of the family of IFNA genes such as IFNA4, 5, 8, 10, and 14, but the expression of the type 1 IFN receptors IFNAR1 and IFNAR2 was not affected by any of the treatments (Fig. 6A). IRF3 can induce a subset of antiviral effector molecules in the absence of IFN?, such as IFIT1 (IFN-induced protein with tetratricopeptide repeats-1/ISG60), ISG15, and IFI44. A hierarchical clustering matrix for these three genes is shown in Fig. 6B, top panel. These IRF3-regulated genes were more strongly induced in C mutant virus infected cells compared to wt HPIV1 infected or IFN? treated cells (see expression profile in Fig. 6B). Since IRF3 dimerization and activation occurs upstream of IFN? and ISGF3 signaling during viral infection, these genes would be induced more rapidly than genes that are 88 only ISGF3-stimulated, but, later in infection, IFN-stimulation would contribute to expression of both sets of genes. A selection of genes that are only ISGF3-stimulated is shown in the lower panel of Fig. 6B, and indeed appears to be induced less rapidly than the IRF3-stimulated genes. For all of these genes, the expression profiles were highly similar in CF170S and P(C-) infected cells. Throughout the time course, wt HPIV1 failed to induce ISGF3-responsive genes to a level comparable to that induced by CF170S or P(C-), consistent with the reduced expression of IFN (Fig. 6B). MX2 expression was completely suppressed for 24 h in the presence of wt C protein whereas CF170S or P(C-) induced high levels of MX2 mRNAs by 24 h p.i. (Fig. 6B). Importantly, wt HPIV1-induced expression of IFN?- and IRF3- responsive genes, including IRF7, ADAR, and PKR, was delayed, diminished, or completely absent even at 48 h p.i. compared to CF170S or P(C-) induced up-regulation (Fig. 6B). Interestingly, CF170S or P(C-) infection up-regulated these IFN?- or IRF3- responsive genes far more than IFN? treatment (Fig. 6B). 89 Figure 6. The CF170S and P(C-) mutant viruses are similar with regard to the expression of IFNs, IFN receptors, and IRF3-responsive genes. A) Hierarchical clustering matrix of genes encoding IFNs and IFN receptors and B) Hierarchical clustering matrix of genes encoding IRF3-responsive (first three entries) and ISGF3-responsive (all of the entries) genes. Treatment conditions and time points are indicated below the hierarchical clustering matrix. On the right, expression kinetics are shown for four representative genes of each group, with the geometric mean gene expression (y-axis) plotted against each treatment condition at the indicated time-points in h p.i. (x-axis), to illustrate the full range of differential gene expression. 90 Changes in host cell transcription do not reflect the striking cytopathic effect induced by P(C-) infection P(C-) is a potent inducer of apoptosis in A549 cells while CF170S and wt HPIV1 are not (9). We sought to examine individual genes in the apoptosis pathway to see if subtle differences in their expression level in CF170S, P(C-), and wt HPIV1 infected cells could be detected that might explain the apoptosis phenotype seen in cells infected with P(C-). To do this, we looked at the kinetics and magnitude of expression of individual genes in the extrinsic and intrinsic apoptosis pathways. Despite this prominent phenotypic difference, CF170S and P(C-) induced remarkably similar transcriptional profiles (Fig. 7). Both C mutant viruses up-regulated the expression of the same death ligands and death receptors examined with highly similar kinetics and magnitude (Fig. 7A and B). In addition, no difference was found in the regulation of apoptosis inhibitors such as BIRC3 and BIRC5 (Baculoviral IAP Repeat-Containing Protein 3 and 5), BCL2A1 (BCL2- Related Protein A1) and MCL1 (Myeloid Cell Leukemia I) (Fig. 7C; Table S4). We also compared gene expression in the intrinsic apoptosis pathway, for instance CASP9, DIABLO, and BAX, and, again, found no difference in gene expression induced by the C mutant viruses (Table S5). As already noted, direct comparison between CF170S and P(C-) at 6, 12, 24, or 48 h did not detect a single gene that differed by 4-fold or more between the two mutants with a P < 0.01 after multiple testing correction. We appreciate that the statistical criteria chosen are somewhat arbitrary and that we may have missed genes that differed between infection with the C mutant viruses due to the stringency of our original selection criteria. To detect subtle differences in gene expression profiles that could 91 explain the marked cytopathic effect seen during P(C-) infection but not during CF170S infection, we tested several fold change cutoffs below the 4-fold level (Table 2). While we were unable to find any genes that were differentially expressed between CF170S and P(C-) infection using a 4-fold cutoff, we found 28 genes that differed significantly (P < 0.01) between the two C mutants by 2-fold if multiple testing correction was performed sequentially and not simultaneously with the fold change cutoff (Fig. S3 and Table S6). Functional analysis revealed that 2 of these 28 genes are known mediators of cell death. Notably, caspase 3 was up-regulated 6.5-fold with P(C-) infection at 24 hours post infection, 2.8-fold greater than with CF170S infection. However, by 48 hours post- infection, caspase 3 expression during CF170S infection increased to 6.3-fold above mock, whereas the up-regulation of caspase 3 induced by P(C-) diminished to 4.1-fold. The second apoptosis-related gene, TNFRSF10B or TRAIL receptor 2 (TRAIL-R2), is responsible for signaling through the extrinsic apoptosis pathway. Similar to caspase 3, TNFRSF10B was up-regulated 4.8-fold with P(C-) infection at 24 hours post-infection, 2.1-fold greater than that seen with CF170S infection. However, at 48 hours post-infection, TNFRSF10B expression during CF170S and P(C-) infection were comparable. At 24 h p.i., TNFSF10 (TRAIL) itself was upregulated 50.9-fold by CF170S infection, 1.95-fold greater than that seen with P(C-) infection, although apoptosis was induced only during P(C-) infection (Fig. 7A). The earlier expression of TNFRSF10B and caspase 3 may contribute to the significant cytopathic effect observed during P(C-) infection at 48 hours post- infection although the induction of TNFSF10 (TRAIL) itself was more pronounced in cells infected with CF170S, the C mutant virus that did not induce apoptosis. 92 Figure 7. The CF170S and P(C-) mutant viruses share remarkably similar expression profiles for genes encoding death ligands, death receptors, and apoptosis inhibitors despite distinct cytopathic phenotypes. Hierarchical clustering matrices and gene expression profiles comparing the gene expression of A) death ligands, B) death receptors, and C) apoptosis inhibitors following infection with wt HPIV1 (WT) or the indicated mutant HPIV1 or following IFN? treatment. A significant difference was not detected between CF170S and P(C-) in any of the apoptosis-related genes depicted. 93 94 Discussion Microarray-based analyses are increasingly being used in virology and have helped to elucidate virus-host interactions for a number of viruses (reviewed in (88)). Here, we performed a comprehensive analysis of the response of a human respiratory epithelial cell line to HPIV1 infection and to IFN? treatment. Microarray analyses have been previously applied to study infection by murine PIV1 (SeV) (39, 178). However, these studies had methodological issues that limited the ability to interpret the results, including: 1) human cells were infected with a murine virus; 2) non-respiratory cells were infected with a 95 respiratory virus; 3) mRNA levels were determined only at a single time point; 4) customized arrays were used that included fewer than 1000 genes that focused on the IFN and antiviral pathways; and 5) the response to viral infection was not compared with the response to IFN treatment (39, 178). In the present study, gene expression kinetics following infection with a human respiratory virus or administration of IFN? were determined across multiple time points in human respiratory epithelial cells, the natural target of HPIV1, using a microarray representing the entire human genome. We chose to analyze only genes whose expression was at least four-fold up- or down-regulated, thereby excluding genes that were more modestly induced or suppressed by viral infection or IFN treatment. In order to avoid a subjective or biased analysis, functional bioinformatics tools were used to group thousands of differentially expressed genes into distinct hierarchical clusters and to identify functional pathways over-represented in each cluster. oPOSSUM Single Site Analysis software was used to examine upstream sequences for predicted transcription factor binding sites within each hierarchical cluster (72, 161, 193), resulting in the identification of several key transcriptional regulatory pathways that are likely involved in the cellular response to HPIV1 infection. As a first step in our analysis, we sought to identify genes that were differentially expressed as a result of wt HPIV1 infection. This set of genes was subsequently compared to the set of genes whose expression was altered by treatment with a physiological concentration of IFN?, allowing us to segregate and separately analyze IFN-responsive from IFN-independent genes in the wt HPIV1 microarray data set. IFN? treatment was chosen for comparison because A549 cells and human airway epithelial 96 cells produce this IFN in response to HPIV1, whereas IFN? is not secreted at all or is barely detectable. In agreement with previous reports that established the central role of the type I IFN response in innate antiviral immunity (35, 159), our clustering analysis indicated that approximately 60% of all genes induced late in infection with wt HPIV1 were also induced by IFN? treatment. Specifically, 209 of the 343 genes were up- regulated by both wt HPIV1 infection and IFN? treatment (Fig. 1, clusters A, B, and D). These 209 genes were over-represented in the IFN signaling, antigen presentation and ubiquitination pathways, as well as in the Toll-like receptor and NF-?B pathways. However, wt HPIV1 up-regulated an additional 129 genes that were not up-regulated by IFN? (Fig. 1, column I, cluster E). Many of these genes were also under the control of the family of IRF and NF-?B transcription factors and were involved in apoptosis signaling and inflammation. This data is in agreement with and expands on a previous report on the dominant role of IRF3 and NF-?B regulated genes in the response of human embryonic kidney cells to SeV infection at 6 h (39). It can be concluded by inspection of the kinetic data that wt HPIV1 is able to silence the innate antiviral response for at least 6 to 12 h p.i., but IFN dependent and IFN independent antiviral pathways are activated at later time points as part of the innate response to infection. Next, we sought to determine the role of the HPIV1 C proteins in suppressing the antiviral response of respiratory epithelial cells by analyzing gene expression patterns following infection of A549 cells with two C mutant viruses. Previous studies had established a role for the HPIV1 C proteins and SeV C proteins as inhibitors of IFN induction and signaling, as well as inhibitors of apoptosis (57, 96, 105). Two mutant 97 viruses with a single point mutation in C or a deletion of all four C proteins, CF170S and P(C-) respectively, were selected for this analysis. Wt HPIV1 inhibits the induction of IFN and signaling of IFN through its receptor, but CF170S and P(C-) each fail to inhibit these two activities. In addition, P(C-) infection induces apoptosis, whereas CF170S and wt HPIV1 do not, and P(C-) is much more restricted in replication in AGMs than CF170S (5, 9, 192). Using the CF170S and P(C-) viruses, we sought to compare the host response following infection with these mutants to that following wt HPIV1 infection. We found that, following infection with either C mutant virus, the number of differentially expressed genes expanded dramatically, implicating a critical role for the HPIV1 C proteins in blunting the host?s antiviral response. Using whole genome microarrays, we found that with CF170S infection, 392 and 2,322 genes were differentially regulated more than 4-fold at 24 and 48 h p.i., respectively, while with wt HPIV1 infection, only 54 and 277 genes were differentially expressed at the same time points. Thus, approximately 1% of the over 41,000 unique probes analyzed in our study were altered by HPIV1-CF170S at 24 h p.i., and only 14% of that subset of genes were altered by wt HPIV1, indicating that the wt HPIV1 C proteins inhibited modulation of the majority of antiviral genes that would otherwise be up-regulated or down-regulated as a result of HPIV1 infection. Importantly, this striking suppression of gene expression was associated with mutation in the C proteins but not with differences in virus replication. CF170S and wt HPIV1 exhibited identical kinetics of replication in vitro, and although P(C-) was approximately 100-fold restricted in replication at 24 and 48 h p.i. compared to wt HPIV1 and CF170S (Fig. S5), P(C-) infection was capable of suppressing gene expression just as well as CF170S. Str?hle et al. examined gene expression in human fibrosarcoma cells 24 h p.i. in 98 response to SeV-wt or SeV-CF170S infection using a customized array designed to probe approximately 150 genes with over-representation of the IFN pathway (178). In their study, infection with SeV-CF170S at a MOI of 20 induced 15 of 150 genes (10%) more than 2-fold at 24 h p.i. while SeV-wt (Ohita M strain) failed to induce the expression of any gene. We also found that the expression of IFN?-inducible genes was greater following infection with CF170S or P(C-) than following treatment with 300 pg/mL of IFN?, the concentration of IFN induced by CF170S infection of A549 cells (192). The greater induction seen with C mutant infection is due in part to direct viral recognition by RIG-I-like receptors (RLR) and subsequent activation of IRFs and NF-?B. Activated IRF3 not only stimulates the IFNB promoter to produce IFN? but also can independently activate IFN stimulated response elements to augment the production of IFN stimulated genes (ISGs). In contrast, the effect of exogenous IFN is limited to signaling through the IFN receptor and activation of the ISGF3 transcription factor complex to up-regulate the expression of antiviral effectors and other ISGs. The wt C proteins likely block the RLR and IFN signaling pathways as evidenced by the lack of IRF3 and STAT activation and IFN production during infection with wt HPIV1, and this antagonism is ablated as a result of mutation or deletion of the HPIV1 C proteins (18, 192). In another similarly designed microarray study, Hartman et al. compared the host response to wt Ebola virus with a highly attenuated Ebola virus containing a single amino acid change, namely, R312A in the IRF3-inhibitory domain of VP35 (65). Like wt HPIV1, wt Ebola was remarkably effective in suppressing the activation of cellular antiviral and IFN- responsive genes. The single VP35R312A mutation in Ebola, however, reversed the inhibition of only 39 genes, whereas the single CF170S mutation in HPIV1 reversed the 99 inhibition of over 2000 genes (Fig. 2). The Ebola VP35 protein confers virulence during infection by inhibiting IRF3 activation and IFN? production, whereas the VP24 protein inhibits STAT1 nuclear translocation and IFN signaling (64, 155). Unlike Ebola which utilizes two separate viral proteins to antagonize IFN production and signaling, HPIV1 encodes the C proteins from a single gene segment that blocks both arms of the viral recognition and IFN pathways and probably suppresses the expression of many more genes compared to VP35 alone. Following comparison of gene expression between wt HPIV1 and the two C mutant viruses, we next compared the cellular response to CF170S versus P(C-) infection to determine whether differences in gene expression profiles were associated with the observed differences in the mutants? apoptosis and attenuation phenotypes. Surprisingly, the cellular response to CF170S infection was comparable to the response to P(C-) infection, both qualitatively and quantitatively (Figure 4). Although the microarray- based analysis may have missed differences between CF170S and P(C-) due to decreased sensitivity compared to RT-qPCR, this limitation occurred only with very highly up- regulated genes where gene induction is already qualitatively clear. For example, IFNB, which was induced 853,062-fold as measured by RT-qPCR and 628-fold by microarray analysis, showed 1358-fold greater sensitivity by qPCR due to saturation of the IFNB probe on the microarray. However, less strongly up-regulated genes such as NFKB1, which was induced 68-fold as measured by RT-qPCR and 11-fold by microarray analysis, showed only 6-fold greater sensitivity in detecting differential expression. The difference is significantly less pronounced since genes that are up-regulated less do not 100 suffer as much from microarray probe saturation. Thus, although the microarray-based analysis may have missed small differences in gene expression between viruses, we have shown that both technologies have comparable sensitivity at lower expression levels. Based on the phenotypic differences between the two mutants, with P(C-) being the more attenuated in vivo and a strong inducer of apoptosis in vitro, we expected to find transcriptional differences in at least the apoptosis pathway. However, not a single mRNA was differentially expressed using our predefined criteria, i.e. 4-fold and P < 0.01. To detect any subtle differences between infection with the two C mutant viruses, we tested several fold-change cutoffs. After relaxing our fold-change cutoff to two-fold and subsequently performing a t-test with multiple testing correction, we identified two genes, caspase 3 and the TRAIL receptor 2 (TNFRSF10B), that could potentially contribute to P(C-)-induced apoptosis. P(C-) infection up-regulated both genes earlier, at 24 h p.i., compared to CF170S infection. However, in both cases, the magnitude of up- regulation during CF170S infection caught up by 48 h p.i., and the TRAIL ligand (TNFSF10) itself was comparably induced by both viruses at 24 h p.i. It was surprising that the CF170S mutant, that contains a single point mutation at amino acid 170 of the C protein, induced a transcriptional profile that was almost indistinguishable from that induced by the P(C-) mutant, a virus that does not express any of the C genes. Although P(C-) infection induces apoptosis much more rapidly and extensively than CF170S (9), the expression kinetics of death ligands, death receptors, or antiapoptotic factors that could account for P(C-) induced cell death did not differ significantly by 4-fold from CF170S at any time (Fig. 7a, b, and c). Since knockout of the 101 C gene causes cell death whereas expression of the wt C gene does not, we hypothesized that the wt HPIV1 and CF170S C proteins prevented cell death through apoptosis inhibition, i.e., either through inhibition of proapoptotic pathways or activation of antiapoptotic pathways. Previous studies on SeV suggested that a delicate balance exists between proapoptotic and antiapoptotic pathways during viral infection. SeV infection activates the cellular phosphatidylinositol 3-kinase (PI3K) pathway and, through AKT activation, prevents apoptosis (145). Thus, the differences in the apoptosis phenotype between the two C mutants likely lies in the interaction of one or more of the C proteins of CF170S and wt HPIV1 with one or more constitutively expressed factors, which results in the suppression of the apoptosis response. Both wt C proteins and point mutated C proteins could potentially activate apoptosis inhibitory proteins or antagonize proapoptotic proteins that would otherwise be triggered during HPIV1 infection. These two possibilities can be tested once the mechanism of interaction with the host apoptosis machinery has been defined. The greater level of attenuation of P(C-) versus CF170S likely reflects a contribution of apoptosis to the decreased replication of P(C-), but the loss of a yet undefined function of C required for replication in vivo may contribute as well. Although wt HPIV1 suppressed the up-regulation of a broad array of cellular genes that were induced by P(C-) and CF170S infection, global shutoff of host transcription did not occur. In fact, only 234 of 2612 differentially expressed genes were down-regulated (Fig. 4, panel II, cluster H). Interestingly, both C mutant viruses down-regulated these genes to a greater extent than wt HPIV1 (Table S2). Thus, the wt C proteins blunted both induction and suppression of a specific set of host genes that would otherwise react to 102 viral infection. This is in stark contrast to what has been described for vesicular stomatitis virus which shuts down transcription globally through M protein-mediated inhibition of the three cellular RNA polymerases I, II, and III (1, 202), or for Rift Valley fever virus which, through inhibition of TFIIH transcription factor complex assembly via its NSs protein, leads to the rapid destabilization of host cellular RNA synthesis (111). However, other members of the order Mononegavirales, such as the Marburg and Ebola filoviruses, suppress the antiviral and type I IFN responses without shutting down host cell functions globally, and the degree of antiviral suppression by these viruses seems to correlate with virulence in vivo (95). Similarly, our data indicate that wt HPIV1 C protein function, i.e., suppression of antiviral pathways early in infection, is an important virulence factor and that the loss of this activity in both CF170S and P(C-) leads to attenuation in vivo. Following global analysis of virus modulated pathways, we attempted a more detailed analysis of the IFN and apoptotic pathways in order to understand the different in vivo and in vitro phenotypes of wt HPIV1, P(C-) and CF170S. We found that by 48 h p.i., wt HPIV1 induced IFNB expression 180-fold, as measured by microarray, and 1200-fold, as measured by qPCR (Fig. 5A). Previously, we reported that IFNB mRNA was not detectable at any time point in response to HPIV1 infection (192). Re-examination of that data identified a 5600-fold induction of IFNB mRNA following CF170S infection versus a 12-fold induction following wt HPIV1 infection at 48 h p.i., i.e., a 458-fold reduction in IFNB transcription following wt HPIV1 infection, as compared to CF170S infection (192). In the present study, CF170S induced 695 times more IFNB mRNA than wt HPIV1 at 48 h p.i., confirming that IFNB mRNA is strongly induced by CF170S but not by wt HPIV1 103 (Table S3). At the protein level, we previously were unable to detect IFN? in the supernatant of wt HPIV1 infected A549 cells by IFN bioassay, not even after 96 h p.i. (192). In contrast, Bousse et al. detected 380 IU/mL of IFN? by ELISA in MRC-5 cells 48 h p.i. with wt HPIV1 strain C-35 (Genbank M74081) (18). This difference in IFN? secretion could be due to the use of different cell lines (MRC-5 versus A549) and/or different virus strains. Protein sequence alignment of the C protein of the C-35 strain of HPIV1 used by Bousse et al. with our Washington 1964 wt HPIV1 strain revealed a CQ38P substitution in the C gene that could potentially be responsible for a distinct IFN phenotype. The CF170S virus serves as an example for the role a single amino acid substitution in the C protein can potentially have on the IFN phenotype. Importantly, the deficient IFN? response of A549 cells to wt HPIV1 infection was also seen in primary human airway epithelial cell cultures, confirming the IFN production deficiency of respiratory epithelial cells infected with wt HPIV1 (11). One aim of this project was to identify cellular pathways that are targeted by the HPIV1 C proteins and to define how the C proteins exert such impressive transcriptional control of the host?s innate immune system. We analyzed the gene expression profiles for all 14 transcription factors identified by bioinformatics analysis (Fig. S6) and found that the expression of 4 genes IRF1, ISGF3G, NF-?B1, and c-Rel were significantly up-regulated increasingly over time, confirming the importance of the IRF3 and NF-?B signaling pathways during parainfluenza infection and furthermore providing a feed-forward mechanism to amplify the host transcriptional response to viral infection. We expected to identify IRF and NF-?B TFBSs over-represented in genes induced by IFN treatment and 104 HPIV1 infection, but other unexpected TFBSs were also identified. For example, FOXD1 and FOXD3 TFBS were identified in genes up-regulated by the HPIV1 C mutant viruses. Only recently has an important role for FoxD1 been identified in the regulation of immune activation through the NF-?B pathway (117). FoxD3 is well known for controlling cellular differentiation but no role has yet been defined for FoxD3 in the regulation of the immune response or the response to viral infection. In future work, it will be important to define how the HPIV1 C proteins modify the activity of FOXD1, FOXD3, and other transcription factors and how C', C, Y1, and Y2 differ in their ability to alter transcriptional activity. In summary, the HPIV1 C proteins exert remarkable control over the cellular transcriptional response to viral infection, indicating that the C proteins are important virulence factors for HPIV1. Mutations within the C gene permit the activation of a broad array of cellular genes involved in the type I IFN, IRF3 and NF-?B pathways that would otherwise be repressed by HPIV1 infection, and these mutations specify an attenuation phenotype in vivo. However, the lack of a clear differential regulation of antiapoptotic and proapoptotic genes in P(C-) and CF170S infected cells allows for two models of P(C-) induced apoptosis that could individually, or in combination, play a key role in determining the apoptosis phenotype of HPIV1 infection: 1) earlier expression of TNFRSF10B and caspase 3 or 2) alterations in post-translational events involving constitutively expressed cellular proapoptotic or antiapoptotic factors. 105 Chapter 4. The C proteins of human parainfluenza virus type 1 limit double stranded RNA accumulation that would otherwise trigger MDA5 and PKR activation Submitted Jim Boonyaratanakornkit1, Emmalene Bartlett1, Henrick Schomacker1, Sonja Surman1, Shizuo Akira2, Yong-Soo Bae3, Peter Collins1, Brian Murphy1, and Alexander Schmidt1 1Laboratory of Infectious Diseases, RNA Viruses Section, NIAID, NIH, Bethesda, MD 20892, USA. 2Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan. 3Department of Biological Sciences, Sungkyunkwan University, Choenchoen-Dong, Jangan-Gu, Suwon, Gyeonggi-Do 440- 746, South Korea 106 Abstract Human parainfluenza virus type I (HPIV1) is an important respiratory pathogen in young children, the immunocompromised, and the elderly. We found that infection with wild- type (WT) HPIV1 suppressed the innate immune response in human airway epithelial cells by preventing not only phosphorylation of IRF3 but also degradation of I?B?, thereby inhibiting IRF3 and NF-?B activation, respectively. Both of these effects were ablated by a F170S substitution in the HPIV1 C proteins (F170S) or by silencing the C open reading frame (P(C-)), resulting in a potent IFN? response. Using murine knockout cells, we found that IFN? induction following infection with either mutant relied mainly on MDA5 rather than RIG-I. Infection with either mutant, but not WT HPIV1, induced a significant accumulation of intracellular dsRNA. These mutant viruses directed a marked increase in the accumulation of viral genome, antigenome, and mRNA that was coincident with the accumulation of dsRNA. In addition, the amount of viral proteins was reduced compared to WT HPIV1. Thus, the accumulation of dsRNA might be a result of an imbalance in the N protein-to-genomic RNA ratio leading to incomplete encapsidation. PKR activation and IFN? induction followed the kinetics of dsRNA accumulation. Interestingly, the C proteins did not appear to directly inhibit intracellular signaling involved in IFN? induction; instead, its role in preventing IFN? induction appeared to be in suppressing the formation of dsRNA. PKR activation contributed to IFN? induction and also was associated with the reduction in the amount of viral proteins. Thus, the HPIV1 C proteins normally limit the accumulation of dsRNA and thereby limit activation of IRF3, NF-?B, and PKR. If C protein function is compromised, as in the case of F170S HPIV1, the resulting PKR activation and reduction 107 in viral protein levels enables the host to further reduce C protein levels and to mount a potent antiviral type I IFN response. Introduction Human parainfluenza virus type 1 (HPIV1) is an important and uncontrolled respiratory pathogen that causes a significant burden of disease, mainly in young children, the immunocompromised, and the elderly (30, 42, 58, 79, 134, 154). HPIV1 is a single stranded, negative sense, nonsegmented RNA virus in the Paramyxoviridae family. The viral genome, 15.6 kb in length, consists of 6 genes (3? ? N-P/C-M-F-HN-L ? 5?) that encode the nucleoprotein (N), phosphoprotein (P), C proteins (C), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and the large polymerase protein (L). Each gene encodes a single major protein with the exception of the P/C gene, which encodes the P protein in one open reading frame and a nested set of four carboxy-coterminal C proteins (C', C, Y1, and Y2) expressed from individual start sites in a second open reading frame. The C proteins play a critical role in HPIV1 virulence by inhibiting apoptosis, regulating type I interferon (IFN) production and signaling, and controlling the transcription of a large number of host genes (9, 17, 18, 192). The C proteins of Sendai virus (SeV), or murine PIV1, have considerable sequence conservation with the HPIV1 C proteins. However, the P/C gene organization of SeV differs from that of HPIV1 in that SeV expresses, in addition to the C proteins, a second accessory protein, the V protein, that also exerts an inhibitory role on the host innate antiviral response (3). In addition, some of the immune evasion activities of SeV and HPIV1 are species- specific (18, 22), indicating that a careful examination of the innate immune response to 108 HPIV1 may yield important clues about the pathogenesis of this human respiratory virus that differ from the murine SeV. Type I IFN is a central mediator of antiviral innate immunity. The induction of IFN synthesis following virus infection depends on a number of pattern-recognition receptors that recognize conserved pathogen-associated molecular patterns and initiate downstream signaling cascades (87). The presence of dsRNA, an intermediate of RNA viral replication, is recognized as a pathogen-associated molecular pattern by Toll-like receptor 3 (TLR3) and two caspase recruitment domain (CARD)-containing RNA helicases, retinoic acid-inducible gene I (RIG-I) and melanoma associated-differentiation gene 5 (MDA5), which act as intracytoplasmic sensors of dsRNA (2, 76, 147, 180, 212, 214). Whereas TLR3 mainly senses extracellular dsRNA on antigen-presenting cells, RIG-I and MDA5 are constitutively present and detect intracellular dsRNA (2, 214). TLR3 signals through an adaptor called TIR domain-containing adaptor inducing IFN? (TRIF), while RIG-I and MDA5 recruit another CARD-containing adaptor called mitochondrial antiviral signaling protein (MAVS, also referred to as IPS-1, Cardif, or VISA) to relay the signal to the kinases TBK1 and IKK?, which phosphorylate IRF3 (interferon regulatory factor 3), and to IKK?, which activates the NF-?B pathway (41, 99, 140, 167, 207). Once activated, IRF3 translocates into the nucleus and binds the positive regulatory domain III (PRDIII) of the IFN? promoter. RIG-I and MDA5 can distinguish between different RNA viruses (98). It was originally reported that IFN production in response to infection with negative-sense RNA viruses, including SeV, was dependent on RIG-I expression whereas the response to infection with positive-sense 109 RNA viruses was dependent on MDA5 expression (98, 195). This model was in agreement with earlier reports that found a substantial accumulation of cytoplasmic dsRNA during infection with positive-sense RNA viruses but not with negative-sense RNA viruses (199). However, a more recent study suggested that MDA5 does contribute in vivo to sustaining the IFN response and restricting SeV infection (53). After the host cell recognizes and signals the presence of foreign RNA, IRF-3 and NF-?B, together with ATF-2/c-Jun (AP-1), form an enhanceosome that binds to the IFN? promoter, inducing transcription of the IFN? gene. Once type I IFN is produced and secreted, it binds to the type 1 IFN receptor on the surface of the infected and neighboring cells to amplify a robust antiviral response (121, 183). One IFN-inducible gene, PKR, is best known for its ability to bind dsRNA and subsequently phosphorylate EIF2A thereby inhibiting translation initiation during viral infection (70). PKR has also been implicated in contributing to NF-?B activation, IRF1 activation, and IFN induction by poly I:C, independent of its kinase activity, as another mechanism of innate antiviral host defense (4, 16, 25, 107, 209, 218). We generated a number of HPIV1 mutants to investigate C protein function as well as to generate novel live vaccines that would be attenuated by disturbing the IFN antagonistic function of the C proteins, a strategy that has been shown to result in attenuation of replication in vivo (5, 6, 192). Previously, a phenylalanine to serine substitution of amino acid 170 of SeV was shown to significantly attenuate a highly virulent SeV strain, increasing the lethal dose 50% (LD50) 20,000-fold in mice (50). This C F170S mutation was introduced into the P/C gene of recombinant HPIV1 by reverse genetics (85, 137). 110 Whereas WT HPIV1 infection prevented IRF3 dimerization, its nuclear translocation, and IFN? production, infection with HPIV1 containing the CF170S mutation (F170S virus) led to IRF3 activation and IFN? production (192). Not unexpectedly, the F170S mutant was attenuated for replication in the respiratory tract of hamsters and African green monkeys (AGMs) (5, 192). We also constructed a HPIV1 mutant, referred to as P(C-), that does not express any of the four C proteins due to point mutations that silence the C ORF, and which was even more attenuated than F170S in vivo (9). In the present study, we found that the C proteins of HPIV1 profoundly suppress the innate immune response in epithelial cells by preventing the activation of both IRF3 and NF-?B. We found that mutation or silencing of the C gene ablated both of these inhibitory effects. In contrast to WT, infection with both F170S and P(C-) HPIV1 resulted in the accumulation of significant amounts of cytoplasmic dsRNA with kinetics that were more rapid for the P(C-) virus. The accumulation of dsRNA coincided with an increase in the accumulation of viral genomic/anti-genomic RNA or mRNA and a decrease in N protein levels, and thus appeared to result from incomplete encapsidation. The enhanced induction of IFN? observed with these mutants relied mainly on MDA5 rather than on RIG-I. PKR activation coincided with dsRNA accumulation, contributed to IFN? induction, and restricted viral C protein expression. Thus, PKR activation provides a mechanism that allows the host cell to eliminate the very protein responsible for evading virus detection. 111 Materials and Methods Cells, viruses, and DNA transfection A549 human respiratory epithelial cells were maintained in F-12 medium supplemented with 0.1 mg/mL gentamicin sulfate, 4 mM L-glutamine, and 10% fetal bovine serum (FBS). Human embryonic kidney 293T cells were maintained in DMEM medium supplemented with 10% FBS and gentamicin sulfate. Mouse embryonic fibroblasts were prepared from RIGI MDA5 +/- +/-, RIGI -/-, and MDA5 -/- mice and maintained in DMEM with 10% FBS and gentamicin sulfate as described (98). Stable shPKR knockdown and shCON control 293A cells were maintained in DMEM with 10% FBS and gentamicin sulfate supplemented with 1 ?g/mL puromycin as described (215). Doxycycline-inducible, stable cells that express WT C? protein tagged at the C-terminus with a c-Myc epitope were generated using Flp-In T-REx 293 cells grown in 100 ?g/mL Hygromycin and 15 ?g/mL Blasticidin (Invitrogen). C expression was induced by incubation for 24 h with 1 ?g/mL Doxycycline (Sigma). Sucrose gradient-purified recombinant WT, F170S, and P(C-) HPIV1 were prepared and titered by limiting dilution assay as reported (17). All viral infections were carried out using a multiplicity of infection (MOI) of 5 TCID50/mL at 32?C in medium containing 1.2% trypsin TrypLE Select (Invitrogen). For type I IFN neutralization, 5000 NU/mL anti-IFN? and 2000 NU/mL anti-IFN? antibodies (PBL InterferonSource) were added at 12 h p.i. For transient transfections, 3 ?L Lipofectamine 2000 (Invitrogen) per 1 ?g total DNA was added for every 400,000 293T cells for 48 h at 37?C. For poly I:C-induced IFN? expression, 3 ?L Lipofectamine 2000 per 1 ?g of poly I:C (Sigma) was added for every 400,000 293T cells for 6 h at 37 ?C. For the preparation of cellular extracts, cells were 112 suspended in lysis buffer (1% Triton X-100, 50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM EDTA) with protease inhibitor cocktail (Sigma) at a concentration of 2000 cells/?L for 30 min at 4?C and centrifuged at 15,000 g for 10 min. Halt protease and phosphatase inhibitor cocktail (Pierce) was added to the lysis buffer whenever phosphorylation states were examined. After centrifugation, the supernatant was used for Western blotting and immunoprecipitation. Real-time RT-PCR Intracellular RNA or virion-associated RNA was extracted and analyzed by real-time RT- PCR as described previously (17). The relative levels of expression of mRNA for human and murine IFN? and GAPDH were determined using TaqMan Gene Expression Assays (Applied Biosystems) for human IFN? (Hs01077958_s1), human GAPDH (Hs99999905_m1), mouse IFN? (Mm00439546_s1), and mouse GAPDH (Mm99999915_g1), with the RT performed on intracellular RNA using random hexamers. Experiments were performed in triplicate. Combined genomic/anti-genomic RNA, genomic RNA, anti-genomic RNA, and IFN? expression in shPKR 293 cells was measured in triplicate and confirmed in two independent experiments. Student?s t-test was used to determine statistical significance. To measure the relative copy numbers of HPIV1 genome plus antigenome in sucrose-purified virus stocks, RT was performed on extracted virion RNA with random hexamers and PCR was performed to amplify genome nucleotides 15,502-15,600, corresponding to the downstream end of the L gene and the adjoining trailer region (forward primer ACCAGACAAGAGTTTAAGAAATATCGA, reverse primer AGAAATCCCTTTAACTGACTCATAAAAACATAGT, probe 113 CAACAGACAAGAGTATTAATAAT). To distinguish between genomic and antigenomic RNA in intracellular RNA, RT was performed with a positive-sense primer representing genome nucleotides 15502-15519 (AGAAATCCCTTTAACTGA) to measure genome or with a negative-sense primer representing genome nucleotides 15583-15600 (CTTAAACTCTTGTCTGGT) to measure antigenome. PCR was then performed to amplify nucleotides 15,502-15,600 of the genome or its complement, using the primer/probe set described above. To measure the relative copy numbers of the N and P mRNAs in intracellular RNA, RT was performed with oligo-dT primer and PCR was performed to amplify, in the case of N, genome nucleotides 917-1014 (forward primer AGCATCTTTCATGAACACCATCAAGT, reverse primer GGCTTCTCAATTTGTTTATATCTGGTCTCA, probe CCGCCCTGACACTAT) and, in the case of P, genome nucleotides 2624-2685 (forward GAAGACCACAACACCAAAACCA, reverse primer GCTGCTGACTCTTCGTTCTTTG, probe ACGGTGGAACTTTTG). Western blotting and antibodies Cells were lysed and clarified as described above for the preparation of cellular extracts, and the resulting extracts (15 ?L) were denatured and reduced in sample loading buffer (0.3M Tris HCl, 5% SDS, 50% glycerol, 0.1M DTT) at 95 ?C for 5 min, separated by SDS-PAGE on 4-20% Tris-Glycine gels (Invitrogen), and transferred to 0.45 ?m PVDF membranes (Invitrogen). Membranes were blocked overnight with 5% milk powder and 1% BSA or, whenever probing with phospho-specific antibodies, with 5% BSA in 0.1% Tween-PBS. Rabbit polyclonal anti-serum was raised against a synthetic peptide 114 corresponding to an internal sequence (amino acids 78?92, TITTKTEQSQRRPK) in the HPIV1 C proteins shared by C?, C Y1, and Y2 by ProSci (Poway, CA), against a synthetic peptide corresponding to an internal sequence (amino acid residues 485?499, RRLADRKQRLSQANN) in the HPIV1 N protein by ProSci, and against a synthetic peptide corresponding to a N-terminal sequence (amino acids 10-24, RDPEAEGEAPRKQES) in the HPIV1 P protein by Spring Valley Laboratories (Woodbine, MD). Membranes were probed with rabbit anti-serum at a 1:1000 dilution with 5% milk and 1% BSA in 0.1% Tween-PBS for 1 hour. Antibodies used included rabbit polyclonal antibodies to human pIRF3 (S396) (Millipore; #07-582), IRF3 (Santa Cruz Biotechnology; sc-9082), I?B-? (Santa Cruz; sc-945), ATF2 (Santa Cruz; sc-187), pP38 (T180/Y182) (Santa Cruz; sc-101759), P38 (Santa Cruz; sc-7149), PKR (Santa Cruz; sc-707), pEIF2? (S51) (Enzo Life Sciences; BML-SA405), EIF2? (Sigma; E0157), MDA5 (Alexis Biochemicals; ALX-210-935), Flag epitope tag (Sigma; F7425), HA epitope tag (Sigma; H6908), Myc epitope tag (Sigma; C3956), rabbit monoclonal antibody to human pPKR (T451) (Epitomics; #2283-1), or mouse monoclonal antibodies to human pATF2 (T71) (Santa Cruz; sc-8398), GAPDH (Sigma; G9295) and V5 epitope tag (Invitrogen; R960-25). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Santa Cruz) was used at a dilution of 1:5000 and horseradish peroxidase- conjugated goat anti-mouse secondary antibody (Kirkegaard & Perry Laboratories) was used at a dilution of 1:2400 for 1 hour. Membranes were washed 3 times for 5 minutes with 0.1% Tween-PBS after primary and secondary antibody incubations. Enhanced chemiluminescent substrate (Pierce) was used to visualize proteins on BIOMAX MR film (Kodak). Relative band intensity was determined by densitometry using ImageJ. 115 Immunoprecipitation Cell lysates extracted from transfected 293T cells were mixed for 16 h at 4?C with anti- Myc agarose (Pierce), washed extensively with wash buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl), eluted with 0.1 M glycine pH 2.8, and separated by SDS-PAGE. Immunofluorescence A549 cells were fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X- 100, and blocked with 10% goat serum and 0.02% BSA for 2 h. Mouse monoclonal J2 anti-dsRNA antibody (English and Scientific Consulting) at a 1:200 dilution or rabbit polyclonal anti-HPIV1 P antibody at a dilution of 1:100 was added in 1% goat serum and 0.002% BSA for 1 h at 37?C. Texas Red-conjugated goat anti-rabbit and FITC- conjugated goat anti-mouse secondary antibodies (Jackson ImmunoResearch) were added at a dilution of 1:250 for 45 min at 37?C. Cells were mounted in ProLong Gold reagent with DAPI (Invitrogen) and visualized on a Leica SP5 confocal microscope. Plasmid constructs A cDNA for HPIV1 C was generated by PCR (forward primer CATCGGTACCGTGGATACCTCAGCATCCAAAACTCTCCTTCCC and reverse primer ATCACATGCGGCCGCTTACAGATCTTCTTCAGAAATAAGTTTTTGTTCTTCTTG TACTATGTGTGCTGCTAGTTTCC) of the full-length antigenomic cDNA for WT HPIV1 and constructed with the insertion of a C-terminal c-Myc tag and KpnI and EagI 116 restriction sequences to allow cloning into pcDNA3.1 (Invitrogen). The nucleotide sequence was confirmed using BigDye Terminator and a DNA Analyzer 3730 (Applied Biosystems). pEF-IRES V5-TRIM25 was kindly provided by Michaela Gack and Jae Jung (45). pEF-BOS HA-Riplet was kindly provided by Hiroyuki Oshiumi and Tsukasa Seya (141). pRK5 Flag-TRAF3 and Myc-TRADD3 were kindly provided by Jurg Tschopp (128). pCMV2 Flag-TRAF6 was kindly provided by Masashi Muroi and Ken- ichi Tanamoto (132). pEF-BOS Flag-RIGI and Flag-MDA5 were kindly provided by Takashi Fujita (213, 214). pcDNA3 Flag-TBK1 was kindly provided by Katherine Fitzgerald and Makoto Nakanishi (41, 190). pCMV2 Flag-IPS1 has been previously described (99). pcDNA3 Flag-BIRC2 and Flag-BIRC4 were kindly provided by Temesgen Samuel (162). pFlag-CMV2 Flag-FADD was kindly provided by Andrew Thorburn (189). Results HPIV1 activation of the interferon induction pathway We found previously that, in stark contrast to WT HPIV1, HPIV1 with a F170S point mutation in the C proteins or in which the C gene was silenced by point mutations induced the transcription of a broad array of cellular genes, in particular genes involved in type I IFN production and the subsequent antiviral response (17, 192). To investigate the mechanism of C-mediated antagonism of the host innate immune response, we used three cell types. A549 cells were used to model infection in human respiratory epithelial cells, 293 human kidney cells were used for transfection and knockdown experiments, and mouse embryonic fibroblasts were used for knockout experiments. We found that 117 the HPIV1-induced IFN phenotype was comparable between the three cell types: specifically, in each cell type, F170S and P(C-) HPIV1 induced similar, abundant levels of IFN? expression mRNA expression, whereas WT HPIV1 did not induce significant IFN? (not shown). IFN? induction is transcriptionally controlled by the coordinate assembly of an enhanceosome consisting of IRF3, NF-?B, and AP-1 at the IFN? promoter (120, 210). We examined the activation of the three components of the IFN? enhanceosome during HPIV1 infection and found that both F170S and P(C-) HPIV1 induced IRF3 phosphorylation and I?B? degradation, which would permit nuclear translocation of IRF3 and NF-?B, respectively, whereas WT HPIV1 effectively blocked IRF3 phosphorylation and I?B? degradation (Fig. 1). On the other hand, WT, F170S, and P(C-) HPIV1 did not induce detectable phosphorylation of P38 or ATF2 (Supplementary Fig. 1). 118 Figure 1. Activation of the IRF3 and NF-?B components of the IFN? enhanceosome by infection with WT and mutant HPIV1. A549 cells were infected with WT, F170S, or P(C-) HPIV1 at a MOI of 5 for 6 or 48 h. Phosphorylated IRF3 (Ser 396), total IRF3, and total I?B? were detected by Western blot. To investigate the basis for the observed antagonism of the host response by the WT HPIV1 C proteins, a selective search for cellular C-interacting proteins was performed among RIG-I/MDA5 pathway components. Cells transfected with expression plasmids expressing a tagged C? protein and various tagged proteins of the RIG-I/MDA5 pathway were lysed and analyzed in co-immunoprecipitation experiments using various conditions in an attempt to sensitively detect any interactions. However, we were unable to detect any cellular C-interacting proteins among those tested, including TRIM25, Riplet, RIGI, MDA5, IPS1, TBK1, TRAF3, TRAF6, BIRC2, BIRC4, TRADD, and FADD, even though, under these same conditions, TRAF3 was immunoprecipitated with TRADD, a known binding partner of TRAF3 (Supplementary Fig. 2). Since we could not identify 119 any cellular C-interacting proteins, we investigated whether supplying the HPIV1 C proteins in trans could block the IFN? induction pathway triggered by infection with respiratory syncytial virus (RSV) or by transfection with poly I:C, which mainly involves activation of RIG-I and MDA5, respectively (118, 174). Infection with WT HPIV1, as a source of C proteins, strongly suppressed IFN? induction in response to super-infection with P(C-) HPIV1, but failed to block IFN? induction in response to super-infection with a RSV mutant that expresses GFP from an added gene and lacks the NS1 and NS2 RSV IFN antagonist genes (?NS1/NS2 RSV) (Fig. 2a). As a control, analysis of GFP expression showed that the efficiency of infection by ?NS1/NS2 RSV was not affected by pre-infection with WT HPIV1 (not shown). To test whether the HPIV1 C proteins could interfere with IFN production triggered through MDA5, a doxycycline-inducible stable human cell line that expresses the HPIV1 C proteins was generated. In this system, expression of the HPIV1 C proteins in trans strongly suppressed IFN? induction by infection with P(C-) HPIV1 (Fig. 2b), confirming the efficient expression of functional C protein. In contrast, expression of the HPIV1 C proteins had no effect on IFN? induction in response to transfection with poly I:C (Fig. 2c). In this last experiment, the cells had also been transfected with a plasmid expressing human MDA5 to provide the homologous sensor for dsRNA prior to the transfection of poly I:C. Taken together, these results indicate that the HPIV1 C proteins supplied in trans can efficiently suppress IFN induction by HPIV1 mutants, but not by heterologous inducers. 120 Figure 2. IFN? induction in A549 cells by heterologous RIG-I and MDA5 stimuli is not affected by expression of the HPIV1 C proteins in trans. (A) A549 cells were pre- infected with WT HPIV1 at a MOI of 5 for 24 h and then super-infected with either P(C-) HPIV1 or GFP-expressing ?NS1/NS2 RSV at a MOI of 5 for another 24 h. IFN? expression was measured by real-time PCR and was normalized to the mock pre-infected, mock super-infected sample. * denotes P < 0.01 compared to mock pre-infected samples. (B) C protein expression was induced in stable C-expressing 293 cells with 1 ?g/mL doxycycline for 24 h. Cells were then mock-infected or infected with P(C-) HPIV1 at a MOI of 5 for another 24 h. IFN? expression was measured by real-time PCR and was normalized to the control treated, mock infected sample. * denotes P < 0.01 compared to control treated cells. (C) C protein expression was induced in stable C-expressing 293 cells with 1 ?g/mL doxycycline for 24 h, then transfected with 2.5 ?g MDA5 expression plasmid for another 18 h, and then transfected with 10 ?g poly I:C for 6 h. IFN? expression was measured by real-time PCR and was normalized to the control treated, control transfected sample. 121 HPIV1-induced IFN? expression is mediated mostly by MDA5 Since we were unable to obtain either direct or indirect evidence that the C proteins blocked IFN production by interacting with a host factor of the RIG-I/MDA5 pathway, we evaluated the involvement of this pathway in IFN? induction by HPIV1 using 122 knockout MEFs. In contrast to WT HPIV1, F170S and P(C-) HPIV1 strongly induced IFN? expression in control MEFs (Fig. 3a, black bars). RIG-I knockout led to a slight decrease (2.3-fold) in IFN? expression in F170S-infected cells, but no effect was observed in P(C-)-infected cells (Fig. 3a). MDA5 knockout led to a greater reduction in IFN? expression in both F170S- and P(C-)-infected cells (4.5-fold and 8.0-fold, respectively) (Fig. 3a). TBK1 and IKK? double knockout led to a dramatic reduction in IFN? expression in both F170S- and P(C-)-infected cells, confirming the requirement for TBK1 and IKK? in IFN induction due to infection with F170S or P(C-) HPIV1 (Fig. 3b). The observation that knock-outs of TBK1 and IKK? led to almost complete inhibition of IFN? mRNA synthesis, whereas knock-out of MDA5 led to only a partial inhibition, raised the possibility that factors in addition to MDA5 are contributing to the induction of IFN? mRNA via TBK1 and IKK?. 123 Figure 3. IFN? expression in RIG-I/MDA5 pathway knockout MEFs infected with WT or mutant HPIV1. (A) RIG-I and MDA5 knockout MEFs and (B) TBK1 and IKK? double knockout MEFs were infected with WT, F170S, or P(C-) HPIV1 at a MOI of 5 for 48 h. IFN? expression was measured by real-time PCR and normalized to mock infected control MEFs. * denotes P < 0.01 compared to control MEFs infected with the same virus. 124 Intracellular dsRNA accumulates in cells infected with C mutant HPIV1 viruses Since knockout of the dsRNA sensor MDA5 strongly reduced IFN? expression during infection with F170S or P(C-) HPIV1, we looked for the presence of dsRNA in HPIV1- infected cells by immunofluorescence using a monoclonal antibody against dsRNA. Transfection of poly I:C was used as a positive control, demonstrating the ability of the dsRNA-specific monoclonal antibody to detect intracellular dsRNA (Fig. 4a). dsRNA was not detected in WT-infected cells at either 24 or 48 h (Fig. 4b, e), similar to observations previously reported for a number of negative sense RNA viruses, including influenza, La Crosse, Sendai, and Newcastle disease viruses (199). In contrast, dsRNA was readily detected in cells infected with either the F170S or P(C-) virus; however, the kinetics of accumulation differed. In the case of F170S HPIV1, less than 10% of infected cells were positive for dsRNA at 24 h, whereas over 60% of infected cells were positive at 48 h (Fig. 4c, e, f). In contrast to F170S HPIV1, the accumulation of dsRNA in response to P(C-) HPIV1 occurred more rapidly, with approximately 70% of infected cells containing detectable dsRNA at 24 h. However, the accumulation of dsRNA in response to the P(C-) virus was also transient: by 48 h, less than 10% of P(C-)-infected cells contained dsRNA (Fig. 4d-f). This difference did not appear to be due to differences in the kinetics or efficiencies of infection: the percent of cells infected with WT, F170S, and P(C-) HPIV1 was similar at 24 and 48 h, as assessed by immunofluorescence using polyclonal antibodies specific to the HPIV1 P protein (Fig. 4g). 125 Figure 4. Immunofluorescence of dsRNA in A549 cells infected with WT or mutant HPIV1. A549 cells were transfected with (A) poly I:C (0.02 ?g/?L) or infected with (B) WT, (C) F170S, or (D) P(C-) HPIV1 at a MOI of 5 for 24 h. HPIV1 P protein (red) was detected using a rabbit polyclonal anti-P antibody and a Texas-Red conjugated secondary anti-rabbit antibody. dsRNA (green) was detected using a mouse monoclonal anti- dsRNA antibody and a FITC conjugated secondary anti-mouse antibody. (E) dsRNA and the HPIV1 P protein was examined as in A-D in A549 cells infected with WT, F170S, or P(C-) HPIV1 at a MOI of 5 for 24 and 48 h. (F, G) Quantitation of the number of cells in D with and without detectable dsRNA (F) or HPIV1 P protein (G) at 24 and 48 h. dsRNA levels are representative of two independent experiments. 126 127 One possible explanation for the increased accumulation of intracellular dsRNA in cells infected with the F170S and P(C-) viruses was that these virus preparations contained disproportionately high levels of defective interfering particles, which, for nonsegmented negative strand RNA viruses, usually have copy-back genomes that can form base-paired panhandles. To investigate this possibility, we assayed a sucrose gradient-purified preparation of each virus for (i) the number of infectious particles, measured by limiting dilution assay, and (ii) the relative copy number of genomic plus antigenomic RNA, measured by real time RT-PCR with primers specific to the trailer region that would be present in both standard and defective genomes. We then calculated the ratio of infectious particles to genome/antigenome copies for each virus, and then normalized these ratios to that of the F170S virus, which was assigned the value of 1.0. The resulting values were: F170S, 1.0; WT, 0.28; and P(C-), 0.09. In other words, F170S HPIV1 had the highest ratio of infectious particles to genome copies, and P(C-) had the lowest, with WT HPIV1 being intermediate. This lack of a consistent trend, i.e. the F170S virus had a higher ratio of infectious particles to genome/antigenome copies than the WT virus and yet the F170S virus led to dsRNA accumulation whereas the WT virus did not, suggests that the dsRNA produced during infection with the F170S and P(C-) viruses probably cannot be attributed to the presence of a disproportionately high content of defective- interfering particles in the initial inoculum. 128 Mutation or deletion of the HPIV1 C proteins results in increased viral RNA synthesis and decreased accumulation of viral proteins Another potential source for intracellular dsRNA could arise from annealing between incompletely encapsidated negative sense genomic RNA and either positive sense antigenomic RNA or mRNA, which might occur if mutation or deletion of the C proteins perturbed viral RNA production. We therefore used real-time RT-PCR to monitor viral RNA accumulation in A549 cells infected with the WT, F170S and P(C-) viruses. The combined amount of genomic plus antigenomic RNA was quantified using primer/probes specific for the trailer region (Fig. 5a), whereas the N and P mRNAs (Fig. 5b and c, respectively) were quantified using oligo-dT for reverse transcription followed by gene- specific primer/probes for PCR. In cells infected with WT HPIV1, the levels of genome/antigenome, N mRNA, and P mRNA rose gradually over the time-course of the experiment (Fig. 5a-c). For the F170S mutant, the levels of these RNAs were similar to those of WT HPIV1 at 24 h, but then increased substantially by 48 h. A very different pattern was observed during infection with P(C-) virus. The amount of genome/antigenome was higher than for the other two viruses at 24 h, and continued to increase to a very high level at 48 h (Fig. 5a). In contrast, the levels of N and P mRNAs were extremely high at 24 h, but thereafter dropped sharply (Fig. 5b). In addition, the relative levels of intracellular genomic versus antigenomic HPIV1 RNA were measured by reverse transcription with positive- or negative-sense primers, to prime genomic or antigenomic RNA, respectively, followed by real-time PCR (Supplementary Fig. 3). This showed that the ratio of genomic RNA to antigenomic RNA (average ratio 17:1) 129 was similar for each virus at each time point (Supplementary Fig. 3) and, thus, was not detectably perturbed by mutation/deletion of the C protein. Figure 5. Viral RNA and protein expression over time during infection with WT and mutant HPIV1. A549 cells were infected with WT, F170S, or P(C-) HPIV1 at a MOI of 5 for 12, 24, or 48 h. Relative amounts of HPIV1 (A) combined genomic and anti- genomic RNA, (B) N mRNA, and (C) P mRNA were measured by real-time PCR. The values were normalized to the 12 h time point for WT HPIV1, which was assigned the value of 1.0. (D) HPIV1 N, C, and P proteins were detected by Western blot from samples collected in parallel to the samples in A-C. Shown is a representative of two independent experiments with similar results. (E) A549 cells were infected with F170S HPIV1 at a MOI of 5 for 24, 30, 36, 42, or 48 h. The C and P proteins were detected by Western blot. P denotes the position of the P protein, n.s. indicates a non-specific band, and * indicates a potential P cleavage or degradation product detected by the polyclonal anti-P antibody. 130 We also monitored the accumulation of cell-associated viral proteins in the time-course experiment described above by Western blot analysis (Fig. 5d). WT-infected cells contained higher levels of N and P protein at both 24 and 48 h compared to the mutant viruses, which was surprising since WT-infected cells contained the lowest levels of N and P mRNA (Fig. 5d). In contrast, although P(C-)-infected cells contained the highest levels of genomic/anti-genomic RNA at both time points and very high levels of N and P 131 mRNAs at 24 h, they contained the lowest levels of N and P protein at both time points (Fig. 5a-d). Similarly, F170S-infected cells contained much less N and P protein than WT-infected cells despite higher levels of N and P mRNA. Lysates from WT-infected A549 cells also contained a major band of C? protein, which is the most abundant of the HPIV1 C proteins. As expected, no C proteins were detected with P(C-) HPIV1 infection. The C? protein also was detected in lysates from F170S-infected cells at 24 h but, as with the N and P proteins, in lower abundance compared to WT HPIV1. Interestingly, however, the C?F170S protein was not detected at 48 h. We further investigated the kinetics of accumulation of the C?F170S protein in additional time course experiments. This showed that the C?F170S protein was present at 12 and 24 h post- infection (not shown). However, beginning at 30 h post-infection, the level of C?F170S protein began to decline until it was no longer detectable by 42 h (Fig. 5e). In this experiment, the level of the P protein expressed by F170S HPIV1 also decreased with time coincident with the appearance of a smaller P-specific band that might be a degradation product (Fig. 5e). These observations indicated that HPIV1 RNA synthesis indeed was perturbed by mutation and, in particular, deletion of the C protein. These observations also indicate that viral protein synthesis was inhibited in cells infected with F170S and P(C-) HPIV1. The increased production of viral RNA by F170S and P(C-) HPIV1, in the context of reduced levels of N and P protein necessary for encapsidation, could provide for the accumulation of incompletely encapsidated genome capable of annealing with positive sense RNA to form the observed dsRNA. 132 HPIV1 infection and dsRNA accumulation induce PKR activation Since dsRNA was highly abundant during infection with P(C-) and F170S HPIV1 at 24 and 48 h respectively, we examined PKR activation at these same time points. When bound to dsRNA, PKR is activated by autophosphorylation. Activated PKR can phosphorylate EIF2A, resulting in an inhibition of translational initiation (70) that potentially could contribute to the reduced accumulation of viral proteins in cells infected with F170S and P(C-) HPIV1. Consistent with this idea, we found that both F170S and P(C-) HPIV1 induced PKR autophosphorylation and EIF2A phosphorylation, with kinetics mirroring those of dsRNA accumulation (Fig. 4e, f and Fig. 6). Specifically, P(C-) HPIV1 infection induced activation of PKR by 24 h, and F170S HPIV1 infection induced activation of PKR by 48 h. In addition, infection with either mutant strongly induced the expression of MDA5, a highly IRF and IFN-inducible gene (Fig. 6). The expression of the C? and C proteins was observed at 24 and 48 h in cells infected with WT HPIV1, and were not expressed by P(C-) HPIV1, as expected. Consistent with the results in Fig. 5d, both species were present in F170S-infected cells at 24h, and their accumulation greatly decreased by 48 h (Fig. 6). Thus, infection with F170S and P(C-) HPIV1 activated PKR and phosphorylated EIF2A, which might contribute to the reduced accumulation of viral proteins. However, this may not be a complete explanation for the reduced accumulation of viral proteins in the case of F170S HPIV1, since there was little activation of PKR and EIF2A at 24 h (Fig. 6), a time when the reduction in protein accumulation was already evident (Fig. 5d and 6). 133 Figure 6. PKR activation during infection with WT and mutant HPIV1. A549 cells were infected with WT, F170S, or P(C-) HPIV1 at a MOI of 5 for 24 or 48 h. PKR phosphorylation, total PKR, EIF2A phosphorylation, total EIF2A, total MDA5, and C proteins were detected by Western blot. PKR contributes to HPIV1-induced IFN induction as well as the reduced accumulation of viral proteins We examined the effects of PKR activation using stable PKR knockdown 293 cells, in which expression of PKR expression was strongly reduced, although not completely blocked, due to the constitutive expression of a shRNA specific to PKR. PKR knockdown significantly reduced IFN? expression in F170S- and P(C-)-infected cells by 6.2 and 10.1-fold, respectively (Fig. 7). This reduction in IFN? expression corresponded 134 with a reduction in IRF3 phosphorylation (3.1 and 3.4-fold, respectively) (Fig. 8a; see Supplementary Fig. 4a for quantitation). I?B? degradation was only slightly reduced (1.3 and 1.6-fold, respectively) in PKR knockdown cells (Fig. 8a; see Supplementary Fig. 4b for quantitation). P38 phosphorylation was similar in control and PKR knockdown cells (Fig. 8a; see Supplementary Fig. 4c for quantitation). In the mock- infected cells, the expression of PKR in the knockdown cells was 25% that of the control knockdown cells (Fig. 8a; see Supplementary Fig. 4d for quantitation). Interestingly, PKR knockdown also reversed the decrease in the accumulation of C?F170S between 24 and 48 h, an effect that was not observed with the wild-type C' protein (Fig. 8a; see Supplementary Fig. 4e for quantitation). These findings indicated that the reduction in viral protein synthesis in normal cells infected with the C mutant viruses (e.g., Fig 5d, e, and Fig. 6) indeed is linked to PKR activity. Since PKR is an IFN-inducible gene product, the induction of additional PKR due to IFN-mediated signaling could contribute to the loss of viral protein expression during infection with the mutant HPIV1 viruses. Indeed, we found that the decrease in C?F170S protein expression could be partially blocked by IFN neutralizing antibodies (Fig. 8b). 135 Figure 7. IFN? expression induced by infection with WT and mutant HPIV1 during PKR knockdown. Stable 293 cells expressing small hairpin RNAs shPKR and control shCON were infected with WT, F170S, or P(C-) HPIV1 at a MOI of 5 for 48 h. IFN? expression was measured by real-time PCR and normalized to mock infected shCON cells. * denotes P < 0.01 compared to control 293A cells infected with the same virus. 136 Figure 8. Effect of PKR on activation of the components of the IFN? enhanceosome and accumulation of the C protein in HPIV1 WT and mutant virus-infected cells. (A) Stable shPKR-expressing and control shCON 293A cells were infected with WT, F170S, or P(C-) HPIV1 at a MOI of 5 for 48 h. Phosphorylated IRF3, total IRF3, total I?B?, phosphorylated P38, total P38, total PKR, and C proteins were detected by Western blot. (B) Type I IFN affects C? protein expression during HPIV1 infection. A549 cells were infected with F170S HPIV1 at a MOI of 5. At 12 h post-infection, neutralizing antibodies against IFN? (5000 NU/mL) and IFN? (2000 NU/mL) were added. C protein was detected by Western blot at 30 h. 137 Discussion We had previously demonstrated that infection with C mutant HPIV1s (F170S and P(C- )), but not WT HPIV1, effectively stimulated IFN? production in epithelial cells (192). F170S, but not WT, HPIV1 triggered IRF3 dimerization and nuclear translocation, indicating that the wild-type C proteins block the activation of IRF3 (192). In the present study, the mechanisms by which the C proteins prevent IFN? induction were explored in more detail. Since IFN? production is induced optimally by the assembly of an enhanceosome comprised of IRF3, NF-?B, and AP-1 at the IFN? promoter, the activation of these transcription factors during HPIV1 infection was examined first. Infection with HPIV1 containing a single point mutation in C (F170S) or lacking expression of the C proteins (P(C-)) triggered IRF3 and NF-?B activation, whereas WT HPIV1 inhibited activation of these two critical components of the IFN? enhanceosome. Although crystal structures of ATF-2 and c-Jun bound to PRD IV of the IFN? promoter are known, their contribution to the induction of a type I IFN response is less well understood (142). ATF-2 and c-Jun can be phosphorylated and activated by P38 and JNK; however, we found that WT, F170S, and P(C-) HPIV1 did not induce P38 phosphorylation or ATF-2 phosphorylation. Numerous examples of viral proteins that antagonize IFN? production by interacting with various proteins in the RIG-I/MDA5 pathway have been described (19). For instance, the influenza NS1 protein inhibits RIG-I activation by binding to TRIM25 and the hepatitis C NS3/4A protease cleaves and inactivates MAVS (44, 116). In this context, we initially hypothesized that the HPIV1 C proteins acted to inhibit IFN? production by binding or 138 otherwise interfering with one or more components of the RIG-I/MDA5 pathway. Surprisingly, however, several observations made during the present study speak against this hypothesis. First, we were unable to immunoprecipitate the HPIV1 C proteins with any of the known members of the RIG-I/MDA5 pathway that we examined. In addition, we did not observe a significant decrease in abundance or a shift in the gel mobility of any of these host proteins, suggesting that the C proteins do not promote their modification or degradation. Finally, the HPIV1 C proteins, supplied in trans, failed to block IFN? induction by two heterologous inducers, RSV and poly I:C, which are known to signal through RIG-I and MDA5, respectively. Specifically, while infection with WT HPIV1 was able to efficiently block IFN? induction in response to a super-infection with P(C-) HPIV1, it was unable to do so against super-infection with a RSV mutant that does not express the RSV NS1 and NS2 IFN antagonist proteins (Fig. 2a). Similarly, expression of the C proteins in an inducible cell line was able to efficiently block IFN? induction in response to super-infection with P(C-) HPIV1, but failed to prevent poly I:C- induced MDA5-mediated IFN? expression (Fig. 2b, c). The inability of the HPIV1 C proteins to prevent induction in response to these heterologous inducers suggested that they do not antagonize the components of the IFN production pathway at or downstream of RIG-I and MDA5. On the other hand, the ability of the C proteins to inhibit IFN? induction in the context of an HPIV1 infection suggested that their effect is specific to HPIV1. Because the C proteins appeared to disrupt type I IFN production without interacting with key members of the RIG-I/MDA5 pathway, we wanted to confirm that IFN? induction by 139 C mutant HPIV1 viruses did indeed depend on this pathway. Using murine knockout cells, we found that knockout of TBK1 and IKK? completely ablated the IFN? response to F170S and P(C-) HPIV1 infection, confirming that the RIG-I/MDA5 pathway mediates IFN? production for these two viruses. In addition, it was found that IFN? induction relied more strongly on MDA5 than on RIG-I. This is in contrast to the original suggestion that recognition of paramyxovirus infection depends mainly on RIG-I whereas MDA5 is dispensable (67, 98, 119, 179). Several more recent studies with SeV indicated that both RIG-I and MDA5 can contribute to IFN? production in vitro or to resistance in vivo (53, 217). However, these previous findings cannot be readily extrapolated to HPIV1, because SeV, but not HPIV1, also expresses a V protein, a known inhibitor of MDA5 (3, 22, 53, 179, 217). Since MDA5 primarily recognizes long dsRNA molecules (or, more specifically, webs of dsRNA and ssRNA (148)), its involvement in IFN? expression during infection with F170S and P(C-) HPIV1 implied that dsRNA was involved. A striking abundance of cytoplasmic dsRNA was observed in both F170S- and P(C-)-, but not WT-, infected cells. Earlier studies had reported large amounts of dsRNA after infection with positive-strand RNA viruses, double-strand RNA viruses, and DNA viruses but not with negative-strand RNA viruses (113, 177, 199, 201). However, more recently, dsRNA accumulation was detected in cells infected with a C deletion mutant of SeV (181). We did not detect appreciable amounts of dsRNA after infection with WT HPIV1, consistent with expectations (62). In contrast, infection with the P(C-) mutant that completely lacks C expression led to a relatively rapid accumulation of cytoplasmic 140 dsRNA that was readily detected by 24 h but subsequently decreased, and infection with the F170S virus that makes a mutated C protein led to a relatively slower accumulation of dsRNA that was detected in only a few cells at 24 h but increased substantially by 48 h. Thus, HPIV1, a negative-strand RNA virus, does indeed have the capacity to produce dsRNA, and functional C proteins are required during infection to prevent the accumulation of dsRNA that would otherwise trigger a potent host innate antiviral response. The appearance of dsRNA was temporally associated with the activation of PKR, which in turn was associated with a reduction in viral protein accumulation. Infection by P(C-) and F170S HPIV1 was accompanied by autophosphorylation of PKR, phosphorylation of EIF2A, and decreased accumulation of the N, P, and C (in F170S-infected cells) proteins. C protein expression in F170S-infected cells was robust at 24 h but diminished at 30 h and was difficult to detect from 36 h onwards. PKR knockdown studies indicated that PKR is a major contributor to the loss of CF170S from 24 h post-infection onwards. The decrease in C protein synthesis late during infection would render the F170S HPIV1 infection functionally similar to a P(C-) HPIV1 infection, i.e. yield a virus unable to accumulate needed amounts of C protein, and this gradual loss of C proteins in F170S HPIV1 infection accounts for the difference in the kinetics of dsRNA formation between these two mutants. In addition to its role in inhibiting translation during viral infection, PKR has also been implicated in NF-?B activation, IRF1 activation, and IFN induction by poly I:C via MAVS, effects that are independent of its kinase activity and that provide another mechanism of innate antiviral host defense mediated by PKR (4, 16, 25, 107, 141 124, 209, 218). Infection with SeV lacking the entire C gene led to the generation of a large amount of dsRNA with subsequent PKR activation and inhibition of translation (181). In addition, infection with a measles virus mutant lacking the C gene required PKR for maximal IFN? induction (125). Our data indicated that PKR contributed significantly to the induction of IFN? and, more specifically, IRF3 phosphorylation following infection with HPIV1 mutants that generated dsRNA. Thus, MDA5 and PKR each contribute to the generation of IFN? during infection with C mutant HPIV1s, and the generation of dsRNA in F170S- and P(C-)-infected cells is the likely trigger for these two RNA sensors. To investigate the mechanisms underlying the generation of dsRNA during infection with the HPIV1 mutants, we monitored viral RNA levels in infected A549 cells. Compared with WT-infected cells at 24 h post-infection, cells infected with the P(C-) mutant had greatly increased accumulation of genomic and anti-genomic RNA and viral mRNA. Similar findings were made for the F170S mutant, although the increases in viral RNA accumulation were delayed, consistent with the observed delay in dsRNA accumulation compared to the P(C-) mutant. These results indicate that deletion or mutation of the HPIV1 C proteins resulted in a substantial perturbation of viral RNA synthesis. We suggest that the increased accumulation of genomic and anti-genomic RNA in the context of reduced availability of N and P protein led to unencapsidated RNAs that could anneal with each other or with excess viral mRNA to form dsRNA. The C proteins of SeV have been previously implicated in inhibiting viral RNA synthesis and regulating the balance between genome and anti-genome levels (33, 80, 81). Interestingly, one study reported 142 that infection of primate LLC-MK2 and Vero cells with a C deletion SeV led to a predominance of anti-genomic RNA compared to genomic RNA and to an increase in viral protein synthesis (81). In contrast, our results differ in two ways: first, the accumulation of viral proteins was decreased rather than increased, and, second, deletion of the HPIV1 C proteins did not alter the ratio of viral genomic RNA to anti-genomic RNA. These findings suggest that the HPIV1 C proteins prevent the host innate response by modulating the production of viral RNA rather than directly inhibiting the RIG- I/MDA5 pathway. These results also may provide an alternative explanation for the observation that a recombinant SeV expressing HPIV1 C in place of SeV C failed to block IRF3 activation in murine cells (22). This originally was interpreted to indicate that the respirovirus C proteins, like the rubulavirus V protein, is a determinant of host range (22). However, it may be that the HPIV1 C proteins do not function efficiently in the context of the heterologous SeV N, P, and L proteins to prevent the accumulation of dsRNA and subsequent IRF3 activation. It is not clear whether PKR- and IFN?-induced inhibition of protein synthesis can fully account for the reduced accumulation of viral proteins during infection with the HPIV1 mutants. For example, in cells infected with the F170S mutant, the levels of N and P protein were reduced compared to WT-infected cells even at 24 h, a time point before the activation of PKR was detected. Also, while the extremely high accumulation of genomic and anti-genomic RNA with the P(C-) mutant could be imagined to outpace the synthesis of N and P protein and thus initiate the dsRNA formation resulting in PKR activation and IFN? induction, this seemed less likely for the F170S mutant, since the 143 increase in RNA accumulation was more gradual. Thus, there may be an additional host inhibitory effect on viral protein accumulation that is unleashed by deletion/mutation of the C proteins or, alternatively, the C proteins may have a positive effect on viral protein synthesis. Further studies to explore these two alternative mechanisms of regulating viral protein synthesis are clearly needed. Our results indicate that the wild-type C proteins regulate viral RNA synthesis to prevent the production of dsRNA that would otherwise activate MDA5 and PKR which, in turn, would activate IRF3 and NF-?B, result in the induction of IFN?, and inhibit protein synthesis. We propose that the following series of events occur during infection with P(C-) HPIV1 (summarized in Table 1). dsRNA is generated early, within the first 24 h of infection, due to the combined rapid increase of mRNA, genomic RNA, and anti- genomic RNA. The dsRNA activates PKR and also induces IFN?. PKR activation and IFN? induction decrease viral protein synthesis, thereby maintaining low levels of N protein. IFN? might also inhibit protein synthesis by the induction of inhibitors such as IFIT1 and IFIT2 (187). In addition, apoptosis is activated early in P(C-)-infected cells, but the mechanism underlying this induction remains undefined (9). The combined effects of PKR activation, IFN? induction, and the initiation of apoptosis are likely responsible for the peak and subsequent decline in viral macromolecular synthesis observed between 24 and 48 h after infection with P(C-) HPIV1. The events occurring during infection with the F170S mutant represent a delayed version of what is observed following infection with P(C-) HPIV1. The more gradual increase in transcription and replication of viral RNA in F170S-infected cells delays the formation of dsRNA until 144 between 24 and 48 h, at which time PKR is activated and IFN? is induced. The PKR- mediated loss of C protein accumulation in F170S-infected cells likely contributes to the increase of transcription seen at 48 h since C is an inhibitor of viral transcription. A more detailed analysis of the kinetics of viral and host cell RNA and protein synthesis in WT and C mutant HPIV1-infected cells is clearly needed to test the above hypotheses. In summary, our initial working hypothesis that the HPIV1 C proteins inhibit IFN? induction via a direct inhibition of the RIG-I/MDA5 pathway was not confirmed. Instead, we found that the HPIV1 C proteins regulate viral RNA synthesis to prevent the formation of dsRNA that otherwise triggers host antiviral responses, such as PKR activation and IFN? induction. MDA5 was found to be a more important sensor than RIG-I for infection with these dsRNA-generating HPIV1 mutants. The kinetics of dsRNA accumulation and decreased expression of mutant C proteins suggest that a race between the evasion of host antiviral responses and the elimination of viral antagonists of dsRNA accumulation is key in determining the outcome of HPIV1 infections. 145 General Conclusion HPIV1 was first discovered by Robert Chanock in 1959, but, after over 50 years, it remains an uncontrolled respiratory pathogen that continues to plague infants, children and even adults, in particular the elderly, nursing home residents, and the immunocompromised (23, 29). There are currently no antiviral agents with proven efficacy against HPIV1. Instead, the management of croup and other more severe manifestations of lower respiratory disease caused by HPIV1 is merely supportive. Furthermore, although vaccine development began in the 1960s, there is currently no licensed vaccine against HPIV1 (24, 133). To date, non-living inactivated and subunit vaccines have failed to confer protection against upper and lower respiratory disease in humans and none are currently in clinical trials (24, 43, 133). However, two live- attenuated vaccine candidates against HPIV1 are currently being tested in phase 1 clinical trials. One candidate is an unmodified Sendai virus that is administered intrasnasally as a HPIV1 vaccine (171). The only other vaccine candidate against HPIV1 (rHPIV1- CR84G/?170 HNT553A LY942A) was developed by this lab using reverse genetics (7). The polymerase mutation LY942A was first identified in HPIV3cp45 and confers a temperature- sensitive phenotype so that virus replication is permissive in the cooler upper respiratory tract but restricted in the warmer lower respiratory tract (126). This HPIV1 mutant virus was restricted in replication by ? 100-fold compared to WT HPIV1 when the temperature was increased from 32?C to 39?C, the viral shut-off temperature (7). The other attenuating mutations, CR84G and C?170, each interfere with the virus? ability to antagonize the host antiviral response (192). Although the HPIV1 C proteins are non-essential for viral replication in vitro, complete ablation of C protein expression results in a virus that 146 barely replicates in non-human primates and is over-attenuated as a vaccine candidate (9). However, the C proteins can be mutated by reverse genetics to incrementally attenuate HPIV1 in order to develop vaccine candidates that possess an acceptable balance between attenuation and immunogenicity (9). The C proteins are important virulence factors that play a key role in modulating the host response to HPIV1 infection. In the studies described here, we sought to examine the mechanisms by which the wild- type HPIV1 C proteins prevent a potent innate antiviral immune response and to investigate the mechanisms by which mutating or deleting the C proteins leads to attenuation. To address these questions, we first created by reverse genetics a virus named P(C-) HPIV1 in which expression of the C proteins was silenced. We found that the complete lack of C protein expression significantly restricted viral replication and induced type I IFN production during infection. Using microarray-based transcriptional profiling, we found that the C proteins controlled the transcription of a broad array of genes in human respiratory epithelial cells. Additionally, we found that the global gene expression profile induced during infection with F170S HPIV1 was surprisingly similar to P(C-) HPIV1. We applied a bioinformatics analysis and identified a significant over-representation of IRF and NF-?B binding sites among many of the C protein-responsive genes. We also determined that the main components of the type I interferon (IFN) response activated upon infection with the C mutant HPIV1 viruses indeed consisted of IRF3 and NF-?B. We initially hypothesized that the C proteins might interact with a host factor in the RIG- I/MDA5 pathway to prevent IRF3 and NF-?B activation and IFN? induction during 147 infection with WT HPIV1. Numerous examples of viral proteins that antagonize IFN? production by this very mechanism had previously been described. For instance, the influenza NS1 protein inhibits RIG-I activation by binding to TRIM25 and the hepatitis C NS3/4A protease cleaves and inactivates MAVS (44, 116). Thus, we attempted to search for a host factor in the RIG-I/MDA5 pathway that might also interact with the C proteins. However, we made two key observations that contradicted this initial hypothesis. First, we were unable to immunoprecipitate the HPIV1 C proteins with any of the known members of the RIG-I/MDA5 pathway that we examined. Additionally, the HPIV1 C proteins, supplied in trans, failed to block IFN? induction by two heterologous inducers, RSV and poly I:C, which are known to signal through RIG-I and MDA5, respectively. The inability of the HPIV1 C proteins to prevent IFN? induction in response to these heterologous inducers suggested that they do not antagonize the components of the IFN production pathway at or downstream of RIG-I and MDA5. On the other hand, the ability of the C proteins to inhibit IFN? induction in the context of an HPIV1 infection suggested that their effect was specific to HPIV1. Even though the C proteins did not appear to interfere with the signaling pathway leading from RIG-I/MDA5 activation to IFN? induction, we sought to determine whether the IFN? induced during infection with the HPIV1 C mutant viruses occurred through the RIG-I/MDA5 pathway. With the use of knockout MEFs, we discovered that IFN? induction by infection with the HPIV1 C mutant viruses relied mainly on MDA5. As a result, we began to suspect that HPIV1-induced IFN? expression might also involve dsRNA, the substrate for MDA5 activation. We decided to test for the presence of 148 dsRNA in cells infected with the HPIV1 C mutants, even though previous reports suggested that dsRNA was absent in cells infected with other negative sense RNA viruses, including influenza, La Crosse, Sendai, and Newcastle disease (199). To our surprise, we discovered a striking accumulation of dsRNA in response to infection with the HPIV1 C mutants. We then began to explore the mechanisms by which the C proteins of WT HPIV1 suppress the accumulation of dsRNA by examining viral RNA and protein synthesis. We were surprised to find that infection with viruses in which C protein function was compromised led to increased levels of viral RNA species and decreased N protein levels, creating an imbalance between the N protein-to-genomic RNA ratio and thereby potentially permitting dsRNA accumulation. The formation of dsRNA would then trigger MDA5 activation and IFN? production and also PKR activation and an inhibition of further protein synthesis (Figure 1). Figure 1. Diagrammatic representation of the steps leading to dsRNA accumulation and activation of the MDA5 and PKR pathways during infection with P(C-) HPIV1 but not with WT HPIV1. The red lines represent the negative sense viral genome and the blue lines represent the positive sense viral antigenome or viral mRNA. On the left panel, WT HPIV1 attaches, enters, and introduces its negative sense genome into the cytoplasm of the infected cell. The antigenome serves as a template for the production of more genome and mRNA. The mRNAs are translated by the ribosome to produce viral proteins, including the C proteins and the N proteins. Encapsidation by the N protein shields genomic and antigenomic RNAs from recognition by intracellular sensors of dsRNA. As a result, the dsRNA binding proteins MDA5 and PKR which are present 149 constitutively at low levels in the cytoplasm remain latent, the transcription factors IRF3 and NF-?B remain in the cytoplasm and do not induce IFN? expression, and EIF2A does not inhibit translation. On the right panel, the absence of C proteins during P(C-) HPIV1 infection leads to dysregulated viral RNA synthesis, i.e. increased production of negative sense genome and positive sense antigenome and mRNA. The formation of dsRNA webs (symbolized by a spider web) triggers MDA5 and PKR activation. MDA5 activates the transcription factors IRF3 and NF-?B and induces IFN? expression. PKR activates EIF2A which inhibits the translation of viral mRNAs and may limit the amount of N protein available to encapsidate the genome and antigenome. The increase in viral RNA levels in the context of decreased N protein levels would further amplify the formation of dsRNA and the activation of the IFN? enhanceosome. 150 The N protein has long been thought to function as a highly stable and ribonuclease resistant nucleocapsid coat that protects the viral genome and antigenome from nuclease digestion (100). In the present study, we also found an association between the amount of N protein present and the accumulation of dsRNA. HPIV1 genome replication and mRNA transcription are normally tightly coupled to the amount of available N protein to allow sufficient encapsidation of the genome and antigenome, and this process may become dysregulated in the absence of the C proteins. The exact molecular mechanisms by which the HPIV1 C proteins maintain a balance between N protein levels and viral RNA synthesis to prevent the formation of immunostimulatory dsRNA remain to be determined. The C proteins of murine PIV1 (SeV) were found to inhibit viral mRNA transcription in an in vitro transcripton system in which the P and L proteins are combined with RNA templates (33, 97, 110). Furthermore, the SeV C proteins suppressed viral RNA synthesis from defective-interfering genomes in a promoter- specific manner (21). In particular, ablation of C protein expression during SeV infection led to a disproportionate increase in positive sense antigenomic RNA compared to negative sense genomic RNA (81). Thus, it was postulated that a ratio of genomic to antigenomic RNA skewed towards greater levels of positive sense antigenomic RNA was a potential catalyst for dsRNA formation (81). However, we found that, for HPIV1, deletion of the C proteins increased viral RNA synthesis but did not alter the ratio of viral genomic to antigenomic RNA. We also observed decreased levels of viral protein, including N protein, during infection with P(C-) HPIV1. Importantly, an infection with HPIV1 that does not express the C proteins induces the formation of dsRNA which triggers PKR and EIF2A activation and inhibits protein synthesis. This suggests that the 151 extremely rapid and high accumulation of viral RNA might outpace the synthesis of N protein thereby triggering and further amplifying dsRNA formation. Notably, one report has suggested that the SeV C proteins suppress viral RNA synthesis via an interaction with the L polymerase (75). We have found preliminary evidence that the C proteins co- localize with the N protein but not the P protein in cells infected with WT HPIV1 (not shown). Whether the HPIV1 C proteins interact with and alter the function of the viral replication complex or with an as yet unidentified host factor remains to be seen. The proximity of the C proteins to the N and L proteins may serve an important role in coupling viral RNA synthesis with encapsidation. The study of the HPIV1 C proteins has yielded unexpected insights into how HPIV1 can cause disease in children. A better understanding of the mechanisms by which the C proteins control the balance between RNA and protein synthesis and interfere with innate immunity will require further study. In summary, the HPIV1 C proteins are clearly multifunctional and much work to define their functions remains to be done. However, significant progress has been made in the understanding of HPIV1 pathogenesis and in the development of a safe and efficacious vaccine against HPIV1. 152 Appendices Supplementary Information for Chapter 3 Figure S1. Venn diagram comparison of the 2421 genes and the 1600 genes significantly differentially expressed compared to mock during CF170S infection or P(C-) infection, respectively. Table 1 shows the expression of 10 representative genes from this Venn diagram that were not shared between the two C mutant infections. The expression of the majority of genes did not differ. 153 Figure S2. Principal component analysis of experimental conditions using all genes on the microarrays and mean centering to examine the level of similarity between each condition. Each point represents a specific condition (virus and time point post-infection). The distance between points indicates the level of similarity between the conditions based on the expression of all genes on the array. All genes were included in this analysis so that this analysis is not biased by any specific fold change or statistical selection criteria. The axes are eigenvectors, and the first eigenvector (x-axis) passes through the mean, minimizes the sum squared error for each point, and describes the direction of maximum variance. The eigenvalues represent the level of variance for each eigenvector. Overall gene expression during P(C-) and CF170S infection was highly similar. Both C mutant viruses induced gene expression patterns that were significantly different compared to WT infection. 154 Figure S3. Hierarchical clustering and functional analysis of 28 genes that differed between the two C mutants by 2-fold followed by P < 0.01 with multiple testing correction. 155 Figure S4. Differential gene expression between CF170S and P(C-) infection of A549 cells. An alternative analysis identified 18 genes by a two-way ANOVA with multiple testing correction (see Table S7) that were significantly different between the two C mutant viruses. Expression ratios between CF170S and P(C-) for these 18 genes were plotted on a log2 scale to give a visual representation of the kinetics and magnitude of any differences in the transcriptional response between the two C mutant viruses. A ratio of 0 indicates that there is no difference, negative values indicate that the expression with CF170S is lower compared to P(C-), and positive values indicate that the expression with CF170S is higher compared to P(C-). The dotted lines denote a 4-fold difference. The majority of genes were only different by slightly greater than 4-fold. However, a kinetic shift in the transcriptional profile can be observed after 24 h p.i. Up to 24 h p.i., gene expression during CF170S infection is, in general, lower than that during P(C-) infection. These differences do not persist at 48 h p.i., and both viruses approach equivalent expression for this set of genes. However, a small but distinct set of genes appears at 48 h p.i. in which gene expression during CF170S infection is, in general, now greater than that during P(C-) infection. 156 157 Figure S5. Replication of wt HPIV1, CF170S, and P(C-) in A549 cells. A549 cells were inoculated with sucrose purified viruses at a MOI of 5 TCID50/cell and supernatant was harvested for virus quantitation at 0, 6, 12, 24, and 48 h p.i. Photomicrographs taken at 48 h p.i. show more extensive cytopathic effect in P(C-) infected A549 cells than in wt HPIV1 or CF170S infected cells. 158 Figure S6. Gene expression profiles for all 14 transcription factors identified by bioinformatics analysis. The expression of 4 genes IRF1, ISGF3G, NF-?B1, and c-Rel highlighted in yellow was significantly up-regulated increasingly over time. 159 Table S1. Table S1. Genes differentially expressed by wt HPIV1 or IFN? compared to mock infection*? Cluster? Symbol Product Agilent IFN 6 hr IFN 24 hr WT 6 hr WT 12 hr WT 24 hr WT 48 hr A APOL1 apolipoprotein L1 isoform b precursor A_24_P87931 6.7 7.6 1.6 1.7 4.2 8.1 APOL4 apolipoprotein L4 isoform 1 A_23_P380857 6.2 7.9 1.4 1.7 5.1 7.2 APOL6 apolipoprotein L, 6 A_24_P941167 8.6 8.3 1.5 1.7 6.3 9 APOL6 apolipoprotein L6 A_23_P155052 13.5 7.2 0.9 1 2.2 6 APOL6 apolipoprotein L6 A_23_P155049 8.1 6.8 1.1 0.9 4.3 8.5 APOL6 A_24_P7594 7.3 10.3 0.9 1.5 6.8 11.3 B2M beta-2-microglobulin precursor A_23_P37441 1.7 5.5 0.7 0.7 1.4 4.6 BATF2 basic leucine zipper transcription factor, ATF-like 2 A_23_P370682 8.8 14.8 0.9 1.1 3.3 8.3 BST2 bone marrow stromal cell antigen 2 A_23_P39465 8.2 21.5 1.3 2.6 3.5 10.6 BTC betacellulin A_23_P135722 2.3 5 1.5 1.4 1.4 2.7 C15orf48 hypothetical protein LOC84419 A_23_P26024 1.6 3.6 0.9 1.1 1.4 4.8 C1orf38 basement membrane-induced gene isoform 1 A_23_P873 14.2 16.6 1.2 1.5 2.1 3.6 CASP1 caspase 1 isoform alpha precursor A_23_P202978 6.2 46.2 0.8 0.9 6.3 17.1 CD38 CD38 antigen A_23_P167328 1.4 6 0.8 1.1 1.3 5.4 CEACAM1 carcinoembryonic antigen-related cell adhesion molecule 1 isoform 1 precursor A_24_P382319 20.4 15.5 1.3 1.7 6.4 13.6 CEACAM1 carcinoembryonic antigen-related cell adhesion molecule 1 isoform 2 precursor A_23_P434118 2.8 4.6 0.9 1 3 7.9 CFB complement factor B preproprotein A_23_P156687 3 7.9 0.6 0.8 1.7 6.2 CTSS cathepsin S preproprotein A_23_P46141 2.1 4.4 0.9 1.1 1.4 3.3 DDX58 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide RIG-I A_23_P20814 28.7 32 1.3 1.6 4.1 27.3 DTX3L deltex 3-like A_24_P941912 6.1 7.1 0.8 1 2.7 4 DTX3L deltex 3-like A_23_P347040 9.8 8.6 0.9 1 2.1 4.1 EIF2AK2 eukaryotic translation initiation factor 2- alpha kinase 2 A_23_P142750 2.7 5.5 1.1 1.1 1.1 1.5 EMR1 egf-like module containing, mucin-like, hormone receptor-like sequence 1 A_23_P27556 1.3 5.2 0.7 1.1 1.4 2.9 EXOC3L exocyst complex component 3-like A_23_P424734 1.3 4.5 0.9 1.3 1.8 4.9 GBP1 guanylate binding protein 1, interferon- inducible, 67kD A_23_P62890 57.4 82.6 1.1 1 14.3 64.2 GBP1 guanylate binding protein 1, interferon- inducible, 67kD A_32_P107372 48 41.4 1 1.3 18.1 70.7 GBP3 guanylate binding protein 3 A_24_P370702 8.4 11.1 1.2 1.1 6.2 14.4 GBP3 guanylate binding protein 3 A_23_P51487 10.8 17.2 0.6 0.7 8.4 17.6 GBP4 guanylate binding protein 4 A_24_P45446 7.2 20.1 0.7 1.3 3.4 32.7 HCP5 A_23_P111126 1 8.2 0.6 1.1 1.4 14.7 HERC5 hect domain and RLD 5 A_23_P110196 4.3 21.4 0.7 1.5 4.1 40 HLA-B major histocompatibility complex, class I, B A_23_P125107 1.5 5.2 0.8 0.9 1.2 4.2 HLA-B major histocompatibility complex, class I, B A_24_P113674 1.6 4.3 1 0.9 1.2 3.9 HLA-C major histocompatibility complex, class I, C precursor A_24_P298409 1.6 4.6 1.1 1.1 1.3 3.6 HLA-C MHC class I antigen A_23_P95917 1.5 4.6 0.9 0.9 1.3 3.5 HLA-C A_23_P113716 1.5 4.3 1.1 1.1 1.3 3.5 HLA-E major histocompatibility complex, class I, E precursor A_32_P460973 2.5 4.3 1 1 1.4 2.5 HLA-E major histocompatibility complex, class I, E precursor A_24_P326082 2.2 4.2 0.8 0.8 1.1 2.8 HLA-F major histocompatibility complex, class I, F precursor A_23_P314024 1.6 5.2 0.9 1 1.3 5.7 160 HRASLS2 HRAS-like suppressor 2 A_24_P364263 2 6.2 1.1 1.1 1.4 4.2 HRASLS2 HRAS-like suppressor 2 A_23_P105012 2.1 9.8 0.8 1.2 1.9 7.3 HSH2D hematopoietic SH2 domain containing A_23_P153372 6 10.6 1 1.2 1.5 3.7 IFI16 interferon, gamma-inducible protein 16 A_23_P160025 11.9 19.3 1 1 1.8 2.9 IFI16 A_23_P217866 11.3 16.7 1.2 1.1 1.6 2.6 IFI27 interferon, alpha-inducible protein 27 A_23_P48513 12.4 20.8 1.1 1.1 2.9 12.3 IFI27 interferon, alpha-inducible protein 27 A_24_P270460 9.8 12.5 1 1.3 2.4 11.3 IFI35 interferon-induced protein 35 A_23_P152782 2.4 7.8 0.8 0.8 1.5 2.1 IFI44 interferon-induced, hepatitis C- associated microtubular aggregate protein A_23_P23074 50.7 65.1 1 7 16.1 55.2 IFI44L histocompatibility 28 A_23_P45871 24.6 78.5 1 0.9 2.2 24.5 IFI6 interferon, alpha-inducible protein 6 isoform c A_23_P201459 14.3 45.5 1 1.4 2.7 10.4 IFIH1 interferon induced with helicase C domain 1 A_23_P68155 16.7 30 1.2 3.3 8 53.8 IFIT1 interferon-induced protein with tetratricopeptide repeats 1 isoform 2 A_23_P52266 32.1 35.9 4.8 14.5 11.2 33.2 IFIT3 interferon-induced protein with tetratricopeptide repeats 3 A_23_P35412 81.5 108.1 0.8 4.1 15.8 73.4 IFIT5 interferon-induced protein with tetratricopeptide repeats 5 A_24_P30194 6.1 6.4 1.1 1.1 1.5 4.3 IFIT5 interferon-induced protein with tetratricopeptide repeats 5 A_23_P63668 5.4 5.5 1.3 1.3 1.5 3.8 IFITM1 interferon induced transmembrane protein 1 (9-27) A_23_P72737 6.7 8 0.9 1.1 2.3 2.2 IL4I1 interleukin 4 induced 1 isoform 2 A_23_P502520 2.5 7.9 1.3 1 1.3 6.2 INDO indoleamine-pyrrole 2,3 dioxygenase A_23_P112026 10.6 39.4 1.1 1 21.3 71 IRF7 interferon regulatory factor 7 isoform d A_24_P378019 8.4 13.6 1 1.3 2 3.7 ISG15 interferon, alpha-inducible protein (clone IFI-15K) A_23_P819 13.5 36.9 1.2 3.3 9.9 25.2 ISG20 interferon stimulated gene 20kDa A_23_P32404 2.2 6.5 0.9 0.9 1.8 8.2 ISGF3G interferon-stimulated transcription factor 3, gamma 48kDa A_23_P65442 14.1 11 1.4 1.9 6.9 6.2 LAMP3 lysosomal-associated membrane protein 3 A_23_P29773 7.8 114.5 1.3 1.4 1.8 7.6 LAP3 leucine aminopeptidase 3 A_23_P18604 2.2 5.4 0.5 0.8 1 1.5 LGALS9 galectin 9 long isoform A_32_P452655 3.2 8.8 1.3 1.2 1.4 2.5 LMO2 LIM domain only 2 A_23_P53126 12.1 9.7 1 0.9 1.1 2.7 MX1 myxovirus resistance protein 1 A_23_P17663 22 30.4 1.3 2.2 7.8 14.4 MX2 myxovirus resistance protein 2 A_23_P6263 18.5 74.3 1.6 1.2 3 5.5 NMI N-myc and STAT interactor A_23_P154235 2.7 6.2 0.9 0.8 1.6 3.1 NT5C3 5'-nucleotidase, cytosolic III isoform 2 A_23_P59547 1.7 4.3 0.9 0.9 1.2 1.5 OAS1 2',5'-oligoadenylate synthetase 1 isoform 2 A_23_P64828 5.1 8.8 1 0.9 1.5 4.1 OAS2 2'-5'-oligoadenylate synthetase 2 isoform 1 A_23_P204087 10.5 30 0.7 0.8 1 3.7 OAS3 2'-5'oligoadenylate synthetase 3 A_23_P47955 3.9 17.6 1 1.1 1.7 5.1 OAS3 2'-5'oligoadenylate synthetase 3 A_24_P335305 2.9 10.3 0.8 0.9 1.4 3.5 OASL 2'-5'-oligoadenylate synthetase-like isoform a A_23_P139786 40.8 49.6 2.4 24.5 33.3 58.9 PARP12 zinc finger CCCH-type domain containing 1 A_23_P111804 4 9.4 1.1 1.1 2 3 PARP14 poly (ADP-ribose) polymerase family, member 14 A_32_P56759 5.2 8 0.7 1.3 3.6 8 PARP14 poly (ADP-ribose) polymerase family, member 14 A_24_P161018 6.5 12.5 1.5 1.3 2.7 7 PARP9 poly (ADP-ribose) polymerase family, member 9 A_23_P69383 10.2 11.3 1.2 1.1 3.8 5.2 PLA1A phospholipase A1 member A A_24_P294408 1.9 8.7 0.9 1.2 2.2 5.7 PLA1A phospholipase A1 member A A_23_P29816 1.4 5.4 1.2 1.3 1.5 5.3 161 PLEKHA4 pleckstrin homology domain containing, family A (phosphoinositide binding specific) member 4 A_23_P67360 4.7 8.6 1 1.4 3.6 11.7 PLEKHA4 pleckstrin homology domain containing, family A (phosphoinositide binding specific) member 4 A_24_P408047 3.9 8.9 1.3 1.7 3.9 16.7 PLSCR1 phospholipid scramblase 1 A_23_P69109 3.7 12.6 0.9 0.8 1.1 3.2 PML promyelocytic leukemia protein isoform 1 A_24_P207139 4.5 5.7 1.3 1 2 1.5 PML promyelocytic leukemia protein isoform 6 A_23_P306148 2.7 4.1 1 1.2 1.6 1.7 PML promyelocytic leukemia protein isoform 6 A_24_P198598 2.7 4.5 1 1.2 1.7 1.6 PNPT1 hypothetical protein A_32_P76035 2.2 5.1 0.8 1.1 1.2 1.2 PNPT1 polyribonucleotide nucleotidyltransferase 1 A_23_P154488 2.4 4.8 0.9 1.1 1.2 1.2 PPM1K protein phosphatase 1K (PP2C domain containing) A_24_P214598 2.5 5.7 0.7 0.9 1.4 6.7 PSMB10 proteasome beta 10 subunit proprotein A_23_P140807 1.5 4.6 1.2 1.2 2.1 4.9 PSMB8 proteasome beta 8 subunit isoform E1 proprotein A_23_P250629 2.4 6.9 0.8 1 2.1 3.5 PSMB9 proteasome beta 9 subunit isoform 1 proprotein A_23_P111000 3.1 12 0.6 0.9 3.8 8.8 RARRES3 retinoic acid receptor responder (tazarotene induced) 3 A_23_P1962 2.1 6 1 1.3 4.4 6.9 RASGRP3 RAS guanyl releasing protein 3 (calcium and DAG-regulated) A_24_P54390 9.8 15.2 0.9 0.8 3.7 3.8 REC8 REC8 homolog A_24_P344087 5.5 4.9 1.6 2.4 3.9 3.9 RGS22 regulator of G-protein signalling 22 A_32_P125771 2 12.6 1 0.9 1.3 3 SAMD9 sterile alpha motif domain containing 9 A_23_P355244 23 26.9 1 1.1 3.7 14.6 SAMD9 sterile alpha motif domain containing 9 A_24_P175187 42.2 25.2 0.9 1.1 3.5 13.4 SAMD9 sterile alpha motif domain containing 9 A_24_P175188 36.7 37 0.9 1.1 5 12.4 SAMD9L sterile alpha motif domain containing 9- like A_23_P145874 35.7 31.7 1.5 1.6 14.5 22.3 SIDT1 SID1 transmembrane family, member 1 A_23_P132515 4.5 9 0.9 1.9 2.7 2.6 SLC15A3 solute carrier family 15, member 3 A_23_P75786 4.9 14.9 0.9 0.8 1.7 7.1 SP100 nuclear antigen Sp100 isoform 2 A_24_P385611 2.9 5 0.8 0.9 1.2 1.9 SP100 nuclear autoantigen A_24_P916816 5.8 7.1 1.8 1.2 1.9 3.4 SP110 SP110 nuclear body protein isoform b A_23_P120002 8.8 14.5 1 0.9 1.8 2.2 SP140 SP140 nuclear body protein isoform 1 A_24_P328504 3.7 6.8 1.6 1.1 2 3.4 STAT1 signal transducer and activator of transcription 1 isoform alpha A_23_P56630 3 8.3 0.6 1 3.7 4.9 STAT1 signal transducer and activator of transcription 1 isoform beta A_24_P274270 4.6 10.1 0.9 1.5 4.7 5.7 STAT2 signal transducer and activator of transcription 2 A_23_P76090 4.9 3.8 0.9 1.1 2.1 2.5 TAP1 transporter 1, ATP-binding cassette, sub-family B A_23_P59005 4.8 9.2 1 1.4 3.8 9.3 TGM2 transglutaminase 2 isoform a A_32_P86763 1.7 4.2 0.6 0.7 1.2 1.5 TLR3 toll-like receptor 3 A_23_P29922 11.9 16.1 0.9 1 5.7 12.7 TMEM140 hypothetical protein LOC55281 A_23_P31177 12.1 8.5 1.2 0.9 3.4 4.4 TMEM140 hypothetical protein LOC55281 A_24_P372134 4.1 3.4 1.2 1 1.2 1.7 TNFRSF14 tumor necrosis factor receptor superfamily, member 14 precursor A_23_P126908 1.9 6.1 1 0.7 3.1 5.3 TNFSF10 tumor necrosis factor (ligand) superfamily, member 10 A_23_P121253 23.5 20.1 1.2 1.1 2.2 6.9 TNFSF13B tumor necrosis factor (ligand) superfamily, member 13b A_23_P14174 6.2 13.1 1 1.2 2.8 4.3 TRIM14 tripartite motif protein TRIM14 isoform alpha A_23_P425752 1.7 4.6 1 1.2 1.2 1.6 TRIM14 tripartite motif protein TRIM14 isoform alpha A_23_P216655 2.1 7.3 0.7 1 1.5 2.6 TRIM14 tripartite motif protein TRIM14 isoform alpha A_24_P197964 2.3 8.7 0.6 1 1.3 2.5 TRIM14 tripartite motif protein TRIM14 isoform alpha A_23_P334083 1.7 4.6 0.6 0.7 1.2 1.9 162 TRIM22 tripartite motif-containing 22 A_23_P203498 16.1 42 0.8 0.9 6.6 19.6 TRIM34 tripartite motif protein 34 isoform 1 A_24_P398323 2.1 4.1 0.8 0.8 1.5 1.4 TRIM69 A_24_P50543 4.1 3.8 1 0.7 1.6 2.9 UBD diubiquitin A_23_P81898 6 12.9 1 0.8 3 41.6 UBE1L ubiquitin-activating enzyme E1-like A_23_P21207 3.5 21.8 0.9 0.8 2.7 7.5 UBE2L6 ubiquitin-conjugating enzyme E2L 6 isoform 2 A_23_P75741 2.8 9.2 0.7 1.1 1.8 2.9 USP18 ubiquitin specific protease 18 A_32_P132206 5.3 9.2 1.2 1.3 1.7 4.2 USP18 ubiquitin specific protease 18 A_23_P132159 4.9 9.6 0.9 1 1.5 3.5 ZNFX1 zinc finger, NFX1-type containing 1 A_24_P23034 5.7 3.2 1 1.2 1.7 3.7 hypothetical protein A_32_P161292 2.9 5.4 1.1 1.1 2.8 4.2 hypothetical protein LOC55337 A_24_P236949 2.3 7.6 0.8 0.8 1.1 1.7 hypothetical protein LOC55337 A_23_P38894 3.8 9.8 1.4 1.2 1.6 2 hypothetical protein LOC55601 A_23_P41470 5.1 13.7 0.4 0.8 3 10.7 hypothetical protein LOC55601 A_24_P334361 5.8 18.1 0.4 0.8 3.9 16.4 hypothetical protein LOC79132 A_23_P38346 11.8 50.6 1.2 2 6 48.6 hypothetical protein LOC93349 A_23_P337753 2.8 4.3 0.9 1.1 2.6 3.2 leukocyte-derived arginine aminopeptidase A_23_P30243 4.3 4.6 1.1 1.3 3.1 5.8 promyelocytic leukemia protein isoform 2 A_23_P358944 5.1 4.5 1.4 1.2 1.4 2.3 Unknown (protein for MGC:105145) A_24_P418044 1.7 5.4 1 1.1 1.4 4.2 XIAP associated factor-1 isoform 1 A_23_P4286 65.7 270 1.6 1.4 7.9 77.7 XIAP associated factor-1 isoform 1 A_23_P4283 52.8 95.9 1.4 0.9 5 38.6 A_23_P354547 11.5 8.7 1.5 3.1 5.1 5 A_32_P171793 6.2 12.8 0.9 1.3 4.2 10.5 A_24_P829156 5.5 4.7 1.5 1.2 3.1 5.1 A_24_P740662 11.5 8.7 1.6 1.9 7.2 10 A_24_P127641 5.6 8.3 1.4 1.3 2.9 7.9 A_23_P384355 63.7 88.6 0.8 1 2.8 24.8 A_32_P156746 8.1 6.2 1.3 1 2.5 3.5 A_24_P868905 4.2 12.9 0.9 1.1 1.4 2.3 A_32_P54553 3.4 5.9 0.6 1 1.3 2.4 A_23_P65174 2.3 4 1 1 1.5 1.8 A_23_P30069 4 17.2 0.9 1.2 2.5 4.6 A_24_P15702 2 4.3 0.5 0.9 1.1 1.4 A_24_P118892 6.3 11.9 0.7 1.5 1.1 1.9 A_32_P99533 5.5 10 1.8 2 6.1 21.4 A_32_P56249 10 18.8 0.9 1 6.2 32 A_32_P154726 2.6 3.4 0.9 0.9 1.5 4.3 A_23_P370707 1.7 5.3 0.8 0.9 1.5 6.3 A_24_P586264 3.3 6.3 0.9 1.2 1.6 8.3 A_23_P250353 4 11.1 0.9 1.1 2 8.5 A_24_P68079 4.6 9.3 1 1.1 2.9 7.7 A_32_P92415 4.1 10 1.3 1.2 2.9 7.9 A_23_P125109 1.5 5 0.9 1 1.2 4.7 A_23_P373126 1.8 6.6 0.8 0.9 1.3 6.2 A_24_P860781 1.5 4.6 0.9 1 1 3.2 A_32_P143980 2 4.3 1.3 1.1 1.4 3.7 A_23_P17837 5.3 10.7 1.1 1.8 3.4 7.9 A_23_P203629 1.5 4.8 1.4 1.1 1.3 2.9 B APOL2 apolipoprotein L2 A_23_P211488 5.6 2.5 1 1.3 3.6 6 163 APOL2 apolipoprotein L2 A_24_P48898 3.7 3.1 1.8 1.8 3.5 5.2 BTN3A3 butyrophilin, subfamily 3, member A3 isoform a A_24_P311917 3.7 4.2 1.1 1.2 4.4 5.4 C20orf141 A_32_P133182 6.2 2.4 1.1 1.1 4.1 13.9 CA13 carbonic anhydrase XIII A_23_P381714 5.4 2.3 1.1 1.3 4.5 3.7 CCL2 small inducible cytokine A2 precursor A_23_P89431 1.9 1.2 1 1 1.1 5 CD274 CD274 antigen A_23_P338479 3.6 2.7 0.8 1 2 8.2 CXorf43 hypothetical protein LOC139324 A_23_P386364 2.8 3.7 0.6 0.8 1.8 4.7 FAS tumor necrosis factor receptor superfamily, member 6 isoform 1 precursor A_23_P63896 1.7 1.5 0.6 0.9 1.3 4.4 GBP5 guanylate-binding protein 5 A_23_P74290 25.7 20.1 1 1.3 16.5 88.5 HOXB4 homeobox B4 A_24_P416370 2.9 2 1.2 0.9 2.1 5.8 ICAM1 intercellular adhesion molecule 1 precursor A_23_P153320 4 1.8 0.7 1 2.5 23.3 IFIT2 interferon-induced protein with tetratricopeptide repeats 2 A_23_P24004 72.6 85.4 2.9 14.5 42.9 531 IL15RA interleukin 15 receptor, alpha isoform 2 A_23_P138680 3.5 2.7 1 1.2 2.8 4.1 IRF1 interferon regulatory factor 1 A_23_P41765 4.8 2.2 0.9 2 3.6 8.5 KLK10 kallikrein-related peptidase 10 preproprotein A_23_P107911 2.3 4.3 1 1.2 2.8 16.1 NLRC5 nucleotide-binding oligomerization domains 27 A_23_P402892 8.9 4.9 0.9 1.1 10.3 7.6 NLRC5 A_23_P26583 15.7 6.8 1 1 10.8 11.9 PMAIP1 phorbol-12-myristate-13-acetate-induced protein 1 A_23_P207999 2 1.4 0.7 1.1 1.8 5.3 SOCS1 suppressor of cytokine signaling 1 A_23_P420196 4.1 2.1 0.9 1.5 1.5 4.2 TNFAIP6 tumor necrosis factor, alpha-induced protein 6 precursor A_23_P165624 1.5 1.2 0.5 0.6 0.7 7 ZC3HAV1 zinc finger antiviral protein isoform 2 A_24_P341938 4.1 1.7 1 1.1 1.9 10.5 ZC3HAV1 zinc finger antiviral protein isoform 2 A_23_P20122 4.3 2.9 1.1 1.1 2.1 15.8 ZNFX1 zinc finger, NFX1-type containing 1 A_23_P68462 5.8 2.9 0.7 1.2 1.4 3.8 A_23_P256487 3.3 3.6 0.8 0.9 2.1 12.9 A_32_P224888 1.8 1.5 1 0.9 2.5 6.3 A_24_P36898 7.2 2.5 0.9 0.7 2.4 3.6 C AGT angiotensinogen preproprotein A_23_P115261 7.5 2.7 0.9 1 1 0.7 GLI3 GLI-Kruppel family member GLI3 A_23_P111531 1.3 4.2 1 0.9 1.2 1 GMPR guanosine monophosphate reductase A_24_P277657 1.4 6.6 0.9 1 1 0.8 IFITM2 interferon induced transmembrane protein 2 (1-8D) A_24_P287043 1.8 5.4 1 1 0.8 0.7 IFITM3 interferon-induced transmembrane protein 3 (1-8U) A_23_P87545 2.6 9.7 1 1 1 1.2 IFITM4P A_24_P16124 2.2 11.5 1 0.9 0.8 0.9 LGALS9 galectin 9 long isoform A_23_P116557 1.7 9.3 1 1 1.4 1 RET ret proto-oncogene isoform c A_24_P343695 0.9 4.6 1 1 1 1 SP100 hypothetical protein A_23_P209712 2.5 6.4 2 1.4 1.6 1.7 TRIM34 tripartite motif protein 34 isoform 3 A_23_P124190 3.4 7 2.2 1.3 1.6 2 similar to galectin 9 short isoform A_23_P101025 1.4 7 1.6 1.4 1.1 1.2 A_24_P254933 2 8.1 1 1 0.9 0.9 A_32_P167592 2.2 7.4 1 1 0.9 0.9 A_24_P15502 1.6 4.4 1 1 0.9 0.8 A_24_P7040 2.8 10.4 1 0.9 0.9 1.2 A_23_P202245 2.2 13.4 1 0.6 0.7 1.4 A_32_P154321 1.9 4.4 1.3 1 1.8 1.7 D ATP4A ATPase, H+/K+ exchanging, alpha polypeptide A_23_P430728 0.8 0.9 1 2.7 13.5 45.9 CCL3 chemokine (C-C motif) ligand 3 A_23_P373017 0.7 1.5 1.6 5.2 8.7 14.6 164 CCL3L3 chemokine (C-C motif) ligand 3-like 3 precursor A_23_P321920 1 1.3 0.8 2 5.8 10.2 CCL3L3 chemokine (C-C motif) ligand 3-like 3 precursor A_24_P228130 0.8 0.7 0.9 2.6 4.8 6.3 CCL5 small inducible cytokine A5 precursor A_23_P152838 1.6 12 1.8 17.5 23 29.9 CXCL10 small inducible cytokine B10 precursor A_24_P303091 0.8 2.2 0.9 3.9 9.3 12.4 GPR109B G protein-coupled receptor 109B A_23_P64721 1.6 2.3 1.5 4.1 10.2 42.3 IFNB1 interferon, beta 1, fibroblast A_23_P71774 0.7 3 0.9 9.3 31.7 180.6 IL28A interleukin 28A A_23_P409438 1.3 6.3 2.1 14.8 21 32.2 IL28B interleukin 28B A_23_P373619 1 3.7 1 18 19.9 21.5 IL29 interleukin 29 A_23_P337800 0.8 3 1.1 6.2 8.7 11.3 TAC3 tachykinin 3 isoform 1 A_23_P2283 1.3 1.2 1.3 2.8 4.5 9.3 E ACAN aggrecan isoform 2 precursor A_23_P307310 1 1.3 1.2 0.9 2.1 14.2 ATF3 activating transcription factor 3 isoform 2 A_23_P34915 1.6 1.1 1.1 1.3 1.7 5.7 BCL2A1 BCL2-related protein A1 A_23_P152002 1.2 1.4 0.9 1.1 1.3 5 BIRC3 baculoviral IAP repeat-containing protein 3 A_23_P98350 1.1 1.9 1 1 1.7 9.2 BTN3A1 butyrophilin, subfamily 3, member A1 A_24_P329065 1.7 3.7 1 1.4 2.7 6.4 C19orf30 hypothetical protein LOC284424 A_23_P353667 1.5 1.4 1 1.5 0.7 6.3 CCL17 small inducible cytokine A17 precursor A_23_P26325 1.1 1.3 1 1.3 2.5 9.3 CCL20 chemokine (C-C motif) ligand 20 A_23_P17065 0.7 0.7 0.8 0.9 0.9 27.4 CCL4 chemokine C-C motif ligand 4 isoform 1 precursor A_23_P207564 0.7 1.4 0.9 2.2 3.3 30.7 CCRN4L CCR4 carbon catabolite repression 4-like A_24_P213794 1.2 1.1 1.3 1.4 1.4 5.2 CENTD1 centaurin delta 1 isoform a A_32_P83784 1.4 1.9 1.3 1 1.3 4.5 CHEK2 protein kinase CHK2 isoform c A_23_P109452 1.1 0.9 1.1 1.1 1.2 4.1 CREB5 cAMP responsive element binding protein 5 isoform alpha A_23_P157117 0.8 1 0.8 1.2 1.4 14.5 CREB5 cAMP responsive element binding protein 5 isoform alpha A_24_P532232 0.7 1.6 0.9 1.4 1.7 12.3 CSAG1 chondrosarcoma associated gene 1 isoform b A_24_P11061 1.8 2.1 1 1.2 1.4 8 CSAG2 CSAG family, member 2 A_24_P567298 1.3 2.1 1.3 1.3 1.6 8.2 CSAG3A CSAG family, member 3A A_24_P79529 1.3 2.8 1.1 1.1 1.7 15.7 CX3CL1 chemokine (C-X3-C motif) ligand 1 A_23_P37727 1.7 2.4 1.4 1.6 2.2 19.7 CXCL1 chemokine (C-X-C motif) ligand 1 A_23_P7144 1 1.1 1.1 0.9 0.9 4.6 CXCL2 chemokine (C-X-C motif) ligand 2 A_23_P315364 1.8 1.5 1.4 1.4 1.8 5.2 CXCL3 chemokine (C-X-C motif) ligand 3 A_24_P251764 1.6 1.2 1.2 1.2 1.4 4.3 CXCL3 chemokine (C-X-C motif) ligand 3 A_24_P183150 1.7 1.3 1.1 1 1.3 6.1 CXCR4 chemokine (C-X-C motif) receptor 4 isoform a A_23_P102000 0.8 1 1.3 1.4 1.7 27.2 DNAJC15 DNAJ domain-containing A_23_P117190 2.2 1.8 1.2 2.3 2.2 6.3 EGR4 early growth response 4 A_23_P380318 0.9 1.1 1.2 1.2 2.5 23.8 EPHA4 ephrin receptor EphA4 A_23_P108501 0.8 0.9 1.4 1.1 1.1 4.1 EPHA4 ephrin receptor EphA4 A_24_P274219 1.1 1.1 0.9 1.2 1.8 7.5 FUT1 fucosyltransferase 1 A_23_P107963 0.6 1.2 1.1 1.6 1.4 5.1 FZD4 frizzled 4 A_23_P64617 0.9 1.3 1.1 0.9 1.4 14.3 GALNTL4 A_24_P924301 1.4 3 4.6 1.7 2 1.9 GP1BA A_23_P152926 1 1.9 2.5 2.1 1.5 5.1 GPBAR1 G protein-coupled bile acid receptor 1 A_23_P400378 0.8 1 1 1 1.3 4.6 HCP5 HLA complex P5 A_32_P85500 0.9 2.3 1.3 0.7 1.3 5.1 HCP5 HLA complex P5 A_24_P17870 1.4 3.4 1.2 1.1 1.3 8.8 HIP1R huntingtin interacting protein-1-related A_24_P229164 0.9 0.9 1.1 1.2 1.4 4.8 HIP1R huntingtin interacting protein-1-related A_23_P398294 1.1 1.2 1.8 1.4 1.8 11 HTR3E 5-hydroxytryptamine receptor 3 subunit E A_24_P32139 0.8 1.6 1 1.1 1.1 5.7 165 IGF2AS insulin-like growth factor 2 antisense A_23_P116435 1.2 0.9 1.3 1.3 1.2 5.3 IL3RA interleukin 3 receptor, alpha precursor A_32_P217750 1.2 1.9 1.4 1 1.3 6.2 IL6 interleukin 6 (interferon, beta 2) A_23_P71037 1.6 1.5 1 1.8 3.6 34 IL8 interleukin 8 precursor A_32_P87013 2.1 1.2 1.1 1.4 2.8 17.7 JAK2 Janus kinase 2 A_23_P123608 1.8 1.2 0.9 1 1.6 5.1 KCNV1 potassium channel, subfamily V, member 1 A_23_P146125 1.3 0.7 1.2 1.2 1.4 11.7 KLF4 Kruppel-like factor 4 (gut) A_23_P32233 1 0.8 0.8 1 2.3 9.2 KLHDC7B kelch domain containing 7B A_24_P117410 1.2 2.6 1.1 1.3 1.9 11.1 LRRN3 leucine rich repeat neuronal 3 A_23_P31376 1 1.7 0.9 1.5 7.3 81.3 LTB lymphotoxin-beta isoform a A_23_P93348 0.8 1.1 0.8 1 1.1 18.7 MBOAT4 FKSG89 A_24_P306824 1.1 1.3 1 0.9 1.8 7.8 MXD1 MAX dimerization protein 1 A_24_P379750 1.2 1.1 1 1.1 1.4 4.1 MXD1 MAX dimerization protein 1 A_23_P408094 1.1 1.2 1.2 1.2 1.3 4.6 NCF2 neutrophil cytosolic factor 2 A_23_P138194 1.1 1 1 1 1.5 5.9 NFKBIA nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha A_23_P106002 1.1 1.3 1 1.1 1.6 7.1 NOV nov precursor A_23_P82929 1 0.7 1.1 1.2 1.6 4.6 NPHS1 nephrin A_23_P5200 0.8 1 1 0.7 1.2 5.2 OTUD1 A_24_P374834 0.8 0.8 0.7 0.5 1 5.3 OTUD1 A_32_P60459 1.1 1 0.9 0.6 1.1 7.1 PLA2G4C phospholipase A2, group IVC A_23_P50508 1 1.1 0.9 1 1.3 8.3 PLCG2 phospholipase C, gamma 2 A_23_P106675 1.4 1.4 1.7 1.2 1.4 6.2 PPP1R15A protein phosphatase 1, regulatory subunit 15A A_23_P90172 0.9 1 1 1.1 1.4 5.7 PRR15 proline rich 15 A_32_P154911 2.4 1.6 1.8 1.5 2.3 6.8 PTGER4 prostaglandin E receptor 4, subtype EP4 A_23_P148047 1.1 0.9 0.7 1.1 2.3 14.4 PTX3 pentraxin-related gene, rapidly induced by IL-1 beta A_23_P121064 0.8 1.2 0.7 0.8 1.4 20.1 RELB reticuloendotheliosis viral oncogene homolog B A_23_P55706 0.9 1.5 1.1 1 0.9 4.4 RGS9 regulator of G-protein signalling 9 isoform 1 A_23_P66881 0.7 0.7 1.2 1 2 5.5 RHCG Rhesus blood group, C glycoprotein A_23_P151975 1 0.9 1.4 1.3 1.5 4.4 SLAMF7 SLAM family member 7 A_24_P353638 0.9 1.4 0.7 1.3 2.2 6.9 SLC12A7 solute carrier family 12 (potassium/chloride transporters), member 7 A_23_P61688 1.6 1.6 1.5 1.3 1.5 5.1 SMPD3 sphingomyelin phosphodiesterase 3, neutral membrane A_23_P163567 1.2 1.5 0.9 0.8 1.1 5.6 SNPH syntaphilin A_23_P102706 0.9 0.9 1.5 1.2 1.2 4.7 SOD2 SOD2 protein A_24_P935819 4 1.3 2 1.5 2.4 10.8 TMC8 FLJ00400 protein A_23_P346093 0.7 1.5 0.8 1.4 1.8 5.3 TNF tumor necrosis factor alpha A_23_P376488 0.7 1.2 1.1 1.1 1 5.9 TNFAIP2 tumor necrosis factor, alpha-induced protein 2 A_23_P421423 0.9 1.1 1.8 1.4 1.3 5.3 TNFAIP3 tumor necrosis factor, alpha-induced protein 3 A_24_P166527 1 0.7 0.9 1 1.5 6.6 TNFAIP3 tumor necrosis factor, alpha-induced protein 3 A_24_P157926 1.4 1.5 1.2 1.2 1.9 13.8 TNFRSF9 tumor necrosis factor receptor superfamily, member 9 precursor A_23_P51936 1.1 1.2 1 1.4 2.3 8.2 TTC9B A_23_P332392 1 1 1 1.2 1.4 7.2 ULBP2 UL16 binding protein 2 A_23_P168259 1.1 1 0.7 1 1.4 4.1 UNC5B unc-5 homolog B A_23_P52336 1 1 0.7 1.2 1.2 6.3 WARS tryptophanyl-tRNA synthetase isoform a A_23_P65651 1.4 2.1 1 1.6 2.4 6.7 ZBTB32 testis zinc finger protein A_23_P131024 0.8 1.6 1.3 1.2 8.2 61 ZC3HAV1 zinc finger antiviral protein isoform 1 A_23_P425224 3.7 0.7 1.2 1.1 1.6 5.7 ZEB2 zinc finger homeobox 1b A_23_P142560 1.5 1.5 1.3 0.9 1 5.9 A_32_P75867 3.3 5.8 1.8 2 7.2 20.1 166 A_24_P126282 1.5 2.9 1.3 1 2.6 5 A_23_P6535 0.9 3.9 1.1 1.4 2 12.6 A_32_P219135 1 1 1.6 1.8 1.8 8.6 A_24_P933828 0.8 0.7 1.2 1.4 1.4 6.2 A_32_P222684 1 1.2 1.2 1.5 1.5 6.3 A_32_P170664 1.1 1 0.8 1.2 1.3 4.3 A_23_P328740 1 1.7 0.9 1.1 1.5 8.6 A_32_P703 1 1.1 1.1 1 1.2 4.8 A_32_P147395 1.2 1.1 1 0.9 1.1 7.8 A_32_P158181 1.4 1.6 1.2 0.9 1.2 5.7 A_32_P68504 1.3 1.4 1.2 1.3 1.4 7 A_32_P217261 1.4 1.2 1 0.9 1.3 4.7 A_32_P45844 1.4 2.5 1.4 1.8 2.1 18.9 A_23_P11980 0.9 1.3 1.8 0.9 1.3 11.2 A_24_P652537 1.6 1.6 0.9 1.1 2.8 15.2 A_24_P101651 1.3 2 1.2 1.8 2.1 7.8 A_32_P104841 1.4 1.5 1.3 1.4 3.3 9.4 A_32_P52632 1 1.6 1.2 0.9 1.3 7.7 A_24_P255384 0.9 2.1 0.9 0.9 0.9 7.3 A_32_P168375 1.1 1.3 1.4 1.3 1.2 4.5 A_24_P174353 0.9 2.1 1.5 1.9 1.1 8.3 A_32_P91773 2.2 1.1 2.3 1.9 2 10.6 A_32_P210038 1.2 1.2 1.3 1.1 1.4 5.1 A_24_P937817 1.5 1.1 0.7 1.1 1.6 4 A_24_P47329 2.7 2 1.4 2.3 2.9 6.3 A_32_P3317 1 0.8 0.6 0.8 3.8 5.2 A_32_P42989 1.5 1.3 1.4 1.7 1.3 4 A_32_P117322 1.2 1.1 1.1 1.3 0.6 4.8 A_32_P118010 2.4 0.7 1.3 1.5 2.4 4.1 A_32_P55987 1.2 1.6 4.1 1.8 1.7 2.9 A_23_P320407 0.6 2.1 2.1 1.3 1.3 4.7 A_32_P184039 0.9 1.5 3.1 1.4 1.6 5.7 A_24_P930337 2.3 1.9 4.9 1.3 1.4 1.2 ?Genes listed in each cluster correspond to the same genes hierarchically clustered in Figure 1. *Rows in bold correspond to the 190 IFN-induced genes. 1 expressed sequence tag A_23_P416305 was down-regulated by IFN? treatment. ?Rows in blue correspond to 66 of the 190 genes that have not been previously described as IFN-responsive. 167 Table S2. Table S2. Genes differentially expressed by wt, CF170S, and P(C-) HPIV1 compared to mock infection and to each other*? Avo nex Avo nex F1 70 S F1 70 S F1 70 S F17 0S P(C-) P(C-) P(C-) P(C- ) WT WT WT WT Clus ter ? Symbol Product Agilent 6 hr 24 hr 6 hr 12 hr 24 hr 48 hr 6 hr 12 hr 24 hr 48 hr 6 hr 12 hr 24 hr 48 hr A ACTR3B actin-related protein 3-beta isoform 1 A_24_P303 594 0.9 0.7 0.5 0.7 0.8 5 0.5 0.8 1.6 3.2 0.6 0.8 0.9 1 ACVR1 activin A type I receptor precursor A_23_P792 21 1.1 1.1 0.6 0.7 2.2 11. 8 0.5 0.7 4.4 7.6 0.7 0.7 1.1 2.2 ADAM23 ADAM metallopeptidase domain 23 preproprotein A_23_P351 667 1 1 1.1 1.1 1.3 6.5 1 1.1 3 5.7 1.1 1.1 1.2 1.5 ADAR adenosine deaminase, RNA- specific isoform a A_23_P200 439 1.8 2.5 0.9 1.3 3.8 12. 3 0.9 1.3 5 7.6 0.9 1 1.6 2.2 AFF1 myeloid/lymphoid or mixed- lineage leukemia trithorax homolog 2 A_23_P298 97 1.2 1.3 1.2 1.1 3.3 21. 6 1.3 1.3 5.7 9.1 1.1 0.7 1.4 3.2 AFF1 myeloid/lymphoid or mixed- lineage leukemia trithorax homolog 2 A_23_P169 619 1.3 1.8 0.9 1.1 3.5 16. 5 1.1 1.1 5.9 8.2 1.1 0.8 1.4 3 AFF4 ALL1 fused gene from 5q31 A_23_P218 977 1.1 1.3 0.9 0.9 2.1 14. 6 0.8 1 3.2 11.5 0.8 0.9 1.3 1.9 AFF4 ALL1 fused gene from 5q31 A_24_P942 636 1.3 1.2 0.7 0.6 2.3 36. 4 0.7 0.8 4 29.7 0.7 0.8 1.4 3.3 AGL amylo-1, 6-glucosidase, 4-alpha- glucanotransferase isoform 1 A_23_P200 298 1.2 1 0.9 1 1.4 7.4 0.8 0.9 2.7 4.2 0.9 1 1.3 1.7 ALOX5AP arachidonate 5-lipoxygenase- activating protein A_23_P279 3 0.9 0.9 0.6 0.7 1.2 8.6 0.6 0.7 1.7 5.9 0.6 0.8 0.9 1.8 ANKS1A ankyrin repeat and sterile alpha motif domain containing 1 A_23_P156 748 1.1 1 1.2 1.1 2.4 17. 6 1 1.1 8.8 9.8 1.1 1.1 1.3 2.1 ARFGEF2 ADP-ribosylation factor guanine nucleotide-exchange factor 2 A_23_P599 5 0.8 0.9 0.9 0.9 1.1 7 0.8 0.9 1.8 5.1 0.9 0.9 1.1 1.3 ARHGAP 24 Rho GTPase activating protein 24 isoform 2 A_23_P110 151 1.1 0.9 0.8 0.7 1.6 8.7 0.7 0.9 2.5 7.8 0.9 0.7 1 1.4 ARHGAP 24 Rho GTPase activating protein 24 isoform 2 A_24_P380 061 1.2 1 0.8 0.9 1.7 8.9 1.2 1 2.8 6.8 1.3 0.7 1 1.5 ARHGAP 26 GTPase regulator associated with the focal adhesion kinase pp125 A_24_P281 65 1.1 1.5 1.7 1.3 2.3 11. 8 1.6 1.4 6.9 6.6 1.3 1.2 1.3 2.6 ARL14 ADP-ribosylation factor 7 A_23_P921 61 0.9 1.4 0.9 0.8 2.4 19. 8 0.7 0.9 7.6 17.9 0.7 0.6 1.1 1 ARL4A ADP-ribosylation factor-like 4A A_32_P806 841 1.1 1.1 0.9 0.8 2.6 10. 5 0.8 0.8 5.1 6.6 1 1 1.2 2.4 ARL4A ADP-ribosylation factor-like 4A A_23_P145 761 1.1 1.1 1 0.8 2.3 9.3 0.9 0.9 5.3 6.1 1 1 1.1 2.1 ARL5B ADP-ribosylation factor-like 8 A_24_P943 997 1.4 1.3 0.7 0.7 6.2 94. 9 0.7 0.8 12.4 79.3 0.7 0.9 1.3 4.4 ARL5B ADP-ribosylation factor-like 8 A_23_P378 588 1.3 1 0.7 0.8 6 50. 1 0.6 1 16.3 38.9 0.7 0.8 1.3 2.7 ARNTL2 aryl hydrocarbon receptor nuclear translocator-like 2 A_23_P533 45 1.1 1.3 0.7 0.8 1.7 14. 8 0.7 0.8 2.2 8.9 0.7 0.8 1.1 2.3 ATF3 activating transcription factor 3 isoform 2 A_23_P349 15 1.6 1.1 1.2 1.3 11. 6 64. 7 1.1 1.9 19.5 58.4 1.1 1.3 1.7 5.7 BANK1 B-cell scaffold protein with ankyrin repeats 1 isoform 1 A_23_P102 32 1.2 1.5 0.9 0.9 1.2 11. 9 0.8 0.7 2.1 5.2 0.9 1 1 1.5 BCL2A1 BCL2-related protein A1 A_23_P152 002 1.2 1.4 0.9 0.8 1.8 29. 3 1 0.9 3.9 13.9 0.9 1.1 1.3 5 BIK BCL2-interacting killer A_23_P404 667 0.9 1.1 1.1 1 1.2 12. 2 1.1 1 3.1 14.2 1.2 0.9 0.9 2.9 BNIP3 BCL2/adenovirus E1B 19kD- interacting protein 3 A_23_P138 635 1.1 1.3 0.9 0.8 1.1 7.4 0.8 0.8 1.6 6.4 0.8 0.8 1.3 1.7 BSN bassoon protein A_23_P297 35 1.4 1.2 1.1 1.3 1.6 7.2 1.1 1.3 2.8 7.5 0.8 0.9 1.1 1.4 BTG1 B-cell translocation protein 1 A_23_P875 60 1.2 1 0.9 0.9 1.8 5.4 0.8 1 2 4.9 0.9 0.8 1 1.3 BTN2A2 butyrophilin, subfamily 2, member A2 isoform b A_24_P346 210 1 1.4 1.4 1.4 2.4 8 1.4 1.5 5.2 5.9 1.4 1.2 1.2 1.9 BTN2A2 butyrophilin, subfamily 2, member A2 isoform b A_24_P337 592 0.9 1.4 1.4 1.3 3.3 27. 3 1.4 1.4 9 19 1.4 1.1 1.3 2.6 BTN2A2 butyrophilin, subfamily 2, member A2 isoform b A_24_P249 072 1.1 1.4 1.2 1.1 2.8 14. 3 1.1 1.2 5.7 10.5 1.1 1 1.2 2 BTN2A2 butyrophilin, subfamily 2, member A2 isoform b A_23_P122 439 0.8 1.4 1.2 1.1 3.1 20. 8 1 1.1 7.4 15.2 1.2 1 1.1 2.3 C10orf118 CTCL tumor antigen L14-2 A_24_P942 694 0.9 0.8 0.7 0.8 1.4 7.4 0.7 0.8 1.9 6.9 0.8 0.6 1.2 1.6 C18orf19 hypothetical protein LOC125228 A_23_P380 978 1.1 1.1 0.6 0.7 1.4 11. 9 0.6 0.8 4.7 16.9 0.6 0.8 1.2 1.6 C19orf12 hypothetical protein LOC83636 isoform 2 A_23_P209 195 1.6 1.2 0.9 0.9 1.6 7 0.8 0.9 2.4 5.2 0.9 1 1.2 1.6 C19orf12 hypothetical protein LOC83636 isoform 2 A_24_P113 341 1.3 1 1 0.9 1.4 8.4 0.9 0.9 2.7 5.6 0.9 0.9 1 1.5 C2orf13 chromosome 2 open reading frame 13 A_32_P163 39 1.1 1.1 0.7 0.9 1.2 8 0.8 0.9 3 7.8 0.9 0.7 0.7 2 168 C3orf19 hypothetical protein LOC51244 A_23_P354 277 0.9 1 1 1 1.6 6.3 1 0.9 2.2 5.1 1 1 1 1.4 C3orf33 hypothetical protein LOC285315 A_23_P301 476 1.2 1 0.9 1 1.5 7.6 0.8 0.9 2.9 4.7 1 1.1 1.2 1.4 C3orf52 TPA-induced transmembrane protein A_23_P580 09 1.1 1.1 0.7 0.9 1.6 6.1 0.8 0.9 1.5 3.7 0.7 1.1 1.3 1.5 C3orf59 hypothetical protein LOC151963 A_32_P117 464 1.5 1.4 0.7 0.7 3.9 17. 3 0.6 0.9 5.8 13.4 0.8 1 1 2.4 C8orf46 hypothetical protein LOC254778 A_23_P353 614 0.7 1.4 1 0.8 3.2 16. 1 0.8 1.1 4.7 12.2 0.7 1 0.7 1.8 CA8 carbonic anhydrase VIII A_23_P838 38 0.9 1 0.7 1 1.7 10 0.9 0.9 3.3 8.7 0.8 1 0.9 2.1 CCDC117 coiled-coil domain containing 117 A_23_P432 034 1 1 0.9 0.8 1.2 5.6 0.9 0.8 2.2 4.1 0.9 0.8 0.9 1.3 CCDC6 A_23_P127 117 1.3 1.3 0.9 0.9 1.4 13 1 1 3.4 12.7 0.8 0.9 0.9 2.1 CCRN4L CCR4 carbon catabolite repression 4-like A_24_P213 794 1.2 1.1 1.4 1.2 9 68. 2 1.4 1.6 20.5 49.6 1.3 1.4 1.4 5.2 CDKN2C cyclin-dependent kinase inhibitor 2C A_23_P854 60 0.9 0.7 1.1 0.8 1.9 12. 4 1 0.9 5.2 8.5 1.1 0.8 0.9 2 CEBPG CCAAT/enhancer binding protein gamma A_23_P356 755 1 1.1 1 1 1.8 9.7 0.9 0.9 2.4 10.8 1 1 1.1 2 CENTD1 centaurin delta 1 isoform a A_32_P837 84 1.4 1.9 1.3 0.9 5.1 45. 9 1 1 14.6 28.2 1.3 1 1.3 4.5 CHEK2 protein kinase CHK2 isoform c A_23_P109 452 1.1 0.9 1.5 1.2 4.4 35. 2 1.1 1.2 14.5 15.8 1.1 1.1 1.2 4.1 CITED2 Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2 A_23_P214 969 0.9 1 0.9 0.8 2.6 14. 8 0.7 0.9 5.7 9.2 0.9 0.7 1 2.8 CLMN calponin like transmembrane domain protein A_23_P257 06 1.2 0.9 0.8 0.8 2 9.6 0.7 0.8 3.2 7.5 0.8 1.1 1.1 1.7 CPEB3 cytoplasmic polyadenylation element binding protein 3 A_23_P468 13 1.3 1.1 1.2 1 7.2 21. 1 1 1.3 17.2 22.7 0.9 1.1 1.4 3 CPEB3 cytoplasmic polyadenylation element binding protein 3 A_23_P468 12 3.2 2.6 1.9 1 5.3 22. 1 1.9 1.9 10.1 16.3 2.4 1.5 1.4 3.3 CREB5 cAMP responsive element binding protein 5 isoform alpha A_24_P532 232 0.7 1.6 0.8 1.3 5 153 .1 0.9 0.7 21.8 176. 7 0.9 1.4 1.7 12.3 CREB5 cAMP responsive element binding protein 5 isoform alpha A_23_P157 117 0.8 1 0.9 1 9 347 .7 0.9 1.1 42.4 280. 9 0.8 1.2 1.4 14.5 CRYM crystallin, mu isoform 1 A_23_P777 31 1.1 0.8 0.9 1 2 7.3 1 1.1 6 6.4 1 1 1.3 1.1 CSRP2 cysteine and glycine-rich protein 2 A_23_P447 24 1.1 1 0.7 0.8 0.9 5 0.9 0.9 1.4 4.1 0.8 0.9 1 0.9 CYLD ubiquitin carboxyl-terminal hydrolase CYLD isoform 1 A_24_P480 78 1.2 1.2 0.8 0.9 2.6 23. 4 0.9 0.9 5.5 18.2 1 0.9 1.1 2.8 CYLD ubiquitin carboxyl-terminal hydrolase CYLD isoform 1 A_23_P659 96 1.1 1 0.5 0.8 2.1 15. 8 0.5 0.6 4 11.2 0.6 0.7 1.2 2.6 DAB2IP DAB2 interacting protein isoform 2 A_24_P149 704 1 1.1 1 0.8 2.1 8.2 1.1 1 3.5 6.3 1.3 0.8 0.8 1.6 DDEF1 development and differentiation enhancing factor 1 A_23_P448 31 1.2 1.2 1.1 1 1.9 11. 9 1.1 1.1 4.2 8.2 1 1 1.3 1.9 DDEF1 development and differentiation enhancing factor 1 A_23_P216 278 1.1 1.2 1 1 1.7 20. 5 1.1 1 5.2 14.8 1 0.9 1 2.1 DDEF2 development- and differentiation- enhancing factor 2 A_23_P400 88 1.1 1 0.6 0.7 0.9 14 0.7 0.8 2.7 9.4 0.6 0.8 0.8 2 DDIT3 DNA-damage-inducible transcript 3 A_23_P211 34 0.9 1.2 0.8 1.3 3.3 8.6 0.9 2.1 6.3 8.7 0.8 1.3 1.3 1.8 DENND4 A c-myc promoter binding protein A_24_P409 265 2 1 0.9 1 1.9 15. 1 0.9 0.9 3.3 9.9 1.1 0.8 1.2 1.8 DENND4 A A_23_P384 329 1.2 1.2 0.7 0.7 1.9 11. 2 0.7 1 3.8 5.8 0.8 0.6 1.1 1.7 DENND4 C A_24_P936 33 1.2 1.4 1.1 0.7 1.5 11. 3 0.8 0.8 2.3 9.3 0.9 0.9 1.4 1.8 DICER1 dicer1 A_24_P221 63 1.1 1.1 1.5 1.1 2.2 25. 7 1.4 1.2 9.5 13.4 1.4 1 1.1 2.7 DNAJB4 DnaJ (Hsp40) homolog, subfamily B, member 4 A_24_P393 958 1 1 0.4 0.6 2.4 9 0.4 0.6 3.9 4.6 0.5 0.6 0.9 1.3 DNAJB4 DnaJ (Hsp40) homolog, subfamily B, member 4 A_23_P513 39 1 0.9 0.5 0.6 2.2 8.2 0.4 0.6 3.9 4.6 0.5 0.7 1 1.2 DNAJB5 DnaJ (Hsp40) homolog, subfamily B, member 5 A_23_P112 241 0.7 0.8 1.4 1.2 4.1 23. 3 1.4 1.6 10.5 18.5 1.3 1.2 1.3 2.9 DOCK11 dedicator of cytokinesis 11 A_23_P148 584 1.1 0.9 0.6 0.7 1.1 8.2 0.5 0.7 2.1 3.4 0.7 0.7 1 1.6 DSCR1 calcipressin 1 isoform a A_23_P166 248 1.4 1 0.7 0.8 2.2 9.1 0.6 0.8 3.2 4.9 0.8 0.8 1 1.6 DUSP10 dual specificity phosphatase 10 isoform a A_23_P518 56 1.3 1.3 1 1 1.4 9.9 1 0.9 3.5 4.6 1.1 0.8 0.9 1.5 DUSP10 dual specificity phosphatase 10 isoform a A_24_P182 494 1.1 1.1 1.1 1 1.3 5.5 1 1.1 2.7 3.2 0.9 0.9 1 1 DUSP16 dual specificity phosphatase 16 A_24_P189 739 1 1.1 0.8 0.9 1.9 5.6 0.6 0.9 2.2 3.2 0.8 1.2 1.1 1.2 DUSP8 dual specificity phosphatase 8 A_32_P982 98 1 1.1 1 1.1 2.7 12. 5 1.1 1 4 4.8 1 1 1 2.3 EBF4 KIAA1442 protein A_23_P109 122 1.1 1.1 1.4 1.3 3.7 20. 3 1.4 1.4 10.7 11.7 1.3 1.3 1.2 2.7 ECM2 extracellular matrix protein 2 precursor A_23_P303 671 1 1.1 0.7 0.7 2.6 28 0.7 1.2 6 48.3 0.7 1.6 1.7 4 169 EFNB2 ephrin B2 A_24_P355 944 1.2 0.8 0.8 0.9 3 29. 5 0.8 0.9 6.8 19.3 1 1.3 1.2 2.4 EIF5 eukaryotic translation initiation factor 5 A_23_P205 265 1.1 1.1 0.6 0.8 1.2 7.2 0.8 0.9 1.2 7.7 0.7 0.8 1.1 1.6 ELOVL7 ELOVL family member 7, elongation of long chain fatty acids A_24_P324 73 1 1.7 0.6 0.7 3.2 41. 4 0.5 0.7 8.3 18.9 0.5 0.9 1.4 4.5 EPHA4 ephrin receptor EphA4 A_24_P274 219 1.1 1.1 0.8 0.9 3.9 98. 4 1 0.8 12.1 52.1 0.9 1.2 1.8 7.5 EPHA4 ephrin receptor EphA4 A_23_P119 899 1.1 1 0.7 1.5 3.3 48. 4 1.3 1.2 10.5 50.1 0.6 2.2 1.2 3.8 EPRS glutamyl-prolyl tRNA synthetase A_23_P976 32 0.9 0.7 0.6 0.8 1.1 10. 1 0.6 0.7 1.4 5.6 0.7 1 1.1 1.3 ETS1 v-ets erythroblastosis virus E26 oncogene homolog 1 A_23_P127 525 1.1 1 1.2 1.2 2.2 15. 4 1.5 1.3 3.4 14.9 1.1 1 1 2.1 EXT1 exostosin 1 A_23_P432 73 1.2 1.8 0.9 0.9 2 12. 6 0.9 1 2.1 4.6 1 0.9 1.1 1.7 FAM126A down-regulated by Ctnnb1, a A_23_P858 2 1.1 0.8 0.7 0.8 1.4 9.3 0.7 0.8 2.1 9.7 0.7 0.9 1.4 1.9 FAM62B family with sequence similarity 62 (C2 domain containing) member B A_23_P874 4 1.1 1.2 1 1 1.5 13. 4 0.9 1.1 2.5 8.8 1 1 1.2 2.4 FAS tumor necrosis factor receptor superfamily, member 6 isoform 1 precursor A_23_P638 96 1.7 1.5 0.6 0.8 2.9 22. 7 0.6 0.7 4.4 11.8 0.6 0.9 1.3 4.4 FGF2 fibroblast growth factor 2 A_23_P218 918 1.1 1.1 1.1 1.1 2.4 13. 4 1.2 1.2 5.3 12.3 1.1 1 1.2 3.5 FNDC3A fibronectin type III domain containing 3A isoform 2 A_23_P255 03 1.1 1.2 1 0.8 2.6 24. 8 0.9 0.8 8 14.2 0.9 0.9 1.4 3.3 FNDC3A A_24_P409 126 1.1 1.8 1.2 1.1 2.5 47 0.9 1 7.4 60.8 1.2 1.3 1.3 3.9 FOLR2 folate receptor 2 precursor A_23_P477 09 1.1 0.9 1 0.8 1.3 6.1 0.7 1 2.5 3 0.8 0.9 0.9 1.3 FRMD6 FERM domain containing 6 A_24_P330 303 0.9 1 0.6 0.7 1.9 5.4 0.6 0.8 2.1 6.2 0.6 0.8 1.1 1.2 FYN protein-tyrosine kinase fyn isoform a A_23_P502 142 1 0.9 1.1 1.1 3.2 17. 4 1.1 1.1 9.4 11 1 1.1 1 2.8 FZD4 frizzled 4 A_24_P278 621 1.4 1.4 1.3 1.3 3.3 50. 4 1.1 0.9 10.6 31.1 1.7 0.9 0.7 6.4 GABARA PL1 GABA(A) receptor-associated protein like 1 A_24_P481 6 0.8 0.8 0.6 0.8 1.5 6.1 0.6 0.7 2 3.9 0.7 0.6 0.8 1.3 GABARA PL1 GABA(A) receptor-associated protein like 1 A_23_P162 640 0.9 1 0.8 0.9 1.8 9 0.8 0.9 2.6 5.7 0.8 0.8 0.9 1.7 GADD45A growth arrest and DNA-damage- inducible, alpha A_23_P232 21 0.9 1.1 0.9 0.9 2.4 8.6 0.9 1 2.9 9.9 0.9 1.2 1.1 1.7 GATA6 GATA binding protein 6 A_23_P304 450 1.8 1.6 1.6 1.2 5.7 17. 3 1.1 1.3 6.6 14.8 1.7 1.3 1.4 2.6 GCA grancalcin, EF-hand calcium binding protein A_23_P284 85 1.4 1.2 1.1 1 2.1 18. 4 0.9 1 4.9 8.1 1.2 1 1 1.7 GLCCI1 A_23_P336 198 1.1 0.9 0.7 0.9 2.3 14. 4 0.8 0.9 7.2 7.6 0.8 0.8 1.2 2.6 GMFG glia maturation factor, gamma A_23_P208 866 1.1 1 0.8 0.8 1 6.4 0.9 0.8 2.1 5.8 0.8 0.9 0.8 1.3 GPBP1 vasculin A_23_P364 465 1 1 0.9 0.9 1.8 7.1 0.8 0.9 3.4 4.6 0.9 0.8 0.9 1.6 GRAMD1 B PTSS3032 A_24_P586 20 1.1 1 0.9 1.1 2.3 12. 8 1 1 4 7.5 0.8 1.1 1.4 2.3 GRB10 growth factor receptor-bound protein 10 isoform c A_23_P122 863 2.5 0.9 1.2 0.9 2.5 19. 1 1.1 1 7.6 15.3 1 0.9 1.4 3.1 HIAT1 hippocampus abundant transcript 1 A_23_P458 51 1.1 1.1 0.7 0.9 3 20. 1 0.7 1 6.2 13.3 0.7 1 1.6 3.4 HOXB3 homeobox B3 A_23_P316 511 1 1.2 1.4 1.2 2.1 7.6 1.1 1.3 3.5 4.7 1.1 1.1 1.2 1.7 HOXB9 homeobox B9 A_23_P270 13 0.9 1 1.1 1.2 2.4 10. 9 1.2 1.2 4.8 8.1 1 1.1 1.1 1.9 HSCB J-type co-chaperone HSC20 A_23_P405 88 1.2 1.2 0.7 0.9 2 11. 4 0.8 0.9 4.3 9.8 0.7 0.9 0.9 1.6 HSD17B7 hydroxysteroid (17-beta) dehydrogenase 7 A_32_P522 82 0.9 0.8 0.6 0.8 1.4 4.9 0.8 1.1 2 4.7 0.7 0.9 1.1 0.9 HSPB8 heat shock 27kDa protein 8 A_23_P162 579 1.1 0.8 0.6 0.8 1.5 10. 5 0.4 0.8 3.5 7.5 0.7 0.9 1.4 1.3 IGF2 insulin-like growth factor 2 A_23_P150 609 1.1 0.8 1.5 1.3 4.5 37. 5 1.2 1.4 21.1 25.9 1.3 1.1 0.8 3.9 IGF2AS insulin-like growth factor 2 antisense A_23_P116 435 1.2 0.9 1.1 1.2 6.4 42. 9 1.3 1.3 20.4 25.9 1.3 1.3 1.2 5.3 ING3 inhibitor of growth family, member 3 isoform 1 A_23_P111 811 1.3 1.3 1.2 1.2 1.7 9 1.2 1.2 3.2 7.9 1.2 1.1 1.1 1.7 INHBA inhibin beta A precursor A_23_P122 924 1 1.7 1.3 1.3 2.6 18. 2 1.1 1.5 5.6 9.1 1.2 1.2 1.2 3 INTS6 integrator complex subunit 6 isoform a A_24_P167 614 1.2 1.3 0.9 0.9 1.8 5.5 1 0.9 2.2 4.2 0.9 1.1 0.9 1.4 ITGA5 integrin alpha 5 precursor A_23_P365 62 0.9 0.8 0.7 0.8 1.9 5.8 0.8 0.8 2.6 4.4 0.7 1 1.3 1.3 JAZF1 JAZF zinc finger 1 A_32_P366 94 1.3 1.2 0.9 0.9 3 33. 3 0.9 0.9 8.3 29 1 1.1 1.5 3.4 KCNG3 potassium voltage-gated channel, subfamily G, member 3 isoform 1 A_32_P800 799 1.1 1.1 1 0.9 2 52. 8 0.9 0.9 6.2 30 0.9 1 1.1 4.5 KCNV1 potassium channel, subfamily V, member 1 A_23_P146 125 1.3 0.7 1 1.1 9.7 101 .6 1 1.3 25.3 120. 8 1.2 1.2 1.4 11.7 KLF6 Kruppel-like factor 6 A_24_P696 54 1.4 1 0.9 1 2.9 7.2 1 1.6 3.8 3.9 0.8 0.8 0.8 1.3 170 KLF6 Kruppel-like factor 6 A_23_P637 98 1.5 1.1 1 1.1 3.5 9.7 1.1 1.7 5.3 5 0.9 0.9 0.8 1.4 KLF7 Kruppel-like factor 7 (ubiquitous) A_23_P679 80 1 1 0.9 0.9 1.9 7.4 0.9 1.1 3.8 7 1 0.9 1 1.4 KTN1 kinectin 1 isoform a A_23_P106 131 1 0.9 0.6 0.8 1.1 5.2 0.7 0.8 2.1 3.7 0.7 0.7 1 1.3 LRIG3 leucine-rich repeats and immunoglobulin-like domains 3 A_23_P478 85 1.1 1 0.9 0.9 1.9 10. 6 0.7 0.9 2.2 3.2 0.9 0.8 1 1.3 LST1 leukocyte specific transcript 1 isoform 1 A_24_P949 16 1.2 1.3 1 1.2 2.3 17. 3 1.2 1.2 9.5 14.4 1.1 1.2 1.5 2.8 MAFB transcription factor MAFB A_23_P173 45 1.1 1.1 0.9 1.1 5 17. 8 1.2 1.2 7.3 9.2 1 1.5 1.1 3.2 MAFF transcription factor MAFF A_23_P103 110 1 1.1 0.9 0.9 2.3 8.2 0.9 0.9 3.7 6.5 0.9 0.7 0.9 1.6 MAL2 MAL2 proteolipid protein A_23_P601 30 1.1 1.3 1 1.7 2.3 80. 1 0.8 0.7 7.3 79.1 1.2 1.4 1.6 6.2 MAOA monoamine oxidase A A_23_P838 57 0.9 0.7 0.8 0.8 1.1 6.5 0.8 0.9 1.9 5.8 0.8 0.8 0.9 1 MAP4K4 mitogen-activated protein kinase kinase kinase kinase 4 isoform 2 A_23_P108 604 1 0.9 0.9 0.9 1.8 13. 2 0.9 0.9 4 8.8 0.9 0.9 1.1 2.2 MAP4K4 mitogen-activated protein kinase kinase kinase kinase 4 isoform 2 A_23_P102 192 0.8 0.7 0.7 0.7 1.6 9.2 0.7 0.8 3.6 5.9 0.6 0.7 0.9 1.7 MCL1 myeloid cell leukemia sequence 1 isoform 1 A_24_P319 635 1.4 1.4 0.6 0.9 3.8 9.2 0.7 1 5.1 8.4 0.7 0.9 1.1 1.6 MLL3 hypothetical protein A_23_P168 419 0.9 0.9 1 0.9 1.5 6.6 0.9 0.9 3.4 4 1 0.9 1 1.4 NAV2 neuron navigator 2 isoform 1 A_23_P527 27 0.8 0.9 1 0.8 1.6 12 0.9 0.9 4.7 7.4 0.9 0.7 1 2.2 NAV2 neuron navigator 2 isoform 1 A_23_P354 791 0.7 0.7 0.7 0.4 1.3 8.8 0.8 0.4 3.8 8.5 0.6 0.5 0.9 1.5 NAV3 neuron navigator 3 A_24_P318 160 0.9 1.1 0.7 0.9 2.3 40. 1 0.8 0.8 7.8 31.2 0.6 0.9 1.9 3.2 NAV3 neuron navigator 3 A_23_P137 40 0.9 1 0.9 1 1.7 12. 9 0.9 0.8 3.8 8.4 0.8 0.9 1.7 2.2 NCALD neurocalcin delta A_23_P215 883 0.8 0.6 0.9 1 3.8 16. 8 0.9 0.7 5.8 14.7 1.3 1.2 1.1 2.3 NCF2 neutrophil cytosolic factor 2 A_23_P138 194 1.1 1 1 1.1 3.1 31. 8 1 1 5.3 25.4 1 1 1.5 5.9 NEFH neurofilament, heavy polypeptide 200kDa A_23_P300 600 0.9 0.8 0.9 0.9 2.5 8.5 1.6 1.1 1.9 9.6 0.8 0.9 1 1.9 NFIL3 nuclear factor, interleukin 3 regulated A_23_P322 53 1.5 1.2 1 1 3.1 6.2 0.9 1 3.1 6.3 1 1.1 1.2 1.5 NFKB1 nuclear factor kappa-B, subunit 1 A_23_P300 24 0.9 1.1 1 1 2.3 10. 6 0.9 1 4.4 7.2 1 1 1 2.5 NFKB2 nuclear factor of kappa light polypeptide gene enhancer in B- cells 2 isoform b A_23_P202 156 0.8 1.4 1.2 1.1 1.9 9.1 1.2 1.1 3.7 7.3 1.1 1.3 0.9 2.9 NLRP3 NLR family, pyrin domain containing 3 isoform a A_23_P988 3 1.5 1 0.7 1.1 6.8 74. 6 1.4 1 18.6 33.2 1.1 1 0.9 7.4 NPPB natriuretic peptide precursor B preproprotein A_23_P627 52 1 1.1 1.3 1.3 2.8 17. 6 1.2 1.3 5.3 14.5 1.1 1.3 1.2 1.4 NTN4 netrin 4 A_23_P204 630 1 1 0.8 0.8 1.4 14 0.6 0.7 3.4 4.6 0.8 0.7 0.7 2.1 OR1F1 olfactory receptor, family 1, subfamily F, member 1 A_23_P318 890 1.2 1.3 1.6 1.4 2.7 13. 3 1.4 1.3 7.1 6.7 1.1 1.1 1.1 3 OTUD1 A_32_P604 59 1.1 1 0.8 0.9 7.2 150 .9 0.7 1.1 29.3 102. 4 0.9 0.6 1.1 7.1 OTUD1 A_24_P374 834 0.8 0.8 0.6 0.6 6.8 85 0.5 0.7 24.1 55.8 0.7 0.5 1 5.3 PAG1 phosphoprotein associated with glycosphingolipid microdomains 1 A_23_P347 070 0.8 0.7 0.7 0.9 2.3 20. 9 0.6 0.7 6.7 16 0.7 1.3 1.8 3 PAM peptidylglycine alpha-amidating monooxygenase isoform a, preproprotein A_23_P213 678 1.1 1.1 1.2 1.1 1.3 5.9 1.1 1 1.9 3.2 1.1 1.1 1.2 1.4 PAPD4 PAP associated domain containing 4 A_23_P314 202 1 1.2 1 0.9 1.8 5.6 0.8 1 3.1 4.5 0.9 1.1 1.2 1.4 PELI1 pellino protein A_23_P120 345 1.4 1.1 1.1 1.3 2.5 13. 8 1.2 1 5.3 8.6 1.1 1.1 1.2 3.5 PHLDB2 pleckstrin homology-like domain, family B, member 2 A_24_P240 166 1.5 1.4 0.9 0.9 2.7 9.6 0.7 0.9 5 5.1 0.9 0.8 1.3 1.7 PLAG1 pleiomorphic adenoma gene 1 A_23_P411 723 0.9 1 1 1.1 1.2 5.4 0.9 1 2.2 6 1 0.8 0.9 1.3 PLCB4 phospholipase C beta 4 isoform a A_23_P288 98 0.9 1.2 1.3 1.7 3.6 22. 5 1.5 1.7 16 23.9 1.1 1.2 1.2 1.9 PLCG2 phospholipase C, gamma 2 A_23_P106 675 1.4 1.4 1.6 1.4 3.5 50. 2 1.4 1.4 11.7 32.6 1.7 1.2 1.4 6.2 PLXNA1 plexin A1 A_23_P690 68 0.9 0.7 0.6 0.7 1.4 6.5 0.9 0.9 3.2 4.2 0.8 1.3 1.3 1.6 PPID peptidylprolyl isomerase D A_23_P386 411 1 0.9 0.8 0.8 1.1 4.4 0.8 0.9 1.5 4 0.7 0.9 1.1 1.1 PPM1K hypothetical protein A_24_P162 532 1.9 0.7 1 1.1 4.7 8.9 1 1 3.5 7.3 1.1 1 0.9 2.7 PRKD1 protein kinase D1 A_23_P106 016 1.2 1 0.9 0.9 1.3 5.3 0.9 0.9 2 3.5 1.1 0.9 1 1.2 PRRG4 proline rich Gla (G- carboxyglutamic acid) 4 (transmembrane) A_23_P127 663 1.8 1.6 1.3 1.4 3.3 16. 5 1.5 1.2 4.9 6.8 1.4 1.1 1.7 3.3 PTEN phosphatase and tensin homolog A_23_P980 85 1 1 1.1 1 1.2 7.5 1.1 1 2.3 6 1 0.9 1 1.5 RAB8B RAB8B, member RAS oncogene family A_23_P375 35 1.4 1.4 1 1 1.9 10. 1 0.9 1 2.6 5.6 1.2 1 1.1 2.2 171 RAB9A RAB9A, member RAS oncogene family A_23_P453 89 1 1 0.8 0.8 1.3 5.6 0.7 0.8 1.8 4.7 0.9 0.8 0.9 1.3 RAPH1 Ras association and pleckstrin homology domains 1 isoform 1 A_24_P929 570 0.9 0.9 0.5 0.7 2.9 22. 2 0.6 0.7 6.6 19.3 0.7 0.9 1.3 2 RASGRP 1 RAS guanyl releasing protein 1 A_23_P124 642 1.1 1.2 1.2 0.9 1.3 21. 5 0.7 0.8 2.7 11.7 1.2 1.1 1.1 2.4 RASSF5 Ras association (RalGDS/AF-6) domain family 5 isoform C A_24_P171 268 1.1 1 0.7 0.8 1.6 19. 8 0.6 0.6 1.6 7.8 1.4 1.3 1.3 2.1 RCOR1 A_23_P205 247 1.2 0.8 0.8 0.8 2.5 12 0.7 0.8 4.1 9.4 0.6 0.5 0.7 1.5 RELB reticuloendotheliosis viral oncogene homolog B A_23_P557 06 0.9 1.5 1.2 1.1 2.4 25. 7 1.1 1 6.2 22.2 1.1 1 0.9 4.4 REV3L REV3-like, catalytic subunit of DNA polymerase zeta A_23_P214 139 1 1.1 1 1.1 1.9 10. 7 1.2 1.1 4.6 5.4 1 1 1.1 1.9 RGAG1 retrotransposon gag domain containing 1 A_23_P306 352 1 0.8 0.6 0.8 5 200 .9 0.9 0.9 57.8 105. 4 1 0.6 1.1 15.4 RGMB RGM domain family, member B isoform 2 precursor A_24_P363 100 1.1 1.2 1.2 1.2 1.9 7.3 1.1 1.3 3.2 5.8 1.1 1.3 1.2 1.7 RGS20 regulator of G-protein signalling 20 isoform a A_23_P730 97 1 1.2 0.8 0.9 1.4 9.4 0.8 1 2.7 7.6 0.8 1 1.2 1.2 RIPK1 receptor (TNFRSF)-interacting serine-threonine kinase 1 A_23_P370 005 1.4 1.2 1.1 1 3 8.1 1.1 1.1 5.3 5.6 1 1 1 1.7 RNF144 ring finger protein 144 A_23_P131 566 1.3 1.3 1.2 0.9 2 12. 3 1 1.2 2.2 9.7 1 1 1.1 2.1 RNMT RNA (guanine-7-) methyltransferase A_23_P555 15 0.9 1 0.6 0.7 1.2 6.6 0.6 0.7 2.1 7.7 0.7 0.8 0.9 1.1 RP2 XRP2 protein A_23_P224 33 1.2 1.2 1.1 1 2.3 7.3 1.1 1 4 4.3 1 0.8 1 1.7 RPS6KA6 ribosomal protein S6 kinase, 90kDa, polypeptide 6 A_23_P125 596 0.8 0.8 0.7 0.9 1.3 5.9 0.7 0.8 2.4 5.7 0.7 0.8 1 1.4 RPS6KC1 ribosomal protein S6 kinase, 52kDa, polypeptide 1 A_23_P104 109 1.3 1.4 1.1 1 2.4 28. 6 1.1 1 7 18.4 1.2 1 1.1 2.6 RSRC2 arginine/serine-rich coiled-coil 2 isoform b A_24_P350 160 1.1 1.2 0.9 1 1.8 8.2 1 1.1 3.7 8.2 1.2 1.1 1.1 1.4 SALL1 sal-like 1 A_23_P328 074 0.8 0.8 1 1.1 1.5 35. 3 1 1 4.5 15.4 1.6 0.8 0.9 3.2 SAMD4A KIAA1053 protein A_23_P335 661 1.1 1.2 1.1 1.1 2.7 14. 2 1.1 1.2 7.4 9.1 1 1 1.2 2.2 SAMD4A sterile alpha motif domain containing 4 A_24_P383 523 1.1 1.2 0.8 1.1 4.5 31. 1 1.1 1.2 11 23.3 0.9 1.1 1.2 3.2 SAMD8 sterile alpha motif domain containing 8 A_23_P638 70 1.2 1.2 0.7 0.9 1.6 7.4 0.7 0.9 1.6 6.9 0.9 0.9 1.3 1.4 SAR1A SAR1a gene homolog 1 A_24_P158 421 1.1 1.1 0.9 0.9 2.1 15. 2 0.9 0.9 3.8 9 0.9 1 1.2 3.2 SAR1A SAR1a gene homolog 1 A_23_P127 175 1 1.2 1.1 1 1.7 29. 3 1.1 1.1 4.5 28.2 1 1 1.1 2.9 SATB2 SATB family member 2 A_32_P174 365 1.2 1.2 1 1 1.2 6.1 1 0.9 2.4 3.1 1 0.9 0.9 1.3 SCN3A sodium channel, voltage-gated, type III, alpha isoform 1 A_23_P678 96 1.8 1.5 1.1 1.3 2.9 130 .8 1.6 1.3 11.4 45.4 1.4 1.1 1.3 1.7 SCYL1BP 1 NTKL-binding protein 1 A_23_P368 145 1.1 1.3 1 1 1.6 7.3 1 1.1 3.3 8.1 1 0.9 1.1 1.7 SDK1 FLJ00148 protein A_23_P937 22 1 1 1.1 1 2.2 14. 3 1.3 1.3 4.9 11.1 1 1.3 1.3 1.6 SEPSECS Sep (O-phosphoserine) tRNA:Sec (selenocysteine) tRNA synthase isoform 1 A_23_P167 201 1.1 1.1 0.8 0.8 1.9 7.5 0.7 0.8 2.2 7.9 0.8 0.6 0.7 1.5 SERTAD1 SERTA domain containing 1 A_23_P218 463 1.2 1.1 1.2 1.1 2.1 6.6 1 1.1 3.6 5.4 1.1 1.3 0.9 1.1 SLA Src-like-adaptor A_23_P216 340 1.3 1.3 1.5 0.7 2.1 25 0.6 0.9 5.4 9.3 1.2 1.3 1 2 SLC12A7 solute carrier family 12 (potassium/chloride transporters), member 7 A_24_P606 34 1 1 1 0.8 2.4 20. 4 0.8 1.1 11.8 17.7 0.9 1.2 1.3 3.1 SLC12A7 solute carrier family 12 (potassium/chloride transporters), member 7 A_23_P251 855 1 0.8 0.7 0.9 2.4 23. 8 0.7 0.9 14.6 16 0.7 0.8 1 3.4 SLC12A7 solute carrier family 12 (potassium/chloride transporters), member 7 A_23_P616 88 1.6 1.6 1.7 1.3 3.6 50. 1 1.5 1.5 26.5 45.1 1.5 1.3 1.5 5.1 SLC25A2 5 solute carrier family 25, member 25 isoform b A_24_P355 267 1.1 1.1 1 1 2.4 6.7 0.9 1.1 3 5.8 1 1.1 1 1.4 SLC2A13 solute carrier family 2 (facilitated glucose transporter), member 13 A_32_P199 801 1 1 0.9 1 2.5 31. 3 1 1.2 7.8 21.8 0.9 0.9 1.1 3.2 SLC2A13 solute carrier family 2 (facilitated glucose transporter), member 13 A_23_P102 11 1.2 0.9 1.1 1 1.5 14 0.9 0.9 3.5 11.3 1.2 0.9 1.4 2.8 SLC30A7 zinc transporter like 2 A_23_P229 15 1.1 1.1 0.8 0.9 1.3 5.3 0.9 1 2.2 4.6 0.8 0.9 1.7 1.3 SLC31A2 solute carrier family 31 (copper transporters), member 2 A_23_P217 109 1 0.8 0.7 0.8 1 9.4 0.6 0.7 2 8.1 0.7 0.7 0.9 1.1 SLC33A1 acetyl-coenzyme A transporter A_23_P144 188 1.3 1.2 0.9 0.9 2.6 12. 1 0.8 1 4.8 8.3 1 1 1.7 2.5 SLC41A2 solute carrier family 41, member 2 A_23_P204 801 1 1.1 0.9 0.9 2.5 19. 2 0.7 0.8 4.7 15 1 0.9 1.1 2.8 172 SLC6A12 solute carrier family 6 (neurotransmitter transporter, betaine/GABA), member 12 A_23_P763 86 1.2 1.3 1.1 1.2 3.2 40. 5 0.7 0.7 8.8 11.1 1.1 1.1 0.6 4.4 SLCO5A1 organic anion transporter polypeptide-related protein 4 A_23_P135 669 0.7 1.4 0.7 0.9 2.3 38. 3 0.9 0.8 8.4 17 0.9 1.2 1 2.6 SMPD3 sphingomyelin phosphodiesterase 3, neutral membrane A_23_P163 567 1.2 1.5 0.8 1.1 3.4 50. 6 1 1.2 4.3 20.6 0.9 0.8 1.1 5.6 SNAPC1 small nuclear RNA activating complex, polypeptide 1, 43kDa A_23_P372 44 1.4 1.3 1.1 1 2 6.3 0.9 0.9 2.8 8.9 1 1.1 1 1.7 SNPH syntaphilin A_23_P102 706 0.9 0.9 1.4 1.2 5.2 34. 9 1.3 1.5 24.9 24.5 1.5 1.2 1.2 4.7 SOD2 manganese superoxide dismutase isoform A precursor A_23_P134 176 2.1 1.9 1 1.1 1.9 11. 4 0.8 0.9 3.1 9.1 1.1 1.1 1.2 2.5 SPAG9 SPAG9 protein A_23_P207 476 1 1.2 0.9 1 2.4 15. 4 1 1.3 6.2 8.2 0.9 1.1 1.2 2.3 SPAG9 sperm associated antigen 9 A_24_P365 025 1 1 0.8 0.9 1.4 8.8 0.8 0.9 3.2 6.5 0.8 0.9 1.2 1.8 SPRY2 sprouty 2 A_23_P128 698 0.8 1.1 0.7 0.8 2.8 20. 1 0.8 1 7.3 14 0.7 0.8 0.7 1.8 SRPK2 SFRS protein kinase 2 isoform b A_23_P406 438 1 1 0.8 0.8 1.5 11. 4 0.7 0.7 2.7 7.8 0.8 0.9 1.1 2.1 SRPK2 SFRS protein kinase 2 isoform b A_23_P215 599 1 0.9 0.9 0.9 1.7 10. 8 0.8 0.9 3.1 7.7 0.9 0.9 1.2 1.9 STARD13 START domain containing 13 isoform alpha A_23_P342 727 1.1 1.1 0.6 0.8 2.9 9.3 0.8 1 4.3 7.8 0.7 0.6 0.9 1.3 STARD13 START domain containing 13 isoform alpha A_24_P210 420 1 0.7 0.7 0.7 2.3 6.5 0.8 0.8 4.1 5.9 0.8 0.6 1 0.8 STK38L serine/threonine kinase 38 like A_23_P647 43 1.3 1.2 1 1.1 2.5 15. 3 1 1.1 4.1 13.4 1.1 1.1 1.3 2.5 STX11 syntaxin 11 A_23_P156 788 0.9 0.8 0.7 0.8 2.4 11 0.7 0.8 3.4 9.5 0.8 1 1.1 2.3 SYDE1 synapse defective 1, Rho GTPase, homolog 1 A_23_P101 796 0.9 0.9 0.6 0.8 1.4 8.8 0.9 0.9 2.5 5.5 0.8 0.9 0.8 1.3 TAL2 T-cell acute lymphocytic leukemia 2 A_23_P409 449 1 0.8 0.9 1.3 2 23. 9 1 1.3 6.4 13.2 0.9 1.5 1.8 2.2 TEP1 telomerase-associated protein 1 A_23_P654 81 1.2 1.4 1.3 1.4 3.9 16. 5 1.2 1.2 8.8 10.9 1.2 1.1 1.2 2.1 TESK2 testis-specific protein kinase 2 A_23_P987 5 1 0.9 1.1 1.3 3 16. 2 1.5 1 5.3 8.3 1.3 1 1 3 THRAP1 mediator of RNA polymerase II transcription, subunit 13 homolog A_23_P272 47 1.2 1 0.9 0.9 2.1 7 0.8 0.9 4.1 5.4 0.9 1 1.2 1.7 TMEM38B transmembrane protein 38B A_23_P602 59 1.1 1 0.7 0.8 1.1 10 0.7 0.8 1.6 7.1 0.7 0.9 1.2 1.7 TMEM38B transmembrane protein 38B A_24_P278 156 1 1 0.6 0.8 1 6.4 0.6 0.7 1.2 5.6 0.7 0.9 1.2 1.2 TMEM47 transmembrane 4 superfamily member 10 A_24_P143 171 1.1 1.1 0.3 0.8 2.2 65. 6 0.3 0.5 3.3 44 0.6 1 1.9 3.6 TNFAIP6 tumor necrosis factor, alpha- induced protein 6 precursor A_23_P165 624 1.5 1.2 0.5 0.5 1 50. 9 0.9 1 4.3 26.5 0.5 0.6 0.7 7 TNFRSF1 0B tumor necrosis factor receptor superfamily, member 10b isoform 1 precursor A_23_P169 030 1 1.2 0.9 1 2.3 11. 5 1.1 1 4.8 8.9 0.9 1 1.2 2.5 TRAF1 TNF receptor-associated factor 1 A_23_P216 970 0.7 1.1 1.2 1.2 4.6 25. 8 1.1 1.3 12.8 18.2 1.5 1.4 1.4 2.7 TRAF6 TNF receptor-associated factor 6 A_23_P759 21 1 1.1 0.8 0.8 1.6 6.1 0.7 0.9 1.9 5.4 0.9 0.9 1 1.4 TRAPPC6 B A_24_P304 581 1.1 1.1 0.6 0.9 1.3 5.6 0.7 1 2.6 6.8 0.6 0.9 1 1.6 TRIM26 tripartite motif-containing 26 A_24_P898 583 1.6 1.4 1.2 1.2 3.8 12. 1 1.2 1.3 6.2 8.4 1.2 1.3 1.3 2.3 TRIM26 tripartite motif-containing 26 A_23_P214 587 1.3 1.2 0.8 0.8 2.4 6.9 0.7 0.9 4 5 0.8 0.9 1 1.5 TRIM69 tripartite motif-containing 69 isoform a A_23_P488 26 1.3 1.4 1.3 1.1 1.8 7.9 1.3 1.2 2.3 3.5 1.2 1.2 1.2 1.6 TRPC4 transient receptor potential 4 A_23_P105 873 0.6 0.4 0.7 0.8 3 107 .7 0.7 0.7 30.2 98.8 0.5 0.5 0.7 4.5 TTC26 tetratricopeptide repeat domain 26 A_24_P897 18 1 0.8 0.6 0.9 1.1 5.4 0.6 1 1.8 3.3 0.7 0.8 0.9 1 TTC28 KIAA1043 protein A_23_P291 85 1.2 1.2 1.2 0.9 1.3 6 1 1 2.5 4.5 1.2 0.9 1 1.4 UBE2Z ubiquitin-conjugating enzyme E2Z (putative) A_24_P378 506 1 1.1 1 1 1.8 10. 4 1 1.1 3.1 7.8 1 1 1 1.7 UBE2Z ubiquitin-conjugating enzyme E2Z (putative) A_23_P834 38 1 1 0.9 0.9 1.7 10. 5 0.9 0.9 2.6 9 0.9 0.9 1 1.7 UTX ubiquitously transcribed tetratricopeptide repeat, X chromosome A_23_P217 304 1 1.1 0.8 0.8 2.4 26. 3 0.8 0.9 6 15.1 0.8 0.7 1 2.3 WDR48 WD repeat domain 48 A_23_P804 09 1.1 1.2 1 1 1.3 5.4 0.9 1 2.1 5.5 0.9 0.9 1 1.2 WHDC1 A_24_P307 827 1.5 1.4 1.3 1.2 4.1 15. 3 1.2 1.3 5.8 10 1.2 1 1.2 1.9 WNK1 WNK1 protein A_24_P511 27 1 1 0.6 0.8 1.4 10. 3 0.5 0.7 1.7 6.3 0.7 0.7 0.8 1.3 WTAP Wilms' tumour 1-associating protein isoform 1 A_23_P371 155 1.5 1.3 1.5 1.3 1.8 11. 1 1.5 1.4 4 9.2 1.4 1.2 1.1 2 WTAP Wilms' tumour 1-associating protein isoform 2 A_23_P215 037 1 0.8 0.4 0.6 0.9 6.4 0.4 0.6 1.1 4.3 0.5 0.7 0.9 1.4 173 XPA xeroderma pigmentosum, complementation group A A_23_P602 83 1.1 1.1 0.8 0.8 1.2 7.2 0.7 0.8 2.2 8 0.8 0.8 0.8 1.2 ZBTB10 zinc finger and BTB domain containing 10 A_23_P385 114 1.1 0.9 0.6 0.7 1.8 19. 3 0.6 0.7 3.4 15.9 0.6 0.8 1 1.8 ZBTB43 A_24_P149 713 1 1 0.8 0.8 1.6 4.7 0.9 1.1 2.2 4.3 0.8 1 1 1.1 ZC3HAV1 zinc finger antiviral protein isoform 1 A_23_P425 224 3.7 0.7 1.2 1.2 7.1 88. 4 1 1.5 24.1 40 1.2 1.1 1.6 5.7 ZEB2 zinc finger homeobox 1b A_23_P142 560 1.5 1.5 1.1 1.1 6.1 90. 6 1 1.1 19.2 62.2 1.3 0.9 1 5.9 ZFY zinc finger protein, Y-linked A_24_P942 743 1 0.9 0.8 0.7 1.5 10. 1 0.6 0.7 2.4 8.5 0.8 0.8 0.9 2.1 ZFY zinc finger protein, Y-linked A_23_P624 65 1.2 1.1 0.8 0.9 1.4 5.4 1 1 2 4.7 0.9 1 1.1 1.2 ZHX2 zinc fingers and homeoboxes 2 A_23_P168 951 1 0.9 0.8 0.9 1.3 11 0.8 0.9 2.1 8.5 0.9 1.2 1.1 2 ZNF134 zinc finger protein 134 A_23_P141 866 1 1.1 0.7 0.9 1.5 4.8 0.8 1 2 5.7 0.7 0.9 1 1.3 ZNF134 zinc finger protein 134 A_23_P325 661 1 1.1 0.8 0.9 1.6 6.2 0.8 1 2.2 6.2 0.9 1 0.9 1.3 ZNF14 zinc finger protein 14 A_23_P101 811 1 1.1 1 1 1.7 6.2 0.9 1 3.9 5.7 0.9 0.9 0.9 1.4 ZNF200 zinc finger protein 200 isoform 1 A_23_P152 356 1.1 1 0.7 0.8 1.5 9.7 0.6 0.7 3.1 7.3 0.8 0.8 0.9 1.4 ZNF24 zinc finger protein 24 (KOX 17) A_24_P339 450 1.2 1 0.8 0.7 1.9 9.1 0.6 0.8 1.9 10.1 0.8 0.9 1.2 1.7 ZNF323 zinc finger protein 323 isoform 1 A_24_P137 713 1 1 0.8 0.9 1.8 5 0.7 0.9 2.5 6 0.8 0.9 0.9 1.4 ZNF416 zinc finger protein 416 A_23_P556 88 0.9 1 0.8 0.9 1.6 3.6 0.9 1.1 2 4.9 0.9 0.9 0.9 1.2 ZNF529 zinc finger protein 529 A_23_P433 676 1.1 1.3 1.2 1.1 1.5 8.2 1 1.2 2.4 8.2 1.1 1 1.1 1.6 ZNF567 zinc finger protein 567 A_23_P354 894 1.1 1.2 1.1 1.1 1.2 9 1 1.1 3 6.2 1.1 1 1 1.3 ZNF597 zinc finger protein 597 A_23_P375 3 1.4 1.2 0.9 1 2.4 7.9 1.2 1.5 3.5 6.5 1 1.2 1.1 1.4 ZNF638 zinc finger protein 638 A_24_P419 177 0.9 1.1 1 1 2 4.7 1 1 2.8 4.6 0.9 1.1 1 1.1 ZNF670 zinc finger protein 670 A_23_P749 81 1.1 1.1 0.8 1 1.7 7.1 0.7 0.9 2.8 6.5 0.8 1 1.1 1.5 ZNF671 zinc finger protein 671 A_23_P502 17 0.7 1 0.8 0.6 1.2 5.1 0.7 0.8 2.7 6.2 0.8 0.7 0.8 1 ZNF697 A_32_P197 16 1 0.9 0.8 0.7 2.8 21. 1 0.5 0.9 5.9 21.4 0.9 0.9 1 2.6 ZNF711 zinc finger protein 711 A_23_P217 297 1 1.1 0.8 0.9 1.8 7.5 0.6 0.8 2 11.3 0.9 1 1 1.8 ZNF79 zinc finger protein 79 (pT7) A_24_P128 312 1.1 1.2 0.9 1 2 5.7 1 1.2 2.5 6.3 1 1.2 1 1.4 ZNF79 zinc finger protein 79 (pT7) A_23_P303 763 1.2 1.3 1 1 2.1 6.3 1.1 1.2 2.1 7.2 1 1.3 1 1.5 ZSWIM3 zinc finger, SWIM domain containing 3 A_23_P165 984 0.9 1.1 0.6 0.7 1.4 4 0.8 0.9 2 4.1 0.7 0.8 0.9 1 adenosylhomocysteinase 3 A_24_P725 18 1.1 1 0.9 0.9 1 7.7 0.7 0.8 1.2 3.8 0.9 1 1 1.5 adenosylhomocysteinase 3 A_23_P252 075 1.2 1.3 1 1.2 2.1 20. 9 1.1 1.3 3.8 13.3 0.9 1 1.1 2.5 hypothetical protein A_23_P389 250 1.4 1.7 1 1.6 3 15. 5 1.2 1.3 6 10.5 1 1.2 1.6 2.4 hypothetical protein A_32_P828 63 1.2 1.2 0.7 0.9 1.7 7.5 0.8 0.8 1.8 13 0.7 1.1 0.9 1.9 hypothetical protein LOC152641 A_24_P354 496 0.9 1.2 0.7 1 1.6 6.8 0.9 1.1 2.8 6.7 0.9 0.8 0.8 1 hypothetical protein LOC79912 A_23_P204 187 1 0.9 0.7 0.8 1.4 5.9 0.6 0.8 1.7 6.6 0.7 0.9 1 1.4 hypothetical protein LOC79912 A_24_P221 407 0.9 0.9 0.7 0.7 2.8 8.1 0.7 0.8 1.6 5.9 0.5 0.8 0.9 1.6 hypothetical protein LOC90634 isoform 1 A_23_P766 58 2 1.4 1.1 1.4 1.9 6.1 1 1.2 6.4 6 1.2 1.3 1 1.3 junction-mediating and regulatory protein A_32_P176 550 1 1.1 1 0.9 2.2 12. 5 1 1 4.2 7.5 1 0.6 0.8 2.1 KIAA2002 protein A_23_P658 51 1 1.1 0.7 1 1.4 5.3 0.9 1 2.6 8.1 0.9 1 1.1 1.5 niban protein isoform 2 A_23_P217 832 1 0.8 0.8 0.9 2.1 18. 6 0.8 0.7 2.2 19.9 0.8 1 1.2 2.7 PALM2-AKAP2 protein isoform 1 A_23_P359 043 1.2 0.8 0.5 0.6 1.5 7.6 0.5 0.6 1.8 4.5 0.6 0.7 1.1 1.6 TRAF-interacting protein with a forkhead-associated domain A_24_P350 686 1.1 1.1 1 1 1.4 9 0.8 1 2.6 10.4 1.1 1 1 2.3 A_32_P998 04 1.7 1.5 1.3 1.3 4.6 29. 7 1.5 1.7 14.2 22.6 1.3 1 1.2 3.6 A_32_P889 05 1.2 1.1 1.2 1.2 2.6 14. 9 1.2 1.3 8.4 8.7 1.2 1 1 2.4 A_24_P682 22 1.1 1.1 1.4 1.2 4.2 32. 6 1.3 1.5 13.5 22 1.3 1.1 1.1 3.6 A_32_P165 407 0.7 1 1.1 1 2.9 19. 9 0.9 1 8.3 8.4 0.9 0.9 1 1.9 A_32_P192 389 1.1 1.3 1.3 1 3.5 31. 8 1 1.1 8.4 23.6 1.1 0.9 1 2.7 A_32_P117 322 1.2 1.1 1 0.9 6.7 171 .2 1 1.2 24 98.1 1.1 1.3 0.6 4.8 A_32_P137 604 0.7 1.1 1.2 1.1 2.5 17. 7 1.2 1.1 6.8 9.4 1.1 0.8 0.9 2.7 A_32_P685 04 1.3 1.4 1.3 1.5 5.4 86. 3 1.3 1.4 26.8 46.3 1.2 1.3 1.4 7 174 A_24_P823 011 1 1 0.7 0.8 1.8 7.6 0.6 0.9 4.5 4.9 0.9 0.9 1 1.9 A_32_P731 43 1 0.7 1.3 1 11. 9 69. 5 0.8 0.9 18.3 58.8 1 1.9 1.8 5.3 A_24_P675 74 1.6 1.4 1.8 1.3 3.8 12. 9 1.7 1.1 7.9 9.9 1.1 1.5 1.9 2.7 A_24_P915 784 1.3 1.2 0.9 0.9 2.2 40. 2 0.8 0.9 5.2 27.6 0.8 1.1 1.3 2.9 A_24_P201 39 1.2 1.3 0.7 0.8 2.7 22. 5 0.7 1 5.7 19.1 0.8 0.9 1.1 2.5 A_23_P368 909 1.2 1.1 0.9 1.1 2 27. 6 0.8 0.8 4.4 17.2 0.7 0.9 1 1.7 A_23_P356 041 0.8 0.7 0.5 0.6 1.4 6.7 0.5 0.7 2.3 4.6 0.5 0.7 1.1 1.4 A_32_P621 96 1 1.3 1.2 1 2.1 16. 9 1.2 1.1 5 12.2 1 1.5 2 2.2 A_32_P104 841 1.4 1.5 1.4 0.9 3.4 64. 6 0.8 1.2 14.4 45.3 1.3 1.4 3.3 9.4 A_32_P531 83 0.8 0.9 0.9 0.9 2.1 11. 2 0.8 1.2 4.4 9.7 0.8 1 1.1 1.4 A_23_P649 62 1.1 0.7 0.9 1 3.6 39. 5 1.5 1.2 7.1 19.9 1.1 1.5 1.2 2.5 A_24_P845 4 0.8 0.8 0.8 0.9 1.8 8.2 0.9 1.2 3.8 5.5 0.8 1 1.2 1.8 A_32_P883 49 1.1 1.8 1.3 1.2 2.7 10. 8 1.4 1.3 5.4 11.2 1.3 1.1 1.1 2.7 A_24_P255 384 0.9 2.1 0.9 1 1.8 52. 3 0.8 1.3 13.5 31 0.9 0.9 0.9 7.3 A_23_P497 25 0.8 2 1.2 1.1 2.4 35. 6 1.6 1.2 8.3 17.4 1.4 0.5 0.7 3.7 A_32_P669 08 1.3 1 0.8 0.8 16. 3 65. 5 0.9 0.9 17 44.2 1.1 0.9 1.2 2.8 A_24_P389 612 1.2 1.2 0.8 0.8 3.6 18. 2 0.7 0.9 8.2 14 0.8 0.8 1.2 2.1 A_32_P118 847 1.2 1.1 0.9 1 2.6 11. 3 0.9 0.9 6 7.5 0.9 0.9 1.1 1.5 A_24_P535 256 1 2.1 0.8 0.6 3.6 14. 8 0.6 1 6.7 7.1 0.6 1.2 1.7 2.5 A_32_P362 80 1.1 1.3 0.9 0.8 2.4 9.5 1.3 1.2 4 8.5 0.8 0.6 0.9 1.6 A_23_P435 029 1.1 1.3 1 0.8 1.8 5.8 1.4 1.2 4.3 4.4 1.2 0.7 0.9 1.4 A_24_P364 066 1.3 0.9 1 1 2 7.7 0.9 1 4 3.6 1 1 1.2 1.8 A_32_P617 2 1 0.9 0.8 0.8 1.5 15. 2 0.9 1 5.4 10.1 0.8 0.9 1 1.7 A_24_P814 096 1 1 0.8 0.9 1.5 5.2 0.8 0.9 2.2 3.8 0.8 0.7 0.8 1.2 A_24_P523 061 1 1 1 1 1.5 5.5 1 1.1 2.6 4 1 0.8 0.9 1.3 A_32_P393 84 1.2 1 0.9 0.9 1.4 7.7 0.8 0.8 2.7 7.6 1 0.8 0.7 1.5 A_32_P148 407 0.9 0.9 1.3 1 1.3 15. 9 1 0.9 3.1 10 1 1.1 1.4 2.5 A_24_P929 835 1 1.5 0.8 1 2 5.9 1.1 1.3 4.2 5.9 0.9 0.9 1.1 1.4 A_24_P222 591 1 1.1 0.7 0.9 1.7 5.5 0.8 1 2.1 6.4 0.8 0.9 1.2 1.3 A_32_P174 110 1.2 1.1 0.9 1.8 1.8 7.9 0.8 1.2 2.8 6 1.2 1 1.3 1.4 A_23_P317 654 1.2 1 0.7 0.9 1.6 5.5 0.8 0.9 2.3 3.2 0.7 0.9 1.1 1.1 A_24_P887 092 2 0.9 0.9 1 1.9 18. 2 0.9 1.1 2.6 12.2 0.9 1 0.7 4.5 A_24_P473 29 2.7 2 1.7 1.4 4.2 39. 6 0.9 1.1 7.5 12 1.4 2.3 2.9 6.3 A_24_P307 395 0.9 0.8 0.5 0.5 1 7.3 0.5 0.6 1.7 3.4 0.5 0.8 0.9 1.5 A_23_P143 02 1.1 1 1.2 1.1 1.9 10. 6 1.3 1.3 2.2 4.8 1.2 1.1 1.5 2.6 A_32_P222 571 1.9 1.5 1.7 1.6 3.7 9.3 1.7 1.9 5.6 7.1 1.4 1.3 1.3 2 A_32_P207 767 1.1 1.2 1 0.8 2 8.2 0.8 0.9 2.9 6.9 0.6 0.8 1 1 A_23_P418 031 1.4 1.5 1.5 1.3 2.5 6.1 1.5 1.4 3.9 4.8 1.5 1.6 1.3 1.4 A_23_P143 016 1.3 1.2 1.1 1.1 2.8 6.5 1.3 1.2 3.6 4.4 1 1.3 0.9 1.2 A_32_P108 567 0.7 0.8 1.2 1 3.9 11. 1 1.2 0.8 3.8 7.3 1.1 0.5 1.1 1.9 B ACAN aggrecan isoform 2 precursor A_23_P307 310 1 1.3 1.4 1.4 12. 4 30. 8 1.4 1.2 19.6 20.3 1.2 0.9 2.1 14.2 ACTN2 actinin, alpha 2 A_23_P115 021 0.8 1.6 0.9 1.1 16. 3 475 .3 1 1 185.3 235. 8 1.1 0.9 1.4 40.8 ATF3 activating transcription factor 3 isoform 2 A_24_P338 95 1.4 0.8 0.5 0.6 7.1 21. 1 0.4 1.4 11.1 19.3 0.5 0.8 1.5 3.4 BAMBI BMP and activin membrane- bound inhibitor precursor A_23_P522 07 0.9 0.7 1.2 1 4.6 18. 9 0.8 1 11.6 9.8 1.3 1.1 1.3 3 BIRC3 baculoviral IAP repeat-containing protein 3 A_23_P983 50 1.1 1.9 1 0.9 6.5 40. 8 0.7 0.9 11.6 34.9 1 1 1.7 9.2 C17orf66 hypothetical protein LOC256957 A_23_P430 792 0.8 0.6 0.9 1.1 34. 5 123 .5 1 1.7 100.1 79.3 0.9 1.3 3.3 40.4 C17orf76 hypothetical protein LOC388341 A_23_P368 484 0.8 1.2 1.2 1.1 2.9 12 1.1 1.2 5.8 7.5 1.2 1.1 0.9 2.6 C19orf30 hypothetical protein LOC284424 A_23_P353 667 1.5 1.4 1.1 1.3 3.7 19. 6 1.9 1.1 10.3 12.8 1 1.5 0.7 6.3 175 C20orf141 A_32_P133 182 6.2 2.4 0.8 1.5 48. 5 166 .7 0.8 3.4 237.7 99.4 1.1 1.1 4.1 13.9 C3orf59 hypothetical protein LOC151963 A_23_P326 963 1.3 1.5 0.9 0.5 4.5 9 0.8 0.8 4 12.9 0.6 1 1.4 1.4 CASP3 caspase 3 preproprotein A_23_P924 10 1.1 1.1 0.9 1 2.3 6.3 1 1 6.5 4.1 1 1 1.1 2.5 CCL20 chemokine (C-C motif) ligand 20 A_23_P170 65 0.7 0.7 1 1.1 4.2 115 .8 0.6 1.8 36.2 69.1 0.8 0.9 0.9 27.4 CCL4 chemokine C-C motif ligand 4 isoform 1 precursor A_23_P207 564 0.7 1.4 1.1 1.8 40. 2 142 .2 1.1 4.3 69.3 81.1 0.9 2.2 3.3 30.7 CSAG1 chondrosarcoma associated gene 1 isoform b A_24_P110 61 1.8 2.1 1.2 1.2 6.7 33. 9 1 1 18.6 21.3 1 1.2 1.4 8 CSAG2 CSAG family, member 2 A_24_P567 298 1.3 2.1 1.1 1.2 7.4 43. 6 1.1 1.5 23.5 30.2 1.3 1.3 1.6 8.2 CSAG3A CSAG family, member 3A A_24_P795 29 1.3 2.8 1.2 1 12. 7 70. 9 1 1.2 40.8 45.9 1.1 1.1 1.7 15.7 CXCL1 chemokine (C-X-C motif) ligand 1 A_23_P714 4 1 1.1 1.1 1 1.9 10. 4 0.7 1.3 4.1 7.2 1.1 0.9 0.9 4.6 CXCL2 chemokine (C-X-C motif) ligand 2 A_24_P257 416 1.5 1.2 1.3 1.2 3.5 7.5 1.1 1.8 5.5 5.3 1.2 1.2 1.3 3.9 CXCL3 chemokine (C-X-C motif) ligand 3 A_24_P251 764 1.6 1.2 1.3 1.2 3.6 8.4 1.1 1.8 5.7 6.1 1.2 1.2 1.4 4.3 CXCL3 chemokine (C-X-C motif) ligand 3 A_24_P183 150 1.7 1.3 1.2 1 4.4 16. 3 0.9 1.9 10.1 11.5 1.1 1 1.3 6.1 CXCR4 chemokine (C-X-C motif) receptor 4 isoform a A_23_P102 000 0.8 1 1.2 1.3 25. 1 273 .6 1 1.3 81.9 72.2 1.3 1.4 1.7 27.2 DCHS1 dachsous 1 precursor A_23_P986 45 0.8 1.4 1.4 1.1 7.4 14. 9 1 1 13.3 10.4 1.5 1.5 1.8 6.9 DENND4 A A_23_P420 092 2.1 0.9 0.7 1.3 1.6 8.7 0.5 0.5 3.6 6 0.6 1.1 1 1.3 DNAJC15 DNAJ domain-containing A_23_P117 190 2.2 1.8 1.8 1.4 10. 2 48. 1 0.8 1.8 22.7 17 1.2 2.3 2.2 6.3 EGR4 early growth response 4 A_23_P380 318 0.9 1.1 1.5 1.3 33. 3 84 1.4 4.4 56.3 46.2 1.2 1.2 2.5 23.8 FUT1 fucosyltransferase 1 A_23_P107 963 0.6 1.2 1.3 1.3 5 25. 6 1.4 0.9 8.8 17.7 1.1 1.6 1.4 5.1 FZD4 frizzled 4 A_23_P646 17 0.9 1.3 1.1 1.1 7.5 75. 2 1.2 1.4 22.8 49.5 1.1 0.9 1.4 14.3 GATA3 GATA binding protein 3 isoform 1 A_23_P750 56 1.2 2.1 1 0.9 3.6 6.9 0.7 1 3.2 7.9 1.2 1 0.8 1.8 HES4 hairy and enhancer of split 4 A_23_P149 448 1.3 2.5 1.4 1.3 4.2 10. 9 1.4 1.5 7.7 7.6 1.3 1.4 1.3 3 HIP1R huntingtin interacting protein-1- related A_24_P229 164 0.9 0.9 1.1 1.2 5.9 14. 7 1.2 1.5 11.1 9.6 1.1 1.2 1.4 4.8 HIP1R huntingtin interacting protein-1- related A_23_P398 294 1.1 1.2 2 1.7 14. 5 103 .1 1.8 2.2 43.5 48.3 1.8 1.4 1.8 11 HOXB4 homeobox B4 A_24_P416 370 2.9 2 1.2 1.6 14. 3 48. 8 1.2 1.7 44 32.1 1.2 0.9 2.1 5.8 HTR3E 5-hydroxytryptamine receptor 3 subunit E A_24_P321 39 0.8 1.6 1.1 1.1 3.8 39. 6 0.8 1 21.8 23.4 1 1.1 1.1 5.7 ICAM1 intercellular adhesion molecule 1 precursor A_23_P153 320 4 1.8 0.9 1 14. 2 75 0.8 1 39.2 57.2 0.7 1 2.5 23.3 IL3RA interleukin 3 receptor, alpha precursor A_32_P217 750 1.2 1.9 1.9 1.1 3.9 15. 2 1.9 1.5 6.9 10 1.4 1 1.3 6.2 IL8 interleukin 8 precursor A_32_P870 13 2.1 1.2 1 1.1 18. 9 112 .8 0.7 2.2 42.3 43.2 1.1 1.4 2.8 17.7 JAK2 Janus kinase 2 A_23_P123 608 1.8 1.2 1 1 9.1 22. 5 0.9 1.1 14.8 13.2 0.9 1 1.6 5.1 KCNN1 potassium intermediate/small conductance calcium-activated channel, subfamily N, member 1 A_23_P119 573 0.9 0.7 1 1.3 11. 7 48. 8 1 1.3 32.6 27.4 1 1 1.7 9.3 KLF2 Kruppel-like factor A_23_P119 196 0.8 0.8 1.1 1.1 5.6 25. 4 1.2 1.4 16.4 11.8 1 0.9 1 3.1 KLF4 Kruppel-like factor 4 (gut) A_23_P322 33 1 0.8 0.7 0.9 11. 2 51. 6 0.6 1.1 24.8 34.7 0.8 1 2.3 9.2 KLHDC7B kelch domain containing 7B A_24_P117 410 1.2 2.6 1.4 1.1 10. 2 56. 3 1.3 1.3 17 27.1 1.1 1.3 1.9 11.1 LRRN3 leucine rich repeat neuronal 3 A_23_P313 76 1 1.7 0.7 1.1 10 9.1 386 .8 0.9 1.8 192.9 193. 2 0.9 1.5 7.3 81.3 LTB lymphotoxin-beta isoform a A_23_P933 48 0.8 1.1 0.9 1.3 3.3 34. 4 1.6 1.5 9.8 28.6 0.8 1 1.1 18.7 MED12L mediator of RNA polymerase II transcription, subunit 12 homolog (S. cerevisiae)-like A_32_P701 268 0.8 0.7 1.9 1.2 4.1 46. 7 1.6 1.2 18.3 23.7 1.4 2.4 1.3 6.6 MXD1 MAX dimerization protein 1 A_23_P408 094 1.1 1.2 1.3 1.2 5 21. 7 1.5 1.5 9.8 10.4 1.2 1.2 1.3 4.6 NFKBIA nuclear factor of kappa light polypeptide gene enhancer in B- cells inhibitor, alpha A_23_P106 002 1.1 1.3 1 1.1 9.7 59 0.8 2 34.7 24.5 1 1.1 1.6 7.1 NFKBIE nuclear factor of kappa light polypeptide gene enhancer in B- cells inhibitor, epsilon A_23_P306 55 0.8 1.2 1.2 1.2 3.9 20. 3 1.1 1.1 7.5 12.4 1.2 1.3 1.1 3.7 PIWIL4 piwi-like 4 A_23_P427 760 1 1.5 1.5 1.6 3.4 8.8 1.3 1.3 7.7 6 1.1 1.3 1.4 3.3 PLA2G4C phospholipase A2, group IVC A_23_P505 08 1 1.1 0.7 1 7.5 60. 9 0.7 1.2 21.1 28.1 0.9 1 1.3 8.3 PMAIP1 phorbol-12-myristate-13-acetate- induced protein 1 A_23_P207 999 2 1.4 0.6 1.1 12. 6 34. 2 0.7 2 24.5 21.1 0.7 1.1 1.8 5.3 PPP1R15 A protein phosphatase 1, regulatory subunit 15A A_23_P901 72 0.9 1 1.1 1.2 10. 5 34. 8 1.2 2.4 20.4 19.2 1 1.1 1.4 5.7 176 PRDM1 PR domain containing 1, with ZNF domain isoform 1 A_23_P350 451 1 1.5 1 1 6.9 7.9 0.9 1.1 5.3 6.2 1 0.9 1 2.9 PRR15 proline rich 15 A_32_P154 911 2.4 1.6 1.6 1.6 9 12. 4 1 1.5 12.2 9.1 1.8 1.5 2.3 6.8 PTGER4 prostaglandin E receptor 4, subtype EP4 A_23_P148 047 1.1 0.9 0.6 0.9 20. 5 146 0.5 2 41.7 72.9 0.7 1.1 2.3 14.4 PTX3 pentraxin-related gene, rapidly induced by IL-1 beta A_23_P121 064 0.8 1.2 0.6 0.7 24. 5 127 .4 0.6 1 67.2 89.1 0.7 0.8 1.4 20.1 RHEBL1 Ras homolog enriched in brain like 1 A_23_P230 7 1.1 1.1 0.7 1 4.2 15. 9 1 1.4 7.8 12.4 0.8 0.8 1.1 3.9 SEMA3D semaphorin 3D A_32_P291 18 0.9 0.8 0.9 1 19. 8 258 .3 1 1.3 73.8 127. 7 1 1 2.2 21 SLC1A3 solute carrier family 1 (glial high affinity glutamate transporter), member 3 A_24_P286 114 0.8 0.8 1 1.1 33. 5 195 .1 1.5 1.4 125.4 105. 4 1.4 0.9 1.8 17.1 TMC8 FLJ00400 protein A_23_P346 093 0.7 1.5 1 1.6 5 21. 8 1.7 1.9 11.7 15.4 0.8 1.4 1.8 5.3 TNFAIP2 tumor necrosis factor, alpha- induced protein 2 A_23_P421 423 0.9 1.1 2 1.6 2.9 19. 5 1.5 1.5 9.6 13.4 1.8 1.4 1.3 5.3 TNFAIP3 tumor necrosis factor, alpha- induced protein 3 A_24_P166 527 1 0.7 0.9 1.2 11. 1 36. 7 1 2.5 29 23 0.9 1 1.5 6.6 TNFAIP3 tumor necrosis factor, alpha- induced protein 3 A_24_P157 926 1.4 1.5 1.1 1.5 17. 1 78 0.8 2.7 34.1 37.5 1.2 1.2 1.9 13.8 TNFRSF9 tumor necrosis factor receptor superfamily, member 9 precursor A_23_P519 36 1.1 1.2 1.1 1.1 4.2 28 1.4 1.1 12.4 19.1 1 1.4 2.3 8.2 TRAF1 TNF receptor-associated factor 1 A_24_P898 91 0.7 1.7 1.4 1.3 5.6 21. 8 1.1 1.4 14.8 14.5 1.1 1.4 1.4 3.6 TTC9B A_23_P332 392 1 1 1 1 8 25. 1 1.2 1.3 15.3 16 1 1.2 1.4 7.2 ULBP2 UL16 binding protein 2 A_23_P168 259 1.1 1 0.7 1 8 26. 5 0.6 1.4 16.2 16.8 0.7 1 1.4 4.1 ULBP2 UL16 binding protein 2 A_24_P149 314 1.2 1 0.6 0.7 6.5 13. 7 0.7 1.1 13.1 8.9 0.9 1.1 1.5 2.8 ULBP2 UL16 binding protein 2 A_23_P145 485 1.1 0.9 0.6 0.8 6.4 16. 6 0.8 1.3 13.4 10.7 0.8 1.1 1.3 3 WISP1 WNT1 inducible signaling pathway protein 1 isoform 1 precursor A_23_P354 694 1.4 2 1.2 1 11. 9 59. 1 1.1 1.4 38.8 18.7 1.2 0.9 1.1 9.1 XBP1 X-box binding protein 1 isoform XBP1(U) A_23_P120 845 1 1 0.8 0.9 5.1 12. 9 0.7 1 7.4 12.7 0.8 1 1.7 2.3 ZC3HAV1 zinc finger antiviral protein isoform 2 A_24_P341 938 4.1 1.7 0.7 1 11. 5 86. 1 0.6 1.6 26 74 1 1.1 1.9 10.5 ZC3HAV1 zinc finger antiviral protein isoform 2 A_23_P201 22 4.3 2.9 1 1.3 14. 7 109 .8 1.1 2 33.6 77.2 1.1 1.1 2.1 15.8 hypothetical protein A_24_P119 678 1.2 0.9 0.9 1.1 11. 7 26. 6 1.4 1.2 21.4 18.9 1.6 0.9 1.4 9.9 A_32_P957 66 0.8 0.7 0.9 1.1 16 125 .9 1 1 84.6 88.1 1 1 1 25.5 A_24_P931 804 0.8 0.7 0.9 1.1 16. 9 91 1 1.5 54 42.6 1 0.8 1.1 10 A_32_P219 135 1 1 1.7 1.6 9.7 26. 6 1.5 1.8 19.2 18.8 1.6 1.8 1.8 8.6 A_24_P933 828 0.8 0.7 1.3 1.1 5.9 27. 4 1.1 1.5 16.4 19.1 1.2 1.4 1.4 6.2 A_23_P506 46 1.3 1.3 1.6 1 11. 4 38. 3 1.2 2 23.2 25.3 1.6 1.4 2 9.1 A_23_P119 80 0.9 1.3 1.3 1.1 10. 8 59. 1 1 1.9 34.3 40.5 1.8 0.9 1.3 11.2 A_32_P703 1 1.1 1.4 1.4 12. 4 64. 7 1.4 2.4 33.9 32.2 1.1 1 1.2 4.8 A_32_P221 437 1.1 1.1 1.2 1.2 4 13. 3 1.4 1.3 8.8 7.2 1.2 1 1.2 2.9 A_32_P308 98 1.1 1.2 1.1 1.4 4.1 18. 8 1.3 1.5 8.2 9 1 1.1 1.6 3.9 A_32_P222 684 1 1.2 1.3 1.4 9.1 46 1.1 1.4 40.7 26.8 1.2 1.5 1.5 6.3 A_24_P401 830 0.9 1.2 1.4 1.5 6.7 18. 2 1.4 1.7 17.8 10.4 1.1 1.2 1.2 3.8 A_32_P170 664 1.1 1 0.9 1.2 5.4 19. 7 1.1 1.4 16.8 8.8 0.8 1.2 1.3 4.3 A_24_P652 537 1.6 1.6 1.4 1.2 30. 8 95 1.4 1.3 55.5 52.1 0.9 1.1 2.8 15.2 A_23_P328 740 1 1.7 0.9 1.3 8.9 36. 8 1.1 1.9 17.7 35.7 0.9 1.1 1.5 8.6 A_32_P224 888 1.8 1.5 2.2 2 15. 2 75. 1 1.1 1.5 44.9 36.2 1 0.9 2.5 6.3 A_32_P122 494 1.2 1 1.6 1.3 4.6 14. 9 1.2 1.3 9.9 8.8 1.7 1.2 2.1 3.3 A_32_P217 261 1.4 1.2 1 1.1 4.5 32. 4 0.9 1 8 17.7 1 0.9 1.3 4.7 A_24_P849 245 1.2 1.1 0.8 1 3.1 10. 2 0.8 0.9 4.5 7 0.8 1 1.1 3.4 A_24_P101 651 1.3 2 1.3 1 8.1 32. 3 1.1 1.3 18.9 21.2 1.2 1.8 2.1 7.8 A_23_P285 82 0.7 1 0.7 0.7 2.2 14 0.9 0.8 5.8 7.7 0.8 0.7 0.8 3.9 A_32_P458 44 1.4 2.5 1.2 1.3 29 77. 3 0.9 1.8 32.1 23.6 1.4 1.8 2.1 18.9 A_24_P734 406 1.3 3.1 0.9 1.9 14. 8 23. 5 1 1.6 14.2 16.4 1.2 1.1 1.8 7.5 A_32_P169 550 0.9 1.3 1.2 0.9 6.2 9.2 0.9 1.4 5.6 6.4 1.1 0.9 0.9 2.9 177 A_23_P157 766 1 1.2 1.3 1 4.9 8 0.8 0.9 5.7 5.1 1.4 1.1 0.9 3.1 A_23_P320 407 0.6 2.1 2.1 2.1 4.1 25. 6 2.8 1.7 10.8 11.4 2.1 1.3 1.3 4.7 A_24_P174 353 0.9 2.1 1.6 1.1 3.9 14. 9 1.5 1.4 9 15 1.5 1.9 1.1 8.3 A_24_P231 483 1.6 3.8 1.3 1.1 8.9 26. 5 0.8 1.3 18.7 17.9 1 1.2 1.2 2.1 A_32_P535 24 1 0.9 0.7 1.3 1.7 9.7 0.9 0.5 4 4 0.9 1.3 1.1 1.9 A_23_P207 774 1 0.9 1.2 1.2 2.7 9.7 0.6 0.6 4.6 4.6 0.8 1 0.7 2.1 C ABCA5 ATP-binding cassette, sub-family A , member 5 A_24_P670 96 1 1 1.8 1.5 1.8 9.1 1.6 1.5 4.4 4.3 1.5 1.1 1.2 1.4 ABCA5 ATP-binding cassette, sub-family A , member 5 A_23_P780 18 1.1 1 1.5 1.4 1.7 7.7 1.5 1.5 3.8 3.5 1.3 1 1.1 1.4 ABL2 v-abl Abelson murine leukemia viral oncogene homolog 2 isoform b A_23_P138 099 1.1 1 1.8 1 3.2 10. 6 1.1 1.2 5.6 7.6 1.6 1.1 1.3 1.6 ACTR3B actin-related protein 3-beta isoform 1 A_23_P123 193 1.1 0.8 1.1 1.1 1.1 7 1 1.1 2.7 3.7 1.1 1.1 1.1 1.2 ALDH1L2 hypothetical protein A_23_P879 41 2.2 1.2 0.9 1.1 2.1 8.8 1.1 0.8 1.6 13.8 0.9 1.1 1.8 1.8 ALOX12P 2 A_32_P440 768 0.9 1.1 1.9 1.3 1.6 3.9 1.3 1.5 2.6 4.3 1.4 1.1 1.3 1.1 AMDHD1 amidohydrolase domain containing 1 A_32_P122 226 1.1 1 1 1.1 2 7.6 0.9 1.1 5.4 4.2 1.1 1 1.1 1.6 ANKRD1 cardiac ankyrin repeat protein A_23_P161 218 0.9 0.9 1.1 1.2 1.3 5.6 0.9 1.1 1.3 4.4 0.8 0.7 0.5 0.9 AP3B2 adaptor-related protein complex 3, beta 2 subunit A_23_P773 04 1.7 1.4 1.4 1.4 1.2 10. 5 1.2 1.2 1.7 6.5 0.9 1.2 1.4 1.1 AQP9 aquaporin 9 A_23_P106 362 0.8 0.8 0.9 1 0.7 22. 8 1 0.9 1.8 26 0.9 0.9 1 1 ARHGAP 9 Rho GTPase activating protein 9 isoform 1 A_23_P646 61 1.6 1.6 1.1 1.1 2.2 12. 8 1 2.8 5.6 14 1.3 1.6 1.1 2.8 ASMTL acetylserotonin O- methyltransferase-like A_23_P159 539 1 0.9 1 1 1.4 4.9 1.3 1.1 3.4 3.2 1 1 0.9 1.1 AVIL A_23_P532 57 1 1.3 1.6 1.4 2.6 6.8 1.9 1.6 7.8 2.8 1 0.8 0.6 1 AXUD1 AXIN1 up-regulated 1 A_23_P121 011 1.1 0.8 0.7 0.9 4.3 3.8 0.7 1.1 4.6 2.9 0.7 1.2 0.9 1.1 BAGE4 B melanoma antigen family, member 4 A_24_P126 262 0.8 0.9 1.5 0.9 2.2 13. 8 1.6 1 4.2 8.7 1.1 1.5 0.8 1.4 BBC3 BCL2 binding component 3 A_24_P305 312 0.8 0.7 0.6 0.8 3.1 6 0.8 1.3 5.6 2.5 0.7 0.9 0.8 1.6 BBC3 BCL2 binding component 3 A_23_P382 775 1.4 1.2 1.5 2 9.8 35. 1 1.9 3.5 28.5 10.3 1.2 1.4 1.2 3.9 BCL6 B-cell lymphoma 6 protein A_23_P578 56 1.7 1 1.3 1.2 4.1 9.3 1.1 1.2 7.8 5.2 1.1 0.8 1.2 1.8 BEXL1 A_24_P405 51 1 0.9 0.9 1 1.1 5.4 0.8 0.9 1.3 5.1 0.9 1 0.8 1.1 BMP7 bone morphogenetic protein 7 precursor A_23_P684 87 1.1 0.9 1.1 1 1.1 7.4 0.9 0.9 1.1 3.3 1 1.1 1 1.1 BOC brother of CDO A_23_P257 763 0.8 0.7 1.3 0.9 1.2 5 1.3 1.3 2.4 2.8 1 0.8 0.8 0.9 C10orf10 fasting induced gene A_23_P355 97 0.8 1.3 0.9 0.8 1.4 8.9 0.9 1.2 2.7 4.5 0.7 0.5 0.3 1 C10orf10 fasting induced gene A_24_P329 795 2.4 1.9 1 1.1 1.3 12. 2 0.9 1.2 2.7 6.1 0.9 0.6 0.3 1.2 C11orf17 chromosome 11 open reading frame 17 A_23_P521 61 1 0.9 1.2 1.1 4 6.3 1.1 1.6 7.5 3.9 1.2 1 0.9 1.7 C12orf28 hypothetical protein LOC196446 A_32_P424 761 0.9 0.6 0.9 1.1 1 10 1 1.1 4.7 7.4 1.3 1 1 1.4 C12orf34 hypothetical protein LOC84915 A_23_P128 375 1 1 1.2 1.2 1.4 8 1.1 1.2 2 6 1.1 1.3 1.2 1.6 C14orf81 A_24_P153 558 0.8 1 1.5 1.3 1.2 8.6 1.4 1.1 3.9 4.1 1.3 1.1 0.8 1.2 C17orf55 hypothetical protein LOC284185 A_23_P426 565 1.1 1 1.4 1.2 2.7 4.9 1.1 1.2 7.7 2.9 1.3 0.9 1.1 2 C19orf28 hypothetical protein LOC126321 isoform c A_23_P345 139 0.8 1.7 0.8 0.9 1.9 11. 1 1 1.1 2.2 9 0.8 0.9 1.1 2.7 C19orf47 hypothetical protein LOC126526 A_32_P505 133 0.9 0.9 1 0.8 1.1 3.6 1 0.9 1.6 5 0.8 0.8 0.8 1.1 C1orf102 oxidored-nitro domain-containing protein isoform 1 A_23_P103 433 1.1 0.9 1.2 1.1 1.3 4.3 1.3 1.2 2.1 4.5 1.1 1 0.9 1.1 C1orf79 A_23_P745 81 1.1 1.3 1.5 1.4 2 6.5 1.3 1.3 2.4 6.2 1.3 1.3 1.1 1.6 C2orf49 hypothetical protein LOC79074 A_23_P165 698 1.1 1.1 0.9 0.9 1.3 5.8 0.9 1 1.6 6.1 0.9 1 0.9 1.4 C3orf19 hypothetical protein LOC51244 A_24_P144 481 1.1 1.3 1.3 1.2 2 8.5 1.4 1.2 4 8 1.2 1.1 1 1.6 C5orf5 chromosome 5 open reading frame 5 A_24_P207 00 1.1 1.3 1.3 1.2 2.7 6.6 1.6 1.6 2.9 5.6 1.3 1.2 1.4 1.4 CAMK1G calcium/calmodulin-dependent protein kinase IG A_23_P974 02 1.2 0.6 1 1.1 3.1 22. 7 0.8 0.8 7.3 23 1 1.1 0.5 3.1 CCDC65 coiled-coil domain containing 65 A_23_P479 04 1.4 0.7 0.7 1.2 1 9.3 0.7 1.4 1.8 11.3 0.7 1.4 0.6 1.3 CCL23 small inducible cytokine A23 isoform CKbeta8-1 precursor A_24_P319 088 0.3 0.9 1 1.2 1.2 9.6 1.1 1.1 2.9 2.9 1.1 1 0.9 1 CD163L1 scavenger receptor cysteine-rich type 1 protein M160 precursor A_23_P614 66 1.2 1.3 0.7 1.1 1.6 5.7 1.3 0.6 2.3 5.2 1 0.8 1.2 1 178 CD70 tumor necrosis factor ligand superfamily, member 7 A_23_P119 202 1.1 1 1.7 1.4 1.3 10. 4 1.6 1.4 3.3 7.8 1.5 1.5 1.2 2 CD83 CD83 antigen isoform a A_23_P706 70 0.8 0.9 0.9 0.9 1.7 10. 9 0.8 0.8 4 7.5 1.1 1 0.8 2 CEBPB CCAAT/enhancer binding protein beta A_23_P411 296 1.5 0.9 1.2 1.3 4.6 7.7 1.3 1.5 9.6 5.9 1.1 1.2 1.2 2.1 CEBPB CCAAT/enhancer binding protein beta A_23_P143 242 1.3 0.9 0.9 1 2.6 3.3 1 1.2 4.4 2.6 0.9 1 1 1.5 CEBPE CCAAT/enhancer binding protein epsilon A_23_P299 0 1.5 0.8 1 1.1 3.3 4.3 1.1 1.2 5.8 3.2 1 1 1 1.7 CHGA chromogranin A precursor A_32_P270 46 1 1 1.3 1.1 1.2 5.2 1.3 1.2 1.7 3.9 1.2 1.1 1.1 1.2 CLEC4M C-type lectin domain family 4, member M isoform 3 A_23_P208 482 1 1.1 1.6 1 1.1 7.1 1.5 1.3 2.7 6.4 1 1.1 0.9 1.2 CLK4 CDC-like kinase 4 A_23_P616 74 0.9 1.1 1.4 1.2 1.6 6.3 1.3 1.4 3.9 4.3 1.2 1.1 1 1.4 CLK4 CDC-like kinase 4 A_24_P123 521 1 1 1.2 1.2 1.4 4.7 1.2 1.3 2.8 2.7 1 1.2 1 0.9 COLEC11 collectin sub-family member 11 isoform b A_23_P120 125 1 1.2 1.6 1.3 2.4 40. 4 1.2 1.3 9 14.8 1.4 1 0.8 2.7 COLEC11 collectin sub-family member 11 isoform b A_24_P388 322 0.9 1.1 1.1 0.9 2 24. 9 1.5 1.2 6.3 12.9 1 1.4 0.8 2.3 CXXC4 CXXC finger 4 A_23_P121 676 1.1 1 1.2 0.7 2.2 35. 4 1.1 1 11.2 30.5 1.8 1.1 1.1 2.8 DENND4 A c-myc promoter binding protein A_24_P136 497 1.5 1 1.7 1.4 2.9 11. 9 1.5 1.3 6.6 4.9 1.3 1 1.1 1.6 DHRS2 dehydrogenase/reductase (SDR family) member 2 isoform 1 A_23_P485 70 1 0.9 1.5 1.5 1.6 8.6 1.1 1.1 2.5 7.5 1.4 1.3 1 2.2 DICER1 dicer1 A_23_P371 11 1.1 1.1 1.8 1.4 2.5 21. 8 1.8 1.5 11.3 7.7 1.6 1.2 1.2 2.6 DIP2B DIP2 disco-interacting protein 2 homolog B A_24_P681 62 1 1.1 1.3 1.2 1.5 5.9 1 1.1 2.4 4.4 1.1 1.1 1.1 1.4 DND1 dead end homolog 1 A_32_P420 54 0.9 1.1 1.6 1.3 2 57. 1 1.7 1.4 8.1 19.2 1.4 1 1 1.3 DPYSL4 dihydropyrimidinase-like 4 A_23_P331 049 1.1 1.1 1.2 1 1.5 6.1 1.3 1 3.9 3.8 1.1 1.1 1.2 1.3 DUSP8 dual specificity phosphatase 8 A_24_P322 867 1.1 1.4 1.8 2 2.7 10 1.7 1.8 7.4 5.2 1.8 1.8 1.1 1.9 DUSP8 dual specificity phosphatase 8 A_23_P390 528 1.1 1.4 2.3 2.1 2.7 10. 6 2.1 2.1 7.9 5.1 2 1.7 1.2 1.8 EBI3 Epstein-Barr virus induced gene 3 precursor A_23_P119 478 1.1 1.1 1.1 1.2 1.1 11. 9 1.3 1.1 1.9 10.4 1 1.1 1.1 2.1 EFNA1 ephrin A1 isoform a precursor A_23_P254 512 1.2 1.3 1.5 1.5 4.9 6 1.1 2.1 10 3.6 1.4 1.2 1 3.3 EFNA1 ephrin A1 isoform a precursor A_23_P113 005 1.1 1.1 1.1 1.2 3.3 3.2 1 1.6 5.5 2.4 1.1 1 0.9 2.4 EGR1 early growth response 1 A_23_P214 080 0.7 1 1.4 1.4 4.4 9.1 1.2 2.4 13.7 4.4 1.4 1.3 1.1 1.3 EGR3 early growth response 3 A_23_P216 225 1 0.9 0.9 1.8 25. 2 15. 4 1.2 1.9 39.9 7.3 1 0.9 1 5.6 ELL elongation factor RNA polymerase II A_23_P279 36 1 1.1 1.1 1 1.4 4.7 1 1 1.7 5 1 0.9 0.9 1.1 EPHA4 ephrin receptor EphA4 A_23_P108 501 0.8 0.9 1.3 1.2 2.6 40. 2 0.9 1 8.5 29.4 1.4 1.1 1.1 4.1 ETV3 ets variant gene 3 A_23_P400 945 1.1 1.1 1.6 1.7 5.1 3.9 1.7 2.5 6.6 2.1 1.5 1.4 1.5 3.1 EZH2 enhancer of zeste 2 isoform a A_23_P259 641 1.1 1 1.2 1.1 1.2 8.2 1.2 1.3 3.5 4.2 1.2 0.9 1 1.1 FAM110A hypothetical protein LOC83541 A_23_P386 241 0.9 0.8 1.1 0.9 1.1 5.3 1.2 1.1 2.3 6 1 0.9 0.8 1.4 FAM90A1 hypothetical protein LOC55138 A_24_P643 028 1.1 0.8 1 1.2 1.2 5.1 1.1 0.8 1.5 4.8 1 1.2 1 1.3 FAM90A1 0 hypothetical protein A_24_P903 680 1 0.9 1.2 1.3 1.2 5.7 1.3 1.2 1.8 4.9 1.1 1.2 1 1.3 FGF9 fibroblast growth factor 9 precursor A_23_P105 803 1 1 0.8 1 1.7 3.2 0.9 0.6 1.6 6.2 1.3 0.9 1.3 1.6 GABPB2 GA binding protein transcription factor, beta subunit 2 isoform gamma 1 A_23_P205 789 1.2 1.3 1.3 1.2 1.4 8.2 1.2 1.1 2 8.5 1.3 1.2 1.9 1.8 GBA3 cytosolic beta-glucosidase A_23_P186 72 1 0.7 1 1.2 1 118 .2 1 1.1 5 113. 2 1 1.4 0.8 2.7 GLCCI1 glucocorticoid induced transcript 1 A_23_P824 02 1.2 1.1 1.3 1.4 3 15. 3 1.5 1.3 9.3 8.1 1.2 1.1 1.3 2.7 GLI3 GLI-Kruppel family member GLI3 A_23_P111 531 1.3 4.2 0.9 1.4 5.5 9.7 1 1.5 5.9 5.1 1 0.9 1.2 1 GOT1 aspartate aminotransferase 1 A_23_P638 25 1.1 1 1.1 1.1 1.4 4.1 1.2 1.2 1.4 6.3 1 1.2 1.3 1.5 GP1BA A_23_P152 926 1 1.9 1.9 1.7 3.7 85. 4 1.5 1.4 17.2 112. 9 2.5 2.1 1.5 5.1 GRIP2 FLJ00218 protein A_24_P930 868 2.5 2.2 1.7 2.4 9.2 19. 9 2.2 1.6 12.3 6.1 1.5 1.8 1.2 4.8 HARS histidyl-tRNA synthetase A_24_P892 84 0.8 0.8 1.2 0.9 0.9 20. 1 1 0.9 1.3 8.2 0.9 1 0.9 0.9 HARS histidyl-tRNA synthetase A_23_P133 227 1 1 1.1 1 1.1 33. 5 1.2 1 1.8 13 1 1.1 1 1 HBG1 A-gamma globin A_23_P531 37 1 0.8 1 0.9 1.2 6.8 1 1 3.3 9.5 1 0.9 1.2 1.4 HCFC2 host cell factor C2 A_23_P254 03 1.3 1.3 1.3 1.1 1.5 5.4 1.1 1.1 2 5 1.3 1.1 1 1.2 HCLS1 hematopoietic cell-specific Lyn substrate 1 A_23_P734 29 1 1.1 1.7 1.3 3.3 18. 8 1.7 1.6 9.8 15.1 1.4 1 0.9 2.2 179 HCP5 HLA complex P5 A_24_P178 70 1.4 3.4 1.4 1.6 1.5 40 1.5 1.6 4.1 18.1 1.2 1.1 1.3 8.8 HCP5 A_23_P111 126 1 8.2 0.8 0.8 3 72. 6 0.8 1.6 3.7 40.2 0.6 1.1 1.4 14.7 HIVEP2 human immunodeficiency virus type I enhancer binding protein 2 A_23_P214 766 1.2 1 1.5 1.3 2.1 14. 2 1.5 1.2 5.1 9.1 1.4 1 0.9 1.9 HOXA4 homeobox A4 A_23_P253 982 0.9 0.9 1.1 0.9 1.3 4.3 1.1 1 2.1 6.2 1 1 1 1 HOXD10 homeobox D10 A_23_P381 368 0.9 0.9 1.3 1.1 2.8 4.6 1.3 1.2 5 2.9 1.2 1.1 1.1 2.2 HRK harakiri A_23_P251 94 1 1.4 1.7 1.5 2.1 7.1 1.6 1.4 2.6 7.9 1.4 1.4 1.1 1.2 HTATIP2 alternatively spliced product of metastasis-suppressor gene CC3 A_24_P307 580 1.4 1.3 1.6 1.4 2.2 8.3 1.3 1.4 5.2 3.8 1.6 1.3 1.2 1.9 IFNA4 interferon, alpha 4 A_24_P403 459 1.4 0.9 1.5 1.5 7.5 191 .3 1.4 1.8 4.5 38.8 1.2 1.4 1.2 2.2 IFNA8 interferon, alpha 8 A_23_P146 539 0.8 0.7 0.9 1 2.7 67 0.9 0.6 1.8 15.7 1.2 0.9 0.9 0.9 IGF2 insulin-like growth factor 2 A_23_P421 379 1 1.2 1.5 0.7 1.6 23. 7 1.5 1.2 9.1 14 0.8 0.9 1 1.3 IGF2 A_32_P566 61 1.3 0.9 1.2 1.8 2.7 26. 7 1.1 0.9 7.6 15.4 1.8 1.4 1.2 1.8 IL23A interleukin 23, alpha subunit p19 precursor A_23_P760 78 0.7 1 1.1 1.2 1.7 11. 4 1.1 1.3 4.9 12.8 0.9 0.9 1 1.7 INTU PDZ domain containing 6 A_32_P118 372 1 0.9 1.9 1.3 2.6 17. 4 1.5 1.3 7.9 9.9 1.6 1.3 1 2.1 ITGAM integrin alpha M precursor A_23_P124 108 1.3 1.4 1.2 1.8 2 15 1.3 1.6 5.9 13.5 1.4 1.3 1.2 2.7 JPH2 junctophilin 2 isoform 1 A_23_P394 395 1 1.1 1 1 1.4 7.2 1.2 1 1.9 6.9 1 1 0.9 0.8 JUN jun oncogene A_23_P201 538 1 1.1 1.8 1.7 7.7 18. 8 1.5 2.5 22.9 8.5 1.7 1.8 1.2 2.9 JUNB jun B proto-oncogene A_23_P482 1 1.2 1.4 1.3 1.5 3.5 6.3 1.2 1.7 6.3 3 1.2 1.3 1.2 1.7 KIAA1267 hypothetical protein LOC284058 A_23_P553 76 1 1.2 1.5 1.2 1.3 5.1 1.5 1.2 2.5 3.4 1.4 0.9 0.9 1.1 KRT17 keratin 17 A_23_P961 58 0.9 1.4 0.9 1.1 2.2 29. 4 0.7 1.2 3.8 25.4 1.3 1.1 1.2 5 KRT34 keratin 34 A_23_P101 054 1 1 0.7 1.3 1.4 14. 1 1 1 1.8 13.8 0.8 1.3 1.4 1.5 LATS2 LATS, large tumor suppressor, homolog 2 A_23_P204 958 1 1.1 1.4 1.1 1.8 4.1 1.3 1.1 2.8 3.3 1.2 1 0.9 0.9 LCE1F late cornified envelope 1F A_23_P411 321 1 1.1 1.4 1.6 1.5 6.5 1.5 1.4 2.3 3 1.3 1.4 1.3 1.6 LPA lipoprotein Lp(a) precursor A_23_P952 21 0.8 1.1 0.8 1 1.1 23 0.9 1 1.5 15.5 1.1 0.8 1.1 2.2 LPXN leupaxin A_23_P871 50 1 1.1 1.2 1.3 1.9 10 1.2 1.3 4.3 9.2 1.1 1 1 1.7 LRRC46 leucine rich repeat containing 46 A_23_P152 949 0.9 1 1 0.9 1.1 3.7 1.2 1.2 1.9 4.2 1 1 0.8 0.9 LRRC48 leucine rich repeat containing 48 A_24_P270 971 1.1 0.9 1.2 1.2 1.5 3.5 1.4 1.3 2.9 4.1 1.3 0.9 0.8 0.9 LRRC49 leucine rich repeat containing 49 A_23_P129 174 1.1 0.8 1.1 1 1.2 7.6 1.1 1.1 2.2 7.3 1.1 1 1 1.5 LST1 leukocyte specific transcript 1 isoform 1 A_24_P103 469 1 0.8 1.3 1 1.8 22. 5 1.1 1.3 7.8 15.5 1.1 1 0.8 2 LYPD1 LY6/PLAUR domain containing 1 isoform a A_32_P101 031 1.3 1.2 1.3 1 1.4 5.3 1.4 1.2 2 4 1.2 1.1 0.9 0.9 MAP3K10 mitogen-activated protein kinase kinase kinase 10 A_24_P284 523 1 1 1.9 1.6 2 10. 2 1.9 1.4 4.3 7.3 1.5 1.2 1.1 1.5 MAP3K7I P3 mitogen-activated protein kinase kinase kinase 7 interacting protein 3 A_23_P305 033 1.2 1.4 1.2 1.2 1.4 8.1 1.5 1.2 1.9 7 1.2 1.1 1.1 1.5 4-Mar membrane-associated ring finger (C3HC4) 4 A_23_P333 228 0.9 1.1 0.9 0.9 1.1 11. 3 1.3 1.1 1.8 14.7 0.7 0.9 0.9 1.6 MARK1 MAP/microtubule affinity- regulating kinase 1 A_23_P424 0.8 0.8 0.7 1.3 1.6 4.2 0.8 0.7 1 7.9 0.9 0.9 1 0.8 MDM4 mouse double minute 4 homolog A_23_P103 503 1 1.4 1.5 1.3 1.9 10. 7 1.5 1.6 3.6 7.6 1.3 1.1 1.1 1.9 MEG3 A_24_P272 993 0.9 1.1 2.2 1.7 1.6 7.7 1.9 1.6 2.6 3.8 1.9 1.3 1.3 1.7 MLL3 myeloid/lymphoid or mixed- lineage leukemia 3 isoform 2 A_24_P840 688 1.2 1.3 1.4 1.3 1.6 10. 4 1.6 1.4 5.1 6.6 1.2 1.3 1.2 1.9 NAV2 steerin2 protein A_24_P933 458 0.9 1 1.9 1.5 2.9 14. 3 2.1 1.8 8.3 7.3 1.4 0.9 1 2 NFKBIZ nuclear factor of kappa light polypeptide gene enhancer in B- cells inhibitor, zeta isoform a A_23_P212 089 1.1 1.2 1.9 1.3 2.4 8.3 1.7 1.9 4.2 3.5 1.5 0.8 0.9 2.2 NLGN3 neuroligin 3 A_23_P622 98 1.1 1.8 1.3 1.3 2.1 5.5 1.9 1.3 2.6 3.5 1.7 0.9 1.1 0.9 NR2F1 nuclear receptor subfamily 2, group F, member 1 A_23_P348 737 1 1.1 2.1 1.3 6.8 37. 5 1.9 1.6 29 17.7 1.7 1.1 1.2 2.8 NSUN6 NOL1/NOP2/Sun domain family, member 6 A_23_P615 80 1.2 1.2 1.8 1.2 2.4 27. 9 2 1.5 8.7 31.6 1.5 1 1 1.9 OGFRL1 opioid growth factor receptor-like 1 A_23_P779 1 1.2 1.1 1.4 1.4 2.1 11. 3 1.6 1.2 2.9 6.5 1.2 1.3 1.1 1.7 OLFM3 olfactomedin 3 A_24_P402 444 0.8 0.6 1 1.5 1 18. 5 1 1 2 19.1 1.7 0.9 1 1 PCDH7 protocadherin 7 isoform c precursor A_23_P378 364 0.7 0.7 1.1 1.3 1.2 15. 9 0.9 0.9 2.4 13.4 0.8 1 0.8 1.1 180 PEA15 phosphoprotein enriched in astrocytes 15 A_24_P250 964 1 1 1.2 1.2 1.5 3.8 1.6 1.4 2 4.9 1.1 1.2 1.1 1.1 PHLDB2 PHLDB2 protein A_24_P178 065 1.4 1 1.3 1.1 3 8 1 1.6 6.5 3.1 1.1 1 1.3 1.3 PHLDB2 pleckstrin homology-like domain, family B, member 2 A_24_P260 122 1.4 1.4 1.3 1.3 3.8 6.4 1.1 1.1 6.1 3.4 1.2 1.1 1.3 1.3 PLA2G4C phospholipase A2, group IVC A_24_P316 439 0.5 0.6 0.7 38. 1 3.9 48. 9 0.7 0.8 19 16.5 0.6 1 0.6 5.6 PLXDC2 plexin domain containing 2 precursor A_23_P161 424 0.7 0.6 1 1 1 7.5 1 1 1.2 17.5 1.1 1.1 1 1 PPP1R10 protein phosphatase 1, regulatory subunit 10 A_23_P156 667 1 1.1 1 1.3 1.3 4.9 1.3 1.2 2.6 2.7 0.9 1.1 0.9 1.2 PRICKLE 1 prickle-like 1 A_23_P408 285 1.1 0.9 1.2 1 2.1 8.6 0.8 1.1 2.8 5.9 1.2 1.2 0.9 1.4 PRR8 PRR8 protein A_23_P417 113 1.3 1.5 1.3 1.2 1.3 6.3 1.4 1.3 1.5 5 1.2 1.1 1 1.5 PSCD3 pleckstrin homology, Sec7 and coiled/coil domains 3 A_23_P111 593 0.9 1.1 2.6 1.1 1.4 3.9 1.4 1.4 2.1 4.8 0.8 0.9 0.9 1 PSORS1 C1 SEEK1 protein A_23_P133 902 1.1 1.5 1.6 1.4 2.5 5.1 1.4 1.4 5 2.9 1.4 1.1 0.9 1.1 PSORS1 C1 SEEK1 protein A_24_P248 48 1.3 1.6 1.1 1.2 2.1 5.2 1.4 1.4 3.9 3 1.1 1.1 2.1 1.2 PTPN14 protein tyrosine phosphatase, non-receptor type 14 A_23_P149 111 0.9 1 1.2 1 1.8 6.9 1.2 1.2 2.7 6.3 1.1 1 0.9 1.3 PTPN21 protein tyrosine phosphatase, non-receptor type 21 A_23_P770 31 1 1.1 1.4 1.2 1.7 7.2 1.2 1.2 2.8 5.9 1.2 1 1 1.2 RAI2 retinoic acid induced 2 A_23_P254 165 1.2 1.2 1.1 1.2 1.3 7.1 1.3 0.9 1.9 6.2 1.5 1.2 1.1 1.1 RANBP3L RAN binding protein 3-like A_23_P391 207 0.8 0.7 0.8 1.1 1.9 21. 5 1 0.9 3 10.4 1 0.9 0.6 1.5 RAPH1 Ras association and pleckstrin homology domains 1 isoform 1 A_24_P924 862 1 1 1.3 1.1 1.8 8 1.2 1.1 4.4 8.4 1.2 1.1 1.1 1.4 RAPH1 Ras association and pleckstrin homology domains 1 isoform 2 A_23_P409 966 1.6 1.2 1.8 1.4 3.9 19. 4 1.6 1.4 14.5 9.1 2.3 1.9 1.3 1.7 RASL10A RAS-related on chromosome 22 isoform b A_23_P166 400 1 1 1.2 1.1 1.4 6.1 1.1 1.2 4.4 3.6 1.1 1.2 1 1.2 RBMS3 RNA binding motif, single stranded interacting protein 3 isoform 2 A_24_P359 942 0.9 1 1.6 1.4 2.9 5.6 1.6 1 5.4 2.9 1.3 0.8 0.9 1.4 RND1 GTP-binding protein RHO6 A_23_P533 70 0.9 0.9 0.7 1 3.2 3.7 0.8 1.8 5.4 2.5 0.8 0.8 1 1.8 RNF180 ring finger protein 180 A_23_P377 996 1 1.2 1.2 1.5 1.3 6.5 1.1 1.1 2.2 5.4 1.2 1.4 1.3 1.4 RSRC2 arginine/serine-rich coiled-coil 2 isoform b A_23_P532 67 1.1 1.1 1.4 1.3 1.9 12. 5 1.3 1.3 4.5 10.5 1.2 1.1 1.2 1.6 RUNX1 AML1a protein A_24_P341 55 1.2 1 1.5 1.6 2.5 12. 6 1.2 1.2 6.3 5.8 1.5 1.4 1.1 1.7 SIPA1L2 signal-induced proliferation- associated 1 like 2 A_23_P137 470 1 0.8 1.4 1.2 1.7 7.7 1.1 1.2 3.8 3.7 1.3 1.1 0.9 1.8 SLC2A6 solute carrier family 2 (facilitated glucose transporter), member 6 A_23_P169 249 0.9 1 1.2 1.2 1.6 8.2 1.2 1.2 3.5 4.5 1.2 1.2 1.5 1.9 SLC39A9 solute carrier family 39 (zinc transporter), member 9 A_23_P380 326 1 0.9 0.8 0.9 0.9 3.7 0.8 0.8 1 5.4 0.8 0.8 0.9 1.1 SLC6A13 SLC6A13 protein A_24_P923 483 1.3 1.3 2 2.4 2.3 11. 7 2 1.6 9.7 3.9 1.7 1.7 1.6 1.7 SNX19 sorting nexin 19 A_24_P816 91 0.9 1.1 1.2 1.1 1.5 4.5 1 1.1 2.6 3.9 1 1 0.9 1.1 SOCS1 suppressor of cytokine signaling 1 A_24_P480 14 2.1 1.2 1.4 1.2 5.6 3.2 1.4 1.3 4.3 2.4 1.3 1.5 1.2 1.3 SOD2 SOD2 protein A_24_P935 819 4 1.3 5 2.7 21. 3 53. 4 2.3 6.7 45.3 20.5 2 1.5 2.4 10.8 SPACA3 sperm acrosome associated 3 A_23_P317 244 1.1 1.2 1.3 0.9 1 15. 3 1.5 1.4 1.8 12.1 1.3 1.8 1.1 1.9 SPHK1 sphingosine kinase 1 isoform 1 A_23_P381 06 1.1 1.2 1.1 1.1 1.5 5.5 1 1.1 2 5.3 1 1.2 1 1.2 SPINK1 serine protease inhibitor, Kazal type 1 A_23_P214 079 1.5 1.1 1.3 1.4 1.6 12 0.9 0.9 1.5 5.7 0.8 0.8 0.7 1.2 SPRR2C small proline-rich protein A_23_P126 089 0.6 1.4 1.1 1 0.9 18. 3 1.3 1.1 1.5 26.2 1.1 1.2 1 1.3 SPRR2D small proline-rich protein 2D A_23_P116 44 1.4 1.1 0.8 1.4 1.3 16. 7 1.2 1.2 1.8 24.3 0.8 1.5 0.9 1.7 STK19 serine/threonine kinase 19 isoform 2 A_23_P194 79 0.9 0.9 1.1 1.1 2.8 10. 6 1.1 1.2 6.4 4.2 1 1 1 1.8 STMN4 stathmin-like-protein RB3 A_23_P317 39 1.6 1.1 1.3 1.1 1.6 12. 7 0.9 1.1 1.2 14.7 1.2 1 0.7 2.1 TDGF1 teratocarcinoma-derived growth factor 1 A_23_P366 376 1.8 1 0.9 1.3 1.5 9.7 0.7 0.8 1 11.1 1.1 0.8 1.1 1.1 THAP2 THAP domain containing, apoptosis associated protein 2 A_23_P105 382 1.1 1 0.9 0.8 0.8 4.8 0.6 0.7 1.1 3.9 1.1 0.8 0.8 0.9 THRAP1 mediator of RNA polymerase II transcription, subunit 13 homolog A_24_P170 77 1.2 1.3 1.5 1.3 2 8 1.5 1.4 3.8 5.7 1.3 1.3 1.3 1.7 TKTL1 transketolase-like 1 A_23_P259 901 0.9 0.9 0.8 1 1.1 3.6 1.4 0.9 1.7 7.3 0.9 1 0.9 1.7 TMEM135 hypothetical protein LOC65084 A_24_P127 075 0.8 0.9 0.8 0.8 1 5.1 0.7 0.6 1.8 4.8 0.6 0.6 1.5 1 TMEM27 transmembrane protein 27 A_23_P339 84 1.4 2.2 0.9 0.9 1.6 8.9 0.8 0.8 3.1 7.8 0.9 0.8 0.7 1.6 TNC tenascin C (hexabrachion) A_23_P157 865 1.3 1.1 0.9 0.6 1.4 7.8 0.8 0.9 2.3 7.1 0.9 1.4 1.3 1.5 181 TNFRSF1 2A type I transmembrane protein Fn14 A_23_P493 38 0.9 1.1 1.2 1.2 1.6 11. 1 1.4 1.3 2.5 11.3 1.1 1.2 1.2 1.4 TSC22D2 TSC22 domain family 2 A_23_P922 30 1.1 1 1 0.8 1 4.8 0.8 0.8 1.7 3.5 1 1 0.8 1.1 TSPAN10 tetraspanin 10 A_24_P213 643 1.1 1 1.2 1.1 2.5 7.1 1.3 1.3 3.7 3.6 1.2 1.1 1.1 1.5 TTC26 tetratricopeptide repeat domain 26 A_23_P257 668 1 0.8 0.7 0.9 0.9 5.7 0.9 0.7 1.5 6.1 0.8 0.8 0.8 0.8 TUFT1 tuftelin 1 A_23_P371 824 0.8 0.9 1.2 1.1 2 4.5 0.9 1 2.3 5 1.2 1 0.9 1.1 UCP3 uncoupling protein 3 isoform UCP3S A_24_P292 470 1 1.2 1.8 1.4 1.7 9 1.6 1.7 5 4.2 1.5 1.3 1.1 1.4 VIP vasoactive intestinal peptide isoform 1 preproprotein A_23_P196 50 0.8 0.7 0.9 1.4 1.8 19. 7 0.9 1.2 1.6 27.2 1 0.9 1 1.6 VNN1 vanin 1 precursor A_23_P255 345 1.2 1.1 1 1.3 0.9 14. 1 0.7 1.2 2.4 12.1 1.4 0.7 0.5 3.7 VPS16 vacuolar protein sorting 16 isoform 2 A_23_P143 303 1 0.9 1 1.1 1.5 5.4 1.1 1.2 3.9 3.3 1 1.1 1 1.1 WBSCR1 9 hypothetical protein LOC285955 A_32_P180 435 1.4 1.2 1.6 1.9 2 9.8 1.4 1.5 4.4 5.9 1.1 1.4 1.1 1.7 WBSCR1 9 hypothetical protein LOC285955 A_32_P177 040 1.4 1.2 1.5 1.7 1.5 9.3 1.3 1.9 3.4 5.9 1.2 1.3 1.4 1.8 WDR42A WDR42A protein A_24_P767 25 1 1.3 1 1 1.1 5.9 1 1 1.5 5 0.9 0.9 1 1 WDR42A A_24_P886 960 0.9 1.1 1.2 1.1 1.7 10. 1 1 1.1 5.8 7 1.1 0.9 0.9 1.3 WDR66 WD repeat domain 66 A_24_P179 504 0.8 0.7 0.9 1 1.2 5.4 1 1 1.2 6.5 0.9 1.2 1.2 1.1 WNT4 wingless-type MMTV integration site family, member 4 precursor A_23_P382 607 1 1 1.2 1.2 1.9 8.7 1.2 1.1 3.4 3.2 1.3 1.2 1.1 1.5 XRN1 5'-3' exoribonuclease 1 isoform a A_23_P132 822 1.1 1.4 0.6 1.2 3.3 9.9 0.7 0.8 3.5 6.5 2.7 0.9 0.8 1.3 ZFP36 zinc finger protein 36, C3H type, homolog A_23_P392 37 1.2 1.1 1.3 1.4 6.4 12 1.2 2.4 15 4.1 1.1 1.2 1.3 2.2 ZFP36L2 butyrate response factor 2 A_23_P101 960 1.3 1.4 2.3 1.5 4.9 17. 6 2.1 2 19.4 10.6 1.8 1.3 1.1 2.3 ZNF121 zinc finger protein 121 A_24_P935 782 1 1.5 0.7 0.7 1.1 4.6 0.9 0.7 1.7 4.7 0.7 1.1 0.8 0.7 ZNF222 zinc finger protein 222 A_24_P165 082 0.9 1.1 0.8 0.9 1.1 4.5 0.8 0.9 1.6 5.6 0.9 0.9 0.9 1.1 ZNF222 zinc finger protein 222 A_23_P218 517 0.9 1.2 0.8 0.9 1.1 5 0.8 0.9 1.6 5.4 0.9 0.9 0.9 1.1 ZNF222 zinc finger protein 222 A_23_P125 042 0.9 1.1 0.7 0.4 1 4.2 0.6 0.7 1.2 4.9 0.6 0.8 0.8 1 ZNF256 zinc finger protein 256 A_23_P160 22 0.9 1.2 1 1 1.4 5 1.2 1.1 2.1 6 0.9 0.9 0.9 1.2 ZNF461 gonadotropin inducible transcription repressor 1 A_23_P674 24 1.1 1 1.3 1.2 1.5 5.7 1.2 1.2 2.9 4.7 1.1 1.1 1.1 1.2 ZNF462 zinc finger protein 462 A_23_P604 99 1.1 1.3 1.7 1.2 1.9 15. 7 1.3 1.3 3.5 9.7 1.3 1.1 1.1 1.6 ZNF503 zinc finger protein 503 A_23_P202 484 1.2 1.4 1.8 1.5 3.4 11. 4 1.8 1.5 8.2 5.5 1.5 1.5 1.2 2 ZNF585A zinc finger protein 585A A_23_P902 20 1 1.1 1 1.1 1.4 4.1 1 1 1.7 5.2 0.9 0.9 1 1.2 ZNF585A A_24_P849 467 1 2.9 0.9 0.9 1.9 11. 6 0.9 0.8 1.6 11.3 0.8 1 0.9 2.4 ZNF697 ZNF697 protein A_23_P322 076 0.9 1 1.9 1.9 1.9 8.2 1.4 1.3 2.5 6.1 1.5 1.2 1.5 1.4 ZNF704 A_24_P505 790 1 1 2.1 1.6 1.9 10. 3 1.7 1.5 4.6 5.5 1.6 1.4 1 1.9 ZNF8 zinc finger protein 8 A_23_P130 466 1.1 1.2 1.3 1.2 1.6 6.1 1.2 1.3 2.7 5.9 1.2 1 1.1 1.4 ZSCAN29 zinc finger protein 690 A_23_P100 092 1.1 1.4 1.6 1.3 1.6 6.5 1.5 1.4 2.7 4.8 1.4 1.3 1.2 1.5 colon cancer-associated antigen AgSK1-2HT-ECS A_32_P779 33 0.9 0.9 1.5 1.4 2.3 22. 3 1.4 1.3 7.7 14 1.3 1.3 1.3 2.7 colon cancer-associated antigen AgSK1-2HT-ECS A_23_P398 275 0.9 0.9 1.5 1.4 2.2 16. 1 1.4 1.3 5.6 10.5 1.4 1.4 1.3 2.3 FLJ00291 protein A_24_P932 648 1.1 1.2 1.2 1.3 1.6 7.6 1.3 1.7 3.5 7.3 1 1.1 1.2 1.8 GA repeat binding protein, beta 2 A_23_P732 08 1 1.1 1 1 0.9 6.6 1.1 0.9 1.1 6 1 0.8 0.8 1.5 golgin-like protein A_23_P106 371 1.3 1.2 1.7 1.3 1.6 9.7 1.7 1.3 4.1 5.7 1.5 1.1 1.2 1.1 HSPC080 A_24_P917 418 1.1 1 1.5 1 2.1 9.3 1.3 1.2 4.5 5.1 1.2 1.2 1 1.6 hypothetical protein LOC147744 A_23_P107 775 0.9 1.1 1.6 1.3 1.4 7.8 1.8 1.5 3.8 4.6 1.4 1 1 1.4 hypothetical protein LOC205860 A_23_P350 005 1 0.9 1.9 1.8 2 11 1.7 1.5 3.7 8.3 1.6 1.7 1.9 1.9 hypothetical protein LOC286006 A_24_P380 679 1.2 1.1 2.3 1.6 2.2 6.4 1.7 1.4 3.9 5.1 1.7 1.7 1.4 1.6 hypothetical protein LOC440321 A_24_P264 166 0.9 1.3 1.7 1.4 2 10. 1 1.3 1.5 4.1 6.6 1.3 1.1 1.2 1.2 hypothetical protein LOC54891 A_23_P142 756 1 1.3 1.3 1.1 1.7 9.2 1.4 1 4 9.2 1.2 0.8 0.8 2.1 hypothetical protein LOC80154 A_23_P129 103 0.9 0.9 1.4 1.4 1.8 8.2 1.3 1.2 3.8 5.8 1.2 1.2 1.2 1.9 KIAA1267 protein A_24_P316 381 1.2 1.4 1.9 1.4 1.8 5.8 1.9 1.7 4 4 1.3 1.3 1.2 1.2 KIAA1267 protein A_32_P210 106 1.1 1.3 1.8 1.9 2 6 1.8 1.8 3.9 4 1.3 1.3 1.4 1.2 KIAA1466 protein A_24_P324 581 1.8 2.1 2 1.3 6.3 8.8 1.7 1.3 6.5 3 1.5 1.4 1.6 1.4 182 KIAA1466 protein A_24_P324 577 1.6 1.9 2.2 1.6 4.6 7.2 2.1 1.6 5.5 3.2 1.9 1.4 1.4 1.5 LOC727751 protein A_23_P320 478 0.9 0.9 1.1 1.2 1.8 7.8 1.1 1.2 4 5.2 1.2 1.3 1.2 2 MGC26597 protein A_23_P389 426 0.7 1.3 0.9 1.2 2.1 5.9 1.1 1.1 2.8 4.7 1.1 1.2 0.7 1.2 Nedd4 binding protein 3 A_23_P587 47 0.7 1.2 1.4 1.4 1.8 14 1.6 1.4 5 13.3 1.2 1.3 1.1 2.5 PRO1995 A_24_P927 090 0.8 1 1.4 1.8 3.6 20. 3 1.7 1.7 9.6 7 1.4 1.6 1.3 2.8 RBP1-like protein A_24_P196 499 1.1 1.9 1 1.1 1.2 10. 1 0.9 1.2 3.1 11.7 1 0.9 1.1 1.4 T-cell activation NFKB-like protein A_23_P383 422 0.9 1.1 1 1.1 0.9 6.5 1.3 1.2 1.8 5.1 1.1 1 0.9 1.3 T-complex protein 1 A_23_P291 10 0.8 0.7 1 1.1 1.1 10. 8 1 1 3.5 5.5 1 0.9 0.8 1.1 testes development-related NYD-SP18 A_23_P134 204 5 1.2 1.1 0.9 1.2 8.2 1.2 1 2.4 6.5 1.4 1.5 1.2 1 A_32_P871 91 1.4 0.7 1.6 1.3 1.2 8.8 1.2 1.1 4 7.9 1.6 0.8 0.6 1.3 A_32_P802 95 1.5 1.3 1.5 1.4 2.1 12. 6 1.4 1.4 5.2 8 1.5 1.3 1.2 2.2 A_24_P399 220 1.3 1.5 1.8 1.6 2.3 11. 3 1.5 1.5 4.8 6.1 1.6 1.2 1.3 1.9 A_32_P356 4 1.2 1.3 1.4 1.5 2.6 7.2 1.6 1.6 3.5 5.1 1.5 1.3 1.2 1.7 A_24_P264 664 1.1 1.3 1.6 1.3 2.2 7 1.5 1.3 3.8 5.5 1.4 1.1 1.2 1.6 A_32_P226 205 1.2 1.6 2.1 1.3 3.2 31. 6 1.3 1.3 15.7 24.2 1.6 1.1 1 2 A_32_P106 925 1 1.3 1.7 1.6 2 11. 4 1.6 1.5 6.8 8.4 1.6 1.4 1.1 1.6 A_32_P614 39 1.3 1.3 1.5 1.2 1.4 12. 1 1.4 1.3 2.9 6.4 1.3 1.1 1.1 1.7 A_32_P209 250 1 1.1 1.2 1 1.3 5.6 1.1 1.1 1.9 4.3 1.1 1 0.9 1.3 A_23_P215 819 1 1 1.1 1.1 1.6 7.8 1.1 1.1 3.3 6 1.1 1 1 1.3 A_32_P126 647 1 1 1.3 1.2 1.3 6.3 1.5 1.2 3.1 5.2 1.1 1 0.9 1.2 A_32_P429 89 1.5 1.3 1.8 2.5 2.5 35. 4 1.4 1.5 8.7 20.2 1.4 1.7 1.3 4 A_32_P744 09 1 1 1.4 1.2 3.8 39. 1 1.1 1.5 6.8 28 1.4 1.4 1.5 4 A_32_P141 184 0.9 0.9 1.3 0.8 2 4.8 1 1 2.2 6 1.3 1 1 1.3 A_32_P592 77 0.9 1.4 2.9 1.9 4 46. 6 2.3 1.7 8.9 13.3 2.2 1.5 1.4 1.8 A_23_P381 979 0.9 1.2 2.2 1.4 1.8 10. 8 1.9 1.3 3.9 5.1 1.8 1.1 1.1 1.7 A_32_P239 2 1.1 1.5 2.3 1.8 2 8.4 2.1 1.7 4.5 5.9 1.9 1.4 1.4 1.3 A_32_P213 948 1.1 1.1 1.8 1.4 1.5 6.1 1.7 1.4 2.5 3.3 1.6 1.2 1.2 1.1 A_24_P272 225 1.1 1.2 1.3 1.1 1.5 5.1 1.3 1.3 2.4 3.2 1.2 1 1.1 1.2 A_24_P510 502 1 1.1 1.6 1.4 1.4 9.1 1.4 1.5 3.3 5.3 1.4 1.2 1.2 1.2 A_32_P199 923 1.1 1.1 1.7 1.1 1.4 9.9 2 1.3 4.5 4.4 1.7 1.3 1 1.5 A_32_P276 6 0.9 1.2 1.6 1.3 1.6 9.6 1.6 1.5 4.8 4.1 1.2 1 0.9 1.1 A_24_P110 141 1 0.9 1.4 1.2 1.5 4.7 1.1 1.4 2.9 3.7 1.1 1 1 1.1 A_23_P209 360 0.9 1.3 1.2 1.2 1.5 4.9 1.2 1.2 2.6 3 1.2 1.1 0.9 1.1 A_32_P234 853 1 1 0.9 1.1 0.9 4.5 1.2 1 2.4 4.4 0.9 1.1 0.9 1 A_24_P399 28 0.9 0.9 1.3 1.1 0.9 6.5 0.9 1.1 2 3.1 0.8 1.1 0.9 1.1 A_32_P128 209 1.1 1.1 1.1 1.2 1.1 6.9 1.1 1.1 2 4 1 1 1.1 1 A_24_P885 873 1 1 0.7 0.9 1.1 9.2 1.1 1.5 3.2 3.6 1 1 1 0.9 A_24_P914 043 0.9 1.1 1 1.4 1.2 19. 8 1 1.4 5 14.6 1.2 1.5 2.1 2.9 A_32_P113 472 0.9 1.1 1.1 1 1.7 6.1 1.1 1.1 2.3 3.8 1.1 0.8 0.7 1.3 A_24_P924 697 1 1.1 1.2 1 1.7 6.6 1.1 1 1.9 4 1.3 0.8 0.7 1.4 A_24_P919 150 0.9 0.7 1.2 1.4 2 10. 5 1.7 1.6 4.3 4.2 1.3 0.9 1.2 1.3 A_24_P718 503 1 1 1.5 1.4 1.7 14. 6 1.6 1.4 3.7 4.4 1.6 1.3 1.1 1.3 A_32_P138 977 1 1.3 1.3 1.6 1.1 9.8 0.9 1 4.8 4.7 1.2 1.2 0.6 1.5 A_32_P644 2 0.9 1 1.6 1.2 2.6 10 1.6 1.5 7.2 3.3 1.4 1.2 1.2 2.4 A_32_P207 802 1 1.2 3.1 2.4 4.8 17. 4 2.6 2 14 5.5 2.1 1.6 1.7 2.5 A_32_P232 883 1 1 1.3 1.4 2.6 10. 5 1.2 1.1 5.2 5.3 1.2 1.2 1.5 1.5 A_32_P224 345 0.8 0.7 0.9 1.1 10. 8 169 .9 1 1 56.1 31.3 1 0.9 1 4.2 A_32_P135 65 1 1 1.2 1.1 3.1 9.5 1.3 1.1 7.3 4.7 1.4 1.2 1.2 1.9 183 A_32_P132 928 1.2 1 2.3 1.5 3.7 23. 5 2.1 1.6 13.2 8 1.9 1.2 1.6 3 A_24_P332 081 1 1.2 2 1.7 3.2 18. 9 1.8 1.7 10.5 8.2 1.7 1.4 1.5 2.4 A_24_P788 878 1 1.4 1.4 1.3 3 15. 4 1.1 1.2 9.2 6.7 1.2 1.1 0.8 2.1 A_23_P812 98 0.8 1 1.1 1 1.8 4.2 1.1 1 3.7 2.6 1 0.8 0.7 1.6 A_24_P323 522 0.8 1 2 1.7 3.9 19. 6 2 1.7 17.3 7.9 1.4 1.2 1.1 2.4 A_23_P335 398 0.9 0.9 1.3 1.1 2 18. 2 0.9 1 6.8 8.5 0.9 0.6 1.3 1.4 A_32_P387 82 1 1.2 2.2 1.3 2.9 12. 4 1.6 1.6 7.9 5.5 1.9 1.4 1.2 1.8 A_23_P984 83 0.9 1.3 1.5 1.2 1.8 6.2 1.3 1.4 4.1 3.1 1.4 1.1 1.1 1.5 A_24_P585 428 0.8 1.3 1.3 1 1.8 5.2 1.2 1.3 5.1 2.7 1.3 0.9 1 1.2 A_32_P127 501 1.1 1.5 2.8 1.9 3 21. 5 2.5 2 12.9 10.4 2.1 1.3 1.2 1.7 A_32_P110 016 0.9 0.9 1.3 1.2 1.4 4.7 1.2 1.4 3.7 2.4 1.3 0.9 0.7 1 A_32_P221 641 1 2.6 2.7 2.3 5.1 29 2.9 1.7 10.9 9 4.5 1.7 1.8 2.6 A_24_P767 972 1.2 1.3 1.1 1.5 3.1 14. 4 1.7 1.3 8 4.6 1.7 1.4 1 1.4 A_32_P184 039 0.9 1.5 4.7 2.5 13. 6 35. 4 2.9 4 33.1 17.6 3.1 1.4 1.6 5.7 A_24_P102 283 1.3 1.5 2.7 1.8 6.2 18. 1 3.5 3 13.4 10 2.5 0.9 1.2 3.8 A_32_P165 823 1.3 1.1 1.8 1.3 2.9 5.9 2.1 1.9 6 5.6 1.3 1.3 1.4 2.1 A_32_P173 955 1.2 1.5 1.4 1.2 2 7.7 2.1 1.7 4.7 3 1.3 1 1.2 1.8 A_32_P463 538 1 0.8 1.1 1.1 1.5 8 0.9 1.2 3.2 3.1 0.8 1.1 1.9 1.3 A_24_P147 76 1.8 0.7 1 1.2 1.6 10. 3 1 1.5 4.3 7.6 1.2 0.9 2.6 1.5 A_32_P677 8 2 1.3 1.2 1.4 2.3 5.2 1.1 1.3 3.7 5.5 1.9 1 2 1.1 A_32_P125 803 1.6 1.6 1.1 0.9 1.3 6 1 1.3 2.7 4.1 1.6 1 1 1.2 A_32_P104 448 1.5 1.3 0.7 0.8 1.4 5.9 0.8 1.2 3.1 6.1 1 0.9 0.9 0.9 A_32_P739 91 0.8 0.7 1.2 1.1 0.8 12. 4 1.4 0.7 1.2 18.3 0.8 1 0.9 1.4 A_32_P220 463 1.1 1.3 1.3 1.4 1.6 6.2 1.5 1.6 1.9 7.2 1.1 1.5 1.2 1.5 A_32_P216 015 0.8 0.7 1 1.1 1.2 5.8 1 1 1.7 7.4 1.1 1 1 1.1 A_32_P593 02 1.3 1.7 1.3 1 1.4 9.4 1.4 1.1 2.2 6.6 1.1 1.1 1.1 1 A_32_P150 81 1.2 1.4 1.1 1 1.3 13. 2 1 1.2 2.2 9.5 1.1 1.2 1 1.5 A_23_P218 1 1 1 1 1 1.1 5.7 0.9 0.9 1.9 5.2 1 1.1 0.9 1 A_32_P518 55 0.9 1.2 1 1 0.9 9.3 1 1.2 2.2 5.7 1.1 0.9 0.8 1.2 A_32_P308 91 1.4 1.1 1.3 1.7 1.5 15. 5 1.8 1.8 3.1 10.9 1.4 2 1.2 1.4 A_32_P206 208 0.9 0.9 1.3 1.1 0.9 6.7 1.3 1.2 1.8 5.1 1.1 1.3 1 1 A_24_P811 397 0.8 0.7 1 1.1 1 35. 8 1 0.9 3 19.9 1.1 0.9 0.9 1.1 A_24_P892 464 0.9 1.1 1.1 1.1 1.6 17. 6 1.5 1.3 3.5 17.5 1.4 1.3 1.2 1.8 A_23_P546 4 1.1 1.1 1.1 1.1 1.3 7.9 1.2 1.2 2.1 6.9 1.1 1.1 1.2 1.5 A_32_P107 6 0.8 0.7 0.8 0.9 1.4 8.2 0.8 0.9 2.2 8.7 1.1 0.7 1 0.9 A_23_P427 68 0.9 1 0.9 1.4 1.5 6.7 1.1 1 1.6 6.7 1.1 1 1.1 1.1 A_32_P170 454 0.9 1.2 1.4 1.4 2.3 7.4 1.3 1.3 2.5 8.3 1.2 1.2 1 1.2 A_32_P156 786 1 1.2 1.2 1.3 2.3 5.7 1.1 1.1 2.2 6 1.2 1.2 1.1 1 A_24_P941 279 0.7 1.1 1.1 1.1 1.7 5.6 1.4 1.1 2.4 5.7 1 1.2 1 1.4 A_24_P882 959 1.3 1.1 0.7 1 1.2 7.8 0.9 1.3 1.3 12 0.7 1 0.7 1.1 A_24_P337 143 1 0.9 0.8 0.8 1.4 6.2 0.7 1.2 1.4 9.5 0.6 0.6 1 1.2 A_32_P147 21 1.7 1.4 1.2 1.8 2 13. 4 1.9 1.4 1.6 9.8 1.3 2.3 1.4 1.4 A_24_P526 190 2.2 1.1 1 1.4 1.2 24. 5 1.2 0.9 3.4 17 1.3 0.9 1.6 2.3 A_23_P413 268 1.3 1 1.1 0.8 1.2 6.5 1.2 1 2.1 3.9 1 1.2 1.6 1.2 A_24_P823 514 1.7 1 1.4 1.2 1.8 4.5 1.2 1.1 2 4 1.3 1.1 0.9 1.1 A_24_P950 70 1 1.4 0.9 1 0.9 5.2 0.7 0.7 1.1 3.1 1 0.8 0.6 1.3 A_32_P174 285 0.9 1.6 1.3 0.9 0.9 8.4 0.9 0.9 3.6 9.5 0.9 1 1 1.6 A_32_P192 354 0.9 1 1.2 1.1 1.7 3.6 1.5 0.8 2.5 4.9 1.2 1.3 1.1 1.2 A_32_P116 957 1.2 1.1 1 1.1 1.1 5 1 1 2.6 10.5 1 1.1 1.1 1.9 184 A_23_P355 46 0.7 0.7 0.9 1.2 1.4 13. 1 1 1 6.1 18.5 0.8 0.8 0.8 2.1 A_24_P772 488 0.7 0.6 0.9 1 1 4.4 1.1 0.9 2.8 6.9 1 0.8 1 1 A_23_P356 965 0.9 1.7 1.6 1.1 1.3 4.5 1.4 1.2 2.2 4.7 1.3 1 1 0.9 A_24_P212 875 0.8 1.2 0.7 0.8 1.8 4.4 0.8 1.2 1.3 4.8 0.8 0.6 0.9 0.7 A_32_P882 5 1.4 0.9 0.9 1.4 3.6 10. 6 1.2 1.2 9.6 7.2 1.3 1.2 0.7 2.9 A_32_P213 002 2.1 2.6 2.3 1.9 7 13. 7 2.3 2.1 13.4 5.9 1.8 1.1 1.5 2.6 A_32_P102 979 1.3 1.2 1.4 1.3 4 5.7 1.5 1.6 9.1 3 1.4 1 1.1 1.4 A_32_P210 038 1.2 1.2 1.6 2.5 11. 7 16. 7 1.7 1.7 16.3 9.5 1.3 1.1 1.4 5.1 A_32_P143 637 1.1 1.1 1.8 1.7 6.5 22. 7 1.3 1.1 11.7 5.4 1.2 0.8 1.2 2.6 A_24_P367 454 0.9 1.6 1.7 2.2 6.4 25. 9 0.9 2.3 22.6 12.9 1.4 0.9 1 4.5 A_32_P111 919 0.7 1.2 1.3 1.6 3.4 10. 9 1.4 1.8 10.3 4.2 1.1 1.4 2.3 2.2 A_32_P201 976 1.4 1 2.3 1.8 3.9 7.8 2.3 2.5 5.8 4.3 1.3 1.2 0.8 1.3 A_24_P252 739 1.3 1 1.5 1.3 3.3 8.3 1.6 2.1 6 4.8 1 1 0.9 1.2 A_32_P139 815 1.2 1 1.5 1.2 1.9 4.1 1.2 2 2.8 2.1 1.1 1.1 1 0.7 A_24_P230 972 1 1.6 1.4 1.2 3.7 4.8 1.3 1.7 7 2 1.7 0.9 1.3 2.7 A_32_P233 405 1.5 2 1.5 1.1 2.6 5.1 1.2 1.5 4.7 3.1 1 1 1.1 1.2 A_24_P924 010 3.6 5.9 1.2 1.4 10. 4 11. 6 1.6 2.2 15.6 9.3 1.3 1.6 1.2 1.3 A_32_P226 556 1.2 2.8 1.9 1.6 2.3 9.5 1.5 1.3 6.9 3.2 1.7 1.4 0.9 1.8 A_23_P653 5 0.9 3.9 1.2 1.6 6.4 20. 8 1.2 0.7 18.8 14.4 1.1 1.4 2 12.6 A_24_P126 406 0.4 0.5 0.9 1.1 18. 2 5.7 0.8 0.8 1.4 7 0.9 1.4 1.4 0.9 D ATP4A ATPase, H+/K+ exchanging, alpha polypeptide A_23_P430 728 0.8 0.9 0.8 5.2 48. 5 150 .5 1.9 13.4 86.6 88 1 2.7 13.5 45.9 CCL2 small inducible cytokine A2 precursor A_23_P894 31 1.9 1.2 0.9 1 5.7 3.5 0.9 1.4 6.5 4.3 1 1 1.1 5 CCL5 small inducible cytokine A5 precursor A_23_P152 838 1.6 12 0.9 19. 6 33. 6 125 .7 5.3 21.8 55.5 90.3 1.8 17.5 23 29.9 CH25H cholesterol 25-hydroxylase A_23_P864 70 1.9 0.9 0.9 6.3 88 8.3 391 9.7 0.9 18.1 2793. 6 2285 .9 1.4 5 31.7 813. 7 CX3CL1 chemokine (C-X3-C motif) ligand 1 A_23_P377 27 1.7 2.4 1.2 1.7 16. 2 27. 8 0.7 2.9 18.8 10.1 1.4 1.6 2.2 19.7 GPR109B G protein-coupled receptor 109B A_23_P647 21 1.6 2.3 1.5 2 85. 5 226 .6 1.3 3.1 37.2 70.3 1.5 4.1 10.2 42.3 IFNB1 interferon, beta 1, fibroblast A_23_P717 74 0.7 3 1.1 13. 3 24 3 627 .9 1 66.2 353.3 321. 6 0.9 9.3 31.7 180. 6 IL28A interleukin 28A A_23_P409 438 1.3 6.3 2.4 21. 9 40. 1 89. 5 6.1 25.5 73.6 65 2.1 14.8 21 32.2 IL6 interleukin 6 (interferon, beta 2) A_23_P710 37 1.6 1.5 1 3.1 55. 5 158 .4 0.8 12.5 71.9 90.7 1 1.8 3.6 34 E ADAD2 adenosine deaminase domain containing 2 A_23_P491 36 1.2 0.9 1.4 1.5 1 14. 3 1.3 1.4 1.6 2.4 1.3 1.1 1.2 1.4 CALCB calcitonin-related polypeptide, beta A_24_P198 178 0.9 0.8 0.7 1 1.3 4.5 0.6 0.8 0.9 5.2 0.7 1.2 1.4 0.9 COL11A2 collagen, type XI, alpha 2 isoform 1 preproprotein A_23_P423 22 1.1 1.1 4 1.7 1.1 14 1.7 1.5 2.4 13.3 1.6 1.3 1.1 1.3 DACT2 dapper, antagonist of beta- catenin, homolog 2 A_24_P289 260 0.9 1.1 0.7 0.5 0.8 2.3 0.8 0.7 2.3 4.6 0.7 1.1 0.8 0.7 FEZ1 zygin 1 isoform 1 A_23_P202 881 1.5 0.9 0.8 0.8 0.9 5.6 0.6 0.8 1.2 4.1 0.8 1.7 1.4 1.3 KIF25 kinesin family member 25 isoform 1 A_23_P594 10 1.6 1.7 1.3 1.8 1.4 5.5 0.8 1.9 1.6 9.7 1.1 1.8 1.8 0.6 LY6K lymphocyte antigen 6 complex, locus K A_32_P138 348 0.9 1 1 1.1 0.8 3.6 1.2 1.1 1 5 1 1 0.8 0.9 LY6K lymphocyte antigen 6 complex, locus K A_23_P397 293 0.8 1 1.3 1.1 0.8 4.1 1.3 1.1 1.3 4.8 1.1 1 0.8 0.9 OXTR oxytocin receptor A_23_P132 619 0.8 0.8 1.5 1.2 0.8 3.4 1.3 1.2 1 5 1.5 1.1 0.7 0.9 RBM35A RNA binding motif protein 35A isoform 1 A_23_P259 127 1.4 1.4 1 1.1 0.8 1.2 0.7 1.1 1.2 5.6 0.9 0.9 0.8 1.6 RERG RAS-like, estrogen-regulated, growth inhibitor A_23_P204 296 1 0.6 1.3 0.7 0.9 9.8 0.8 1.1 0.7 2.3 1.3 1.4 1 0.7 SIRT4 sirtuin 4 A_23_P992 26 0.8 0.8 1 0.8 0.8 3.2 0.5 0.8 0.9 4.6 0.6 0.9 1 0.9 SLC22A1 solute carrier family 22 member 1 isoform b A_23_P145 569 1 0.9 0.9 1.1 0.8 1.7 1.3 1 0.9 4.1 0.8 1.1 1.2 1 SRD5A2 3-oxo-5 alpha-steroid 4- dehydrogenase 2 A_23_P281 86 0.9 1 1 1.8 1.7 6.4 0.6 1.8 1.3 5.5 1.2 1.6 1.6 0.7 TMEM16F transmembrane protein 16F A_23_P536 41 1.1 1.1 0.8 0.9 1.1 4.1 0.7 1 0.8 5.5 0.6 1 1.1 0.8 UPK2 uroplakin 2 A_23_P642 38 1.4 0.9 1.1 1.1 1.2 6.1 1.2 1 1.7 2.3 1.3 1.1 1 1.1 ZNF396 zinc finger protein A_24_P393 565 1.2 1.3 1.1 0.9 1.8 5.3 0.8 1.3 2.3 4.2 0.8 1.6 1.7 1.2 185 ZNF545 zinc finger protein 545 A_23_P416 813 1.1 1.3 0.7 0.5 1.1 1.8 0.9 0.6 1.3 5.3 0.7 1.2 1.2 0.9 A_32_P834 726 0.8 1.2 1.3 1.7 1.1 8.7 0.7 1.5 1.4 3.9 1.5 2.3 0.9 0.9 A_23_P931 09 0.7 0.7 1 0.9 0.5 4.4 0.9 0.9 0.5 6 1.4 1 0.9 0.8 A_23_P247 1.6 0.9 0.9 1.1 0.7 7.3 1 0.7 0.6 19.3 0.9 0.7 1.1 0.9 A_24_P570 583 1 1 1 0.9 1.2 2.2 1 1 1.3 4.9 1 0.9 0.9 0.9 A_23_P637 36 0.9 0.9 1 0.9 0.9 2.4 0.7 1.1 1.2 6.9 0.8 1 1.2 0.8 A_32_P523 86 0.9 1.4 1.5 2.7 1.5 5.8 2.1 1.4 2.3 3.4 1.5 2 1.9 1.1 A_32_P158 019 1.4 2.3 1.1 0.9 2.1 2 1 0.8 1.5 6.3 1 0.9 0.6 0.8 F APOL1 apolipoprotein L1 isoform b precursor A_24_P879 31 6.7 7.6 1.8 1.7 19. 9 38 1.6 2.3 31.5 28.2 1.6 1.7 4.2 8.1 APOL2 apolipoprotein L2 A_23_P211 488 5.6 2.5 1 1.4 14. 9 25. 7 1 1.8 20.6 16.8 1 1.3 3.6 6 APOL4 apolipoprotein L4 isoform 1 A_23_P380 857 6.2 7.9 1.5 2.6 14. 3 22. 5 1.5 2.5 18.2 15 1.4 1.7 5.1 7.2 APOL6 apolipoprotein L, 6 A_24_P941 167 8.6 8.3 1.5 1.9 14. 2 9.8 1.4 2.6 16.7 8.5 1.5 1.7 6.3 9 APOL6 apolipoprotein L6 A_23_P155 049 8.1 6.8 1.2 1.6 11. 8 8.3 0.9 1.2 9.5 8.5 1.1 0.9 4.3 8.5 APOL6 A_24_P759 4 7.3 10.3 0.8 2.7 19. 2 10. 2 0.6 6.3 17.4 6.9 0.9 1.5 6.8 11.3 B2M beta-2-microglobulin precursor A_23_P374 41 1.7 5.5 0.6 0.8 2.8 12. 6 0.6 0.9 3.6 12.7 0.7 0.7 1.4 4.6 BATF2 basic leucine zipper transcription factor, ATF-like 2 A_23_P370 682 8.8 14.8 0.9 1.6 24. 3 17. 4 1.2 1.7 20.3 9 0.9 1.1 3.3 8.3 BST2 bone marrow stromal cell antigen 2 A_23_P394 65 8.2 21.5 1.1 1.9 16. 2 24. 8 1.5 2.5 18 20.9 1.3 2.6 3.5 10.6 BTC betacellulin A_23_P135 722 2.3 5 1.7 1.5 3.5 1.9 1.4 1.5 3.8 2.2 1.5 1.4 1.4 2.7 C1orf38 basement membrane-induced gene isoform 1 A_24_P857 75 14 14.8 0.7 1.3 27. 2 7.8 1.5 1.3 16.8 7.3 0.9 0.7 1.5 2.7 C1orf38 basement membrane-induced gene isoform 1 A_23_P873 14.2 16.6 1.2 2.4 40. 9 15. 5 1.5 3.4 27.7 10.2 1.2 1.5 2.1 3.6 CA13 carbonic anhydrase XIII A_23_P381 714 5.4 2.3 1 1.2 10. 5 22 1.2 0.9 7.9 15.7 1.1 1.3 4.5 3.7 CASP1 caspase 1 isoform alpha precursor A_23_P202 978 6.2 46.2 0.9 1.1 27. 6 7.8 0.8 1.1 17.8 23 0.8 0.9 6.3 17.1 CD274 CD274 antigen A_23_P338 479 3.6 2.7 0.8 0.9 25. 7 38. 6 0.9 1.1 17 12.6 0.8 1 2 8.2 CD38 CD38 antigen A_23_P167 328 1.4 6 0.8 0.9 4.7 13. 5 0.7 0.9 3.7 6.5 0.8 1.1 1.3 5.4 CEACAM 1 carcinoembryonic antigen- related cell adhesion molecule 1 isoform 1 precursor A_24_P382 319 20.4 15.5 0.8 2.6 49. 7 12. 8 1 3.3 44.3 7.9 1.3 1.7 6.4 13.6 CEACAM 1 carcinoembryonic antigen- related cell adhesion molecule 1 isoform 2 precursor A_23_P434 118 2.8 4.6 1.4 1.1 8.9 15. 5 0.7 1 10 7.5 0.9 1 3 7.9 CFB complement factor B preproprotein A_23_P156 687 3 7.9 0.7 0.9 26. 2 38. 5 0.8 1.8 42.5 15.6 0.6 0.8 1.7 6.2 CTSS cathepsin S preproprotein A_23_P461 41 2.1 4.4 0.9 1 2.7 3.7 0.9 0.9 1.9 3.4 0.9 1.1 1.4 3.3 CXCL11 small inducible cytokine B11 precursor A_23_P125 278 2.9 10 1 1.9 14 7.8 643 .8 1 9.4 321.1 284. 5 1.1 2.4 10.1 96 CXorf43 hypothetical protein LOC139324 A_23_P386 364 2.8 3.7 0.5 0.6 10 33. 1 0.3 0.6 8.3 22.3 0.6 0.8 1.8 4.7 DDX58 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide RIG-I A_23_P208 14 28.7 32 1.3 2.9 83. 4 583 .7 1.6 5.7 195 315. 1 1.3 1.6 4.1 27.3 DNAH3 dynein, axonemal, heavy polypeptide 3 A_24_P419 286 2 1.8 0.7 1.7 7.7 2.9 1 1.6 4.2 1.9 0.9 0.7 0.7 0.9 DTX3L deltex 3-like A_24_P941 912 6.1 7.1 0.9 1.5 7 9.4 0.7 1.7 7.6 6.3 0.8 1 2.7 4 DTX3L deltex 3-like A_23_P347 040 9.8 8.6 0.9 1.3 11. 6 21. 6 0.8 1.3 9.1 15 0.9 1 2.1 4.1 EIF2AK2 eukaryotic translation initiation factor 2-alpha kinase 2 A_23_P142 750 2.7 5.5 1.3 1.2 4.4 5.5 1.4 1.5 4.6 3.5 1.1 1.1 1.1 1.5 EMR1 egf-like module containing, mucin-like, hormone receptor- like sequence 1 A_23_P275 56 1.3 5.2 0.6 0.7 2.8 2.5 0.8 0.9 1.9 3.3 0.7 1.1 1.4 2.9 EXOC3L exocyst complex component 3-like A_23_P424 734 1.3 4.5 1.2 1.3 9.7 11. 1 1.1 1.2 8.1 5.8 0.9 1.3 1.8 4.9 FAM46A hypothetical protein LOC55603 A_23_P706 60 3 2.4 1.5 1.5 8.8 13. 8 1.2 1.6 14.4 10.2 1.4 1.5 1.9 2.7 GBP1 guanylate binding protein 1, interferon-inducible, 67kD A_32_P107 372 48 41.4 1.2 1.9 12 8.6 340 .5 1.5 1.7 136 90.2 1 1.3 18.1 70.7 GBP1 guanylate binding protein 1, interferon-inducible, 67kD A_23_P628 90 57.4 82.6 1 1.7 50. 8 426 .7 1 2.7 122.2 140. 5 1.1 1 14.3 64.2 GBP3 guanylate binding protein 3 A_24_P370 702 8.4 11.1 1.2 1.5 20. 9 12. 9 1.2 1.6 17.9 10 1.2 1.1 6.2 14.4 GBP3 guanylate binding protein 3 A_23_P514 87 10.8 17.2 0.4 0.9 23. 4 13 0.6 0.8 19.1 10.7 0.6 0.7 8.4 17.6 GBP4 guanylate binding protein 4 A_24_P454 46 7.2 20.1 1 1.2 39. 5 147 1.2 1.6 45.4 73.6 0.7 1.3 3.4 32.7 186 GBP4 guanylate binding protein 4 A_23_P103 496 49.2 143. 4 0.8 2.3 22 5.8 303 .3 1 4.5 260.1 197. 8 1.4 1 17.9 187. 6 GBP5 guanylate-binding protein 5 A_23_P742 90 25.7 20.1 0.7 2.4 93. 8 256 .7 1.4 3.4 140.5 160 1 1.3 16.5 88.5 GMPR guanosine monophosphate reductase A_24_P277 657 1.4 6.6 1.1 0.5 3.7 1.3 0.8 0.7 2.5 2.6 0.9 1 1 0.8 HERC5 hect domain and RLD 5 A_23_P110 196 4.3 21.4 0.7 1.5 61. 6 420 .3 0.8 3.5 147.3 261 0.7 1.5 4.1 40 HLA-B major histocompatibility complex, class I, B A_23_P125 107 1.5 5.2 0.7 0.8 4.1 18. 4 0.7 0.9 5.1 10.7 0.8 0.9 1.2 4.2 HLA-B major histocompatibility complex, class I, B A_24_P113 674 1.6 4.3 0.8 0.9 4 16. 2 0.8 1 5.2 10.4 1 0.9 1.2 3.9 HLA-C major histocompatibility complex, class I, C precursor A_24_P298 409 1.6 4.6 1.1 1.1 3.3 15. 2 1 1.2 5 11 1.1 1.1 1.3 3.6 HLA-C MHC class I antigen A_23_P959 17 1.5 4.6 0.8 0.9 3.7 13. 9 0.7 0.9 4.4 8.8 0.9 0.9 1.3 3.5 HLA-C A_23_P113 716 1.5 4.3 1.1 1.1 3.5 16 1 1.2 5 11.5 1.1 1.1 1.3 3.5 HLA-E major histocompatibility complex, class I, E precursor A_24_P326 082 2.2 4.2 0.8 0.8 3.4 11. 1 0.8 0.9 3.4 6.7 0.8 0.8 1.1 2.8 HLA-E major histocompatibility complex, class I, E precursor A_32_P460 973 2.5 4.3 1 1 3.7 5.1 1 1.1 3.4 4.5 1 1 1.4 2.5 HLA-F major histocompatibility complex, class I, F precursor A_23_P314 024 1.6 5.2 0.9 0.9 5.9 28. 2 0.9 1 7 14 0.9 1 1.3 5.7 HRASLS2 HRAS-like suppressor 2 A_23_P105 012 2.1 9.8 1.2 1.3 11. 3 13. 1 1.1 1.4 9.7 11.1 0.8 1.2 1.9 7.3 HRASLS2 HRAS-like suppressor 2 A_24_P364 263 2 6.2 0.9 1 4.3 6.6 1.1 1.1 3.6 6.2 1.1 1.1 1.4 4.2 HSH2D hematopoietic SH2 domain containing A_23_P153 372 6 10.6 1 1.9 20 33. 8 1 1.6 23.1 11.3 1 1.2 1.5 3.7 IBRDC3 IBR domain containing 3 A_23_P321 388 1.6 1.8 1.5 1.5 4.8 2.2 1.6 1.4 4 2 1.1 1.1 1 2.2 IFI16 interferon, gamma-inducible protein 16 A_23_P160 025 11.9 19.3 1.1 1.6 17. 1 20. 7 1 2 12.4 9.4 1 1 1.8 2.9 IFI16 A_23_P217 866 11.3 16.7 1.2 1.6 16. 3 19. 3 1.1 1.8 12.2 9.2 1.2 1.1 1.6 2.6 IFI27 interferon, alpha-inducible protein 27 A_24_P270 460 9.8 12.5 1.2 1.5 17. 7 32. 5 1.3 4.3 22.6 21.1 1 1.3 2.4 11.3 IFI27 interferon, alpha-inducible protein 27 A_23_P485 13 12.4 20.8 1.2 1.8 25. 6 59. 3 1.5 3.1 34 34.8 1.1 1.1 2.9 12.3 IFI35 interferon-induced protein 35 A_23_P152 782 2.4 7.8 0.7 0.9 6.3 3.7 0.8 1.1 5.7 3.3 0.8 0.8 1.5 2.1 IFI44 interferon-induced, hepatitis C- associated microtubular aggregate protein A_23_P230 74 50.7 65.1 1 12. 2 88. 7 173 .6 1.4 21 128.9 111. 9 1 7 16.1 55.2 IFI44L histocompatibility 28 A_23_P458 71 24.6 78.5 0.9 1.2 79. 7 172 .2 1.1 3 127.3 95.2 1 0.9 2.2 24.5 IFI6 interferon, alpha-inducible protein 6 isoform c A_23_P201 459 14.3 45.5 1.2 2.8 37. 6 56. 4 1.7 4.8 43.3 43.7 1 1.4 2.7 10.4 IFIH1 interferon induced with helicase C domain 1 A_23_P681 55 16.7 30 1.1 4.3 79. 8 288 .2 2 8.7 150.9 191. 8 1.2 3.3 8 53.8 IFIT1 interferon-induced protein with tetratricopeptide repeats 1 isoform 2 A_23_P522 66 32.1 35.9 5.6 24. 4 58 121 .2 16.2 35.2 94.6 76.7 4.8 14.5 11.2 33.2 IFIT2 interferon-induced protein with tetratricopeptide repeats 2 A_23_P240 04 72.6 85.4 2.4 20. 5 92 6.4 610 0.4 11.9 81.2 2569. 2 3777 .9 2.9 14.5 42.9 531 IFIT2 interferon-induced protein with tetratricopeptide repeats 2 A_24_P304 071 76.7 110. 6 1 17. 1 34 0.4 714 .3 3.4 46.5 411.3 548. 7 1.4 14.9 27.5 327 IFIT3 interferon-induced protein with tetratricopeptide repeats 3 A_23_P354 12 81.5 108. 1 0.9 7.3 22 6.3 421 .9 2.2 21.2 353.2 224. 7 0.8 4.1 15.8 73.4 IFIT5 interferon-induced protein with tetratricopeptide repeats 5 A_24_P301 94 6.1 6.4 1.1 1.3 8.3 24. 6 1.1 1.7 10.6 13.5 1.1 1.1 1.5 4.3 IFIT5 interferon-induced protein with tetratricopeptide repeats 5 A_23_P636 68 5.4 5.5 1.2 1.5 8.3 26 1.2 1.8 12.2 14.8 1.3 1.3 1.5 3.8 IFITM1 interferon induced transmembrane protein 1 (9-27) A_23_P727 37 6.7 8 0.8 2.3 10 9.8 1.3 3.6 13.4 9.9 0.9 1.1 2.3 2.2 IFITM2 interferon induced transmembrane protein 2 (1-8D) A_24_P287 043 1.8 5.4 1.1 1 3.6 1.9 1.2 1.1 4 2.6 1 1 0.8 0.7 IFITM3 interferon-induced transmembrane protein 3 (1-8U) A_23_P875 45 2.6 9.7 1.1 1.1 6.4 5.9 1.3 1.4 8.1 6.8 1 1 1 1.2 IFITM4P A_24_P161 24 2.2 11.5 0.9 0.9 4.4 2.5 1.1 1.1 4.9 4.1 1 0.9 0.8 0.9 IL4I1 interleukin 4 induced 1 isoform 2 A_23_P502 520 2.5 7.9 1.4 1.3 5.9 16. 5 1.1 1.4 7 11.2 1.3 1 1.3 6.2 INDO indoleamine-pyrrole 2,3 dioxygenase A_23_P112 026 10.6 39.4 0.9 1.1 87 110 .2 1 1.6 93.1 89.3 1.1 1 21.3 71 IRF1 interferon regulatory factor 1 A_23_P417 65 4.8 2.2 0.9 1.8 16. 1 22. 4 0.9 2.2 18.3 16.7 0.9 2 3.6 8.5 IRF7 interferon regulatory factor 7 isoform d A_24_P378 019 8.4 13.6 1 3.9 17. 7 25. 2 1.3 4.9 22.2 13.9 1 1.3 2 3.7 ISG15 interferon, alpha-inducible protein (clone IFI-15K) A_23_P819 13.5 36.9 1.2 5.2 35. 2 94 2 10.2 58.8 76.7 1.2 3.3 9.9 25.2 ISG20 interferon stimulated gene 20kDa A_23_P324 04 2.2 6.5 1 1 9.4 38. 6 1 1.3 20.4 30.4 0.9 0.9 1.8 8.2 187 ISGF3G interferon-stimulated transcription factor 3, gamma 48kDa A_23_P654 42 14.1 11 1.9 3.3 14. 4 14. 9 3.2 5.2 15.3 11.4 1.4 1.9 6.9 6.2 KIAA1618 hypothetical protein LOC57714 A_24_P350 124 1.7 3.5 1.6 1.5 4.6 2.9 1.6 1.7 5.6 2.2 1.5 1.2 1.3 1.6 KLK10 kallikrein-related peptidase 10 preproprotein A_23_P107 911 2.3 4.3 1.4 1 14. 9 19. 9 0.9 1.8 14.7 16.4 1 1.2 2.8 16.1 LAMP3 lysosomal-associated membrane protein 3 A_23_P297 73 7.8 114. 5 1.2 1.3 14. 8 83 0.8 0.8 10 84.9 1.3 1.4 1.8 7.6 LAP3 leucine aminopeptidase 3 A_23_P186 04 2.2 5.4 0.5 0.8 4.2 2.6 0.6 0.9 2.8 2.5 0.5 0.8 1 1.5 LGALS9 galectin 9 long isoform A_32_P452 655 3.2 8.8 1.3 1.5 6.8 8.2 1.3 1.5 7.8 7.6 1.3 1.2 1.4 2.5 LGALS9 galectin 9 long isoform A_23_P116 557 1.7 9.3 1.1 0.9 4.2 2.2 1.1 1.4 2.7 2.6 1 1 1.4 1 LMO2 LIM domain only 2 A_23_P531 26 12.1 9.7 1 1.2 25 9.3 0.8 1.1 15.8 5.2 1 0.9 1.1 2.7 MX1 myxovirus resistance protein 1 A_23_P176 63 22 30.4 1.3 12. 3 28. 2 51. 8 1.9 17.2 41.2 32 1.3 2.2 7.8 14.4 MX2 myxovirus resistance protein 2 A_23_P626 3 18.5 74.3 0.8 2 81. 2 81. 1 1.3 3.4 82.9 41.8 1.6 1.2 3 5.5 MYD88 myeloid differentiation primary response gene (88) A_23_P362 659 2.7 3.5 1 1 4.3 1.2 0.9 1.2 3.3 1.3 1.1 1 1.1 1.2 NCOA7 nuclear receptor coactivator 7 A_24_P124 35 2.9 1.9 1 1.2 6.6 14. 8 0.8 1.5 9.2 7.1 1.1 1.1 1.2 3.1 NCOA7 nuclear receptor coactivator 7 A_23_P156 957 3.4 2.4 1 1.2 7.7 15. 9 1 3.2 12.2 7.2 1 1.1 1.2 2.7 NLRC5 nucleotide-binding oligomerization domains 27 A_23_P402 892 8.9 4.9 1 1.1 16. 2 3.7 0.9 1.7 17.5 3.2 0.9 1.1 10.3 7.6 NLRC5 A_23_P265 83 15.7 6.8 1.1 1.3 18. 5 7.2 0.9 1.6 22.3 3.6 1 1 10.8 11.9 NMI N-myc and STAT interactor A_23_P154 235 2.7 6.2 0.8 0.9 4.2 21 0.7 0.8 4.6 10.6 0.9 0.8 1.6 3.1 NT5C3 5'-nucleotidase, cytosolic III isoform 2 A_23_P595 47 1.7 4.3 0.9 1 3.7 2.9 0.9 1 2.8 2.4 0.9 0.9 1.2 1.5 OAS1 2',5'-oligoadenylate synthetase 1 isoform 2 A_23_P648 28 5.1 8.8 0.9 1.3 7.3 10. 6 0.9 1.8 6.9 7.5 1 0.9 1.5 4.1 OAS2 2'-5'-oligoadenylate synthetase 2 isoform 1 A_24_P343 929 7.4 44 0.9 1.1 17. 5 26. 7 0.9 1.1 20 23.4 1 1.1 1.2 7.5 OAS2 2'-5'-oligoadenylate synthetase 2 isoform 1 A_23_P204 087 10.5 30 1 1.2 13. 5 19. 9 1 0.7 8.2 12 0.7 0.8 1 3.7 OAS3 2'-5'oligoadenylate synthetase 3 A_24_P335 305 2.9 10.3 0.8 1.4 8.1 12. 5 0.9 1.9 8 9.6 0.8 0.9 1.4 3.5 OAS3 2'-5'oligoadenylate synthetase 3 A_23_P479 55 3.9 17.6 0.9 1.5 9.3 18. 2 1.2 2.1 10.3 15.2 1 1.1 1.7 5.1 OASL 2'-5'-oligoadenylate synthetase- like isoform a A_23_P139 786 40.8 49.6 2.1 29. 7 77 187 .9 11.5 41.5 114.8 135. 5 2.4 24.5 33.3 58.9 PARP12 zinc finger CCCH-type domain containing 1 A_23_P111 804 4 9.4 1.1 1.4 12. 7 11. 9 1 1.7 12.9 6.2 1.1 1.1 2 3 PARP14 poly (ADP-ribose) polymerase family, member 14 A_24_P161 018 6.5 12.5 1.6 1.9 15. 3 72. 8 1.5 2.4 29.6 36.8 1.5 1.3 2.7 7 PARP14 poly (ADP-ribose) polymerase family, member 14 A_32_P567 59 5.2 8 1.1 1.3 16. 8 62 0.7 2 30.2 27.4 0.7 1.3 3.6 8 PARP9 poly (ADP-ribose) polymerase family, member 9 A_23_P693 83 10.2 11.3 1 2.2 13. 9 11. 9 1 3.4 14.5 7.4 1.2 1.1 3.8 5.2 PLA1A phospholipase A1 member A A_24_P294 408 1.9 8.7 1 1.1 9.9 22. 3 0.9 0.8 7.1 10.7 0.9 1.2 2.2 5.7 PLA1A phospholipase A1 member A A_23_P298 16 1.4 5.4 1.9 1.4 9.9 24. 7 1.5 1.6 5.4 9 1.2 1.3 1.5 5.3 PLEKHA4 pleckstrin homology domain containing, family A (phosphoinositide binding specific) member 4 A_24_P408 047 3.9 8.9 1.4 2.2 23. 9 79. 3 1.8 4.1 43.2 46.7 1.3 1.7 3.9 16.7 PLEKHA4 pleckstrin homology domain containing, family A (phosphoinositide binding specific) member 4 A_23_P673 60 4.7 8.6 1.4 1.2 14. 4 31. 6 0.9 2.1 21.9 20.1 1 1.4 3.6 11.7 PLSCR1 phospholipid scramblase 1 A_23_P691 09 3.7 12.6 0.9 1.4 8.9 14. 5 0.8 1.8 8.7 8.2 0.9 0.8 1.1 3.2 PML promyelocytic leukemia protein isoform 1 A_24_P207 139 4.5 5.7 1.3 1 6.6 4.5 0.8 1.5 6.3 3.1 1.3 1 2 1.5 PML promyelocytic leukemia protein isoform 6 A_24_P198 598 2.7 4.5 1 1.1 4.8 3.6 1 1.3 3.9 2.9 1 1.2 1.7 1.6 PML promyelocytic leukemia protein isoform 6 A_23_P306 148 2.7 4.1 1 1.1 5 3.8 1 1.4 4.2 2.9 1 1.2 1.6 1.7 PNPT1 hypothetical protein A_32_P760 35 2.2 5.1 0.8 1 4.5 2.8 0.9 1 3.4 1.9 0.8 1.1 1.2 1.2 PNPT1 polyribonucleotide nucleotidyltransferase 1 A_23_P154 488 2.4 4.8 1 1 5.2 7.8 1 1.1 5.1 3.1 0.9 1.1 1.2 1.2 PPM1K protein phosphatase 1K (PP2C domain containing) A_24_P214 598 2.5 5.7 0.7 1.2 18. 3 46. 7 1 1.3 28.7 27.7 0.7 0.9 1.4 6.7 PSMB10 proteasome beta 10 subunit proprotein A_23_P140 807 1.5 4.6 1.3 1.2 3 2.4 1.4 1.1 3.1 3.3 1.2 1.2 2.1 4.9 PSMB8 proteasome beta 8 subunit isoform E1 proprotein A_23_P250 629 2.4 6.9 0.7 0.9 4.6 1.8 0.7 0.9 3.1 2.2 0.8 1 2.1 3.5 188 PSMB9 proteasome beta 9 subunit isoform 1 proprotein A_23_P111 000 3.1 12 0.5 0.8 9.4 3.6 0.8 1.1 6.1 4.9 0.6 0.9 3.8 8.8 RARRES 3 retinoic acid receptor responder (tazarotene induced) 3 A_23_P196 2 2.1 6 1.2 1.3 7.4 10. 4 1.3 1.5 8 8.3 1 1.3 4.4 6.9 RASGRP 3 RAS guanyl releasing protein 3 (calcium and DAG-regulated) A_24_P543 90 9.8 15.2 0.8 0.9 25. 7 12. 4 0.6 1.8 14.7 7.3 0.9 0.8 3.7 3.8 REC8 REC8 homolog A_24_P344 087 5.5 4.9 2.1 4.3 6.6 8.3 3.6 4.7 9.3 5.2 1.6 2.4 3.9 3.9 RGS22 regulator of G-protein signalling 22 A_32_P125 771 2 12.6 0.8 0.7 3.9 2.1 1.2 1.5 2.1 4.4 1 0.9 1.3 3 RNF213 chromosome 17 open reading frame 27 A_24_P923 884 1.9 3.5 1.7 1.6 5.4 2.1 1.5 1.3 5.9 2.2 1.7 1.4 1.3 1.5 RSAD2 radical S-adenosyl methionine domain containing 2 A_24_P316 965 7.8 29.5 1.2 1.2 92. 9 409 .1 1 2.9 194.4 302. 7 1 1.2 6.1 61.4 SAMD9 sterile alpha motif domain containing 9 A_24_P175 188 36.7 37 0.8 1.9 49. 1 50 0.8 3.2 35.6 34.8 0.9 1.1 5 12.4 SAMD9 sterile alpha motif domain containing 9 A_24_P175 187 42.2 25.2 0.8 2.1 28. 6 55. 3 0.8 3.4 34.1 37.7 0.9 1.1 3.5 13.4 SAMD9 sterile alpha motif domain containing 9 A_23_P355 244 23 26.9 1 2.2 48. 5 76. 1 0.9 3.2 41.2 53.3 1 1.1 3.7 14.6 SAMD9L sterile alpha motif domain containing 9-like A_23_P145 874 35.7 31.7 1.3 4.1 87. 9 55. 6 1.4 5.9 82.9 37.6 1.5 1.6 14.5 22.3 SAT1 spermidine/spermine N1- acetyltransferase A_23_P378 722 1.3 1 0.6 0.9 3.5 4.3 0.6 0.9 4.9 2.8 0.6 0.8 1.2 1.8 SAT1 spermidine/spermine N1- acetyltransferase A_23_P137 016 1.4 1.2 0.7 1 4 5.3 0.7 1 6.3 3.4 0.7 1 1.2 2 SIDT1 SID1 transmembrane family, member 1 A_23_P132 515 4.5 9 1 1.1 15. 1 2.9 1.3 1.8 9.1 1.2 0.9 1.9 2.7 2.6 SLC15A3 solute carrier family 15, member 3 A_23_P757 86 4.9 14.9 1 0.4 15. 2 33 0.7 0.7 12.9 17.5 0.9 0.8 1.7 7.1 SOCS1 suppressor of cytokine signaling 1 A_23_P420 196 4.1 2.1 1.2 1.4 14. 3 10. 6 1.3 1.9 13.8 6.3 0.9 1.5 1.5 4.2 SP100 hypothetical protein A_23_P209 712 2.5 6.4 2.8 1.8 3.5 4.5 2.5 1.9 4.2 2.6 2 1.4 1.6 1.7 SP100 nuclear antigen Sp100 isoform 2 A_24_P385 611 2.9 5 0.9 1 4.4 5.1 0.8 1.1 3.8 3.2 0.8 0.9 1.2 1.9 SP100 nuclear autoantigen A_24_P916 816 5.8 7.1 2.6 3.3 10. 8 11. 7 2.6 3.7 11.8 5.6 1.8 1.2 1.9 3.4 SP110 SP110 nuclear body protein isoform b A_23_P120 002 8.8 14.5 0.9 1.2 15. 3 10 0.9 1.8 10.9 5.8 1 0.9 1.8 2.2 SP140 SP140 nuclear body protein isoform 1 A_24_P328 504 3.7 6.8 1.3 1.5 5.9 5.5 0.9 1.2 4.9 3.5 1.6 1.1 2 3.4 STAT1 signal transducer and activator of transcription 1 isoform alpha A_23_P566 30 3 8.3 0.5 1 11 13. 1 0.5 1.1 6.8 7.7 0.6 1 3.7 4.9 STAT1 signal transducer and activator of transcription 1 isoform beta A_24_P274 270 4.6 10.1 0.9 2.3 14. 6 11. 1 0.9 2.9 15.3 7.6 0.9 1.5 4.7 5.7 STAT2 signal transducer and activator of transcription 2 A_23_P760 90 4.9 3.8 1.3 1.3 8.6 4.2 1 1.6 8.3 2.4 0.9 1.1 2.1 2.5 TAP1 transporter 1, ATP-binding cassette, sub-family B A_23_P590 05 4.8 9.2 1 1.4 13. 6 12. 8 1.1 1.4 11.5 9.5 1 1.4 3.8 9.3 TAPBPL TAP binding protein-like A_23_P367 00 1.3 2.7 0.7 0.7 3.4 15. 7 0.6 0.9 7.6 11.1 0.7 0.8 1.5 3.4 TDRD7 tudor domain containing 7 A_23_P123 672 2.7 3.1 0.9 1 5.8 20. 3 0.8 1.3 9.4 12.3 1 0.8 1 2.1 TGM2 transglutaminase 2 isoform a A_32_P867 63 1.7 4.2 0.6 0.6 2.2 1.2 0.5 0.7 1.4 1.2 0.6 0.7 1.2 1.5 TLR3 toll-like receptor 3 A_23_P299 22 11.9 16.1 1.3 1.3 29. 3 6.6 0.9 1.8 12 16.1 0.9 1 5.7 12.7 TMEM140 hypothetical protein LOC55281 A_24_P372 134 4.1 3.4 1.1 1 4.3 4.1 1 0.9 2.3 3.4 1.2 1 1.2 1.7 TMEM140 hypothetical protein LOC55281 A_23_P311 77 12.1 8.5 0.9 1.1 23. 9 14 1.1 1.6 14.1 10.7 1.2 0.9 3.4 4.4 TNFSF10 tumor necrosis factor (ligand) superfamily, member 10 A_23_P121 253 23.5 20.1 5.6 1.3 50. 8 11. 7 1.1 1.6 26.1 11.5 1.2 1.1 2.2 6.9 TNFSF13 B tumor necrosis factor (ligand) superfamily, member 13b A_23_P141 74 6.2 13.1 0.9 1.5 17. 8 19. 7 1.2 1 14.5 14.1 1 1.2 2.8 4.3 TRIM14 tripartite motif protein TRIM14 isoform alpha A_24_P197 964 2.3 8.7 0.5 1 6.4 9.4 0.7 1.2 5.6 6.5 0.6 1 1.3 2.5 TRIM14 tripartite motif protein TRIM14 isoform alpha A_23_P216 655 2.1 7.3 0.6 1.1 7 8.6 0.8 1.5 6.3 5.6 0.7 1 1.5 2.6 TRIM14 tripartite motif protein TRIM14 isoform alpha A_23_P425 752 1.7 4.6 1 1.2 4.9 4.9 1.1 1.3 5 3.5 1 1.2 1.2 1.6 TRIM22 tripartite motif-containing 22 A_23_P203 498 16.1 42 0.9 1.4 57. 3 41. 8 1 1.3 63.1 36.6 0.8 0.9 6.6 19.6 TRIM25 tripartite motif-containing 25 A_23_P153 26 2.5 2.2 1.1 1.1 4.5 11 1.2 1.2 4.8 8.3 1.1 1 1.2 1.8 TRIM25 tripartite motif-containing 25 A_24_P936 767 3.5 2.9 0.7 1.1 7.5 5.9 0.7 1.3 5.2 4.3 0.7 0.9 1.2 1.8 TRIM34 tripartite motif protein 34 isoform 1 A_24_P398 323 2.1 4.1 0.9 0.9 4 1.4 0.8 0.9 2.3 1.5 0.8 0.8 1.5 1.4 TRIM34 tripartite motif protein 34 isoform 3 A_23_P124 190 3.4 7 2.4 1.8 5.2 2 1.6 1.6 5.1 1.8 2.2 1.3 1.6 2 TRIM38 tripartite motif-containing 38 A_24_P294 851 3.6 3.3 0.7 1.2 6.4 2.3 0.9 1.3 6 1.9 0.7 1.1 1.4 1.8 189 TRIM69 A_24_P505 43 4.1 3.8 0.9 0.9 5.1 4.4 0.7 1 3.1 3 1 0.7 1.6 2.9 UBD diubiquitin A_23_P818 98 6 12.9 1 1.2 7.7 20. 3 0.8 1.1 5.5 19.2 1 0.8 3 41.6 UBE1L ubiquitin-activating enzyme E1- like A_23_P212 07 3.5 21.8 1 0.8 19 8.4 0.9 0.8 24.4 8.9 0.9 0.8 2.7 7.5 UBE2L6 ubiquitin-conjugating enzyme E2L 6 isoform 2 A_23_P757 41 2.8 9.2 0.6 0.8 4.8 2 0.6 0.9 3.4 2.6 0.7 1.1 1.8 2.9 USP18 ubiquitin specific protease 18 A_32_P132 206 5.3 9.2 1.2 1.5 14. 7 25. 8 1.1 1.8 18.7 18 1.2 1.3 1.7 4.2 USP18 ubiquitin specific protease 18 A_23_P132 159 4.9 9.6 0.8 1.2 18. 5 29. 6 0.8 1.6 22.2 21.1 0.9 1 1.5 3.5 ZNFX1 zinc finger, NFX1-type containing 1 A_24_P230 34 5.7 3.2 1 1.3 7.1 15. 3 0.9 1.6 9.9 11.4 1 1.2 1.7 3.7 ZNFX1 zinc finger, NFX1-type containing 1 A_23_P684 62 5.8 2.9 0.5 1.3 10. 5 18. 1 0.6 1.6 9.8 9.7 0.7 1.2 1.4 3.8 hypothetical protein A_32_P161 292 2.9 5.4 0.6 0.9 10. 8 25. 3 1 1.5 14.5 17.8 1.1 1.1 2.8 4.2 hypothetical protein A_32_P662 8 1.6 3.5 1.1 1 4.4 3 1 1.1 4.7 1.7 1 1 1 1.1 hypothetical protein LOC55337 A_23_P388 94 3.8 9.8 1.6 1.6 12. 7 9.7 1.3 1.9 11.9 5 1.4 1.2 1.6 2 hypothetical protein LOC55337 A_24_P236 949 2.3 7.6 0.7 0.9 6 8.3 1 1.4 6.9 4.7 0.8 0.8 1.1 1.7 hypothetical protein LOC55601 A_24_P334 361 5.8 18.1 0.3 1.1 23. 9 104 .2 0.4 1.5 50.4 75.9 0.4 0.8 3.9 16.4 hypothetical protein LOC55601 A_23_P414 70 5.1 13.7 0.4 0.9 16. 4 66. 9 0.4 1.4 38.5 51.5 0.4 0.8 3 10.7 hypothetical protein LOC79132 A_23_P383 46 11.8 50.6 1.4 2.9 62. 4 323 .3 1.4 5.5 146.3 183. 8 1.2 2 6 48.6 hypothetical protein LOC93349 A_23_P337 753 2.8 4.3 1.2 1.1 6.1 2.7 0.8 0.8 4.3 2.8 0.9 1.1 2.6 3.2 leukocyte-derived arginine aminopeptidase A_23_P302 43 4.3 4.6 1.1 1.4 8.3 11. 6 1.2 1.6 8.8 11.9 1.1 1.3 3.1 5.8 promyelocytic leukemia protein isoform 2 A_23_P358 944 5.1 4.5 1.6 1.7 9.5 7.6 1.6 1.9 9.8 4.4 1.4 1.2 1.4 2.3 similar to galectin 9 short isoform A_23_P101 025 1.4 7 1.4 1.2 3.4 3 1.7 1.4 2.7 2.5 1.6 1.4 1.1 1.2 Unknown (protein for MGC:105145) A_24_P418 044 1.7 5.4 0.9 1 4.2 17. 9 0.8 1 5.2 11.4 1 1.1 1.4 4.2 XIAP associated factor-1 isoform 1 A_23_P428 6 65.7 270 1.2 2.3 27 7.1 345 .9 1.2 3.7 225.6 209. 2 1.6 1.4 7.9 77.7 XIAP associated factor-1 isoform 1 A_23_P428 3 52.8 95.9 0.9 1.5 12 0.5 184 .2 1.1 2.7 143.6 90.8 1.4 0.9 5 38.6 A_32_P995 33 5.5 10 1.6 3.6 29. 8 72. 2 1.5 6.7 42.5 36.9 1.8 2 6.1 21.4 A_24_P927 166 6.4 4.2 1.5 3.3 60. 2 236 .6 1 6.9 126.8 103. 5 1.6 1.4 6.5 28.9 A_32_P924 15 4.1 10 1.3 1.6 16. 3 85. 2 1.2 2.1 33.9 42.8 1.3 1.2 2.9 7.9 A_24_P586 264 3.3 6.3 0.7 1.2 22. 3 47. 7 1.1 1.7 34.9 33.3 0.9 1.2 1.6 8.3 A_32_P154 726 2.6 3.4 0.8 0.9 7.5 20. 4 0.7 1 9.2 12.6 0.9 0.9 1.5 4.3 A_24_P127 641 5.6 8.3 1.4 1.8 16. 3 36. 6 1.3 2 20 16.6 1.4 1.3 2.9 7.9 A_24_P680 79 4.6 9.3 1.7 1.6 14. 4 19. 2 1 2 16.5 12.9 1 1.1 2.9 7.7 A_23_P250 353 4 11.1 0.7 1.2 12. 2 26 0.8 1.8 16.8 22 0.9 1.1 2 8.5 A_32_P114 831 2.3 4.1 1.2 1.8 7 10. 5 1.7 1.4 8 12.7 1 1.3 1.6 4.3 A_32_P220 897 2.8 2.4 1 1.1 3.6 6.8 0.8 1.5 5.4 5 1.1 0.8 1.1 2 A_24_P829 156 5.5 4.7 2.6 2.5 12. 2 16. 2 1.5 2.4 13.7 7.3 1.5 1.2 3.1 5.1 A_23_P256 487 3.3 3.6 0.8 0.9 24. 5 52. 4 0.9 1.3 20.9 27.3 0.8 0.9 2.1 12.9 A_32_P758 67 3.3 5.8 1.7 1.7 22. 2 23. 5 1.2 3.1 19.3 13.6 1.8 2 7.2 20.1 A_32_P143 980 2 4.3 1.2 1.3 4 11. 1 1.3 1.3 4.6 8.1 1.3 1.1 1.4 3.7 A_23_P338 113 1.2 3.3 0.7 0.8 3.2 14. 1 0.7 0.8 3.9 8.2 0.8 0.8 0.9 3.4 A_23_P125 109 1.5 5 0.8 0.9 4.3 22. 9 0.8 0.9 5.7 13.9 0.9 1 1.2 4.7 A_23_P373 126 1.8 6.6 0.8 0.9 6.9 27 0.8 1 7.8 14.7 0.8 0.9 1.3 6.2 A_23_P370 707 1.7 5.3 0.8 0.9 7 23 0.8 1 7.4 12.9 0.8 0.9 1.5 6.3 A_24_P860 781 1.5 4.6 0.9 1 2.5 12. 2 0.9 0.9 3.4 8.4 0.9 1 1 3.2 A_23_P350 295 1.1 2.3 0.9 0.9 2 10. 2 0.8 0.9 2.9 7.1 0.9 0.9 1 2.5 A_32_P545 53 3.4 5.9 0.7 0.9 8.8 14. 6 0.6 1.4 9.5 9.9 0.6 1 1.3 2.4 A_23_P300 69 4 17.2 1 1.5 24. 9 38. 8 1 2.2 34.5 22.3 0.9 1.2 2.5 4.6 A_23_P651 74 2.3 4 1 1.1 4.8 6.5 1.1 1.3 4.5 3.7 1 1 1.5 1.8 A_23_P178 37 5.3 10.7 0.9 1.9 11. 9 22. 3 1.8 2.9 24.5 18.4 1.1 1.8 3.4 7.9 A_23_P384 355 63.7 88.6 0.7 2.8 94. 9 338 .2 0.6 4.5 81.5 170. 7 0.8 1 2.8 24.8 A_24_P368 98 7.2 2.5 0.8 1 6.1 12. 8 0.9 0.7 5.9 5.8 0.9 0.7 2.4 3.6 190 A_24_P740 662 11.5 8.7 2.2 4.9 19. 6 26 2.1 8.1 28.9 15.7 1.6 1.9 7.2 10 A_24_P118 892 6.3 11.9 0.9 2 14. 2 13. 3 0.7 1.9 9.6 9.4 0.7 1.5 1.1 1.9 A_24_P868 905 4.2 12.9 1 1.4 9.5 10. 3 1.4 1.6 12.2 9.7 0.9 1.1 1.4 2.3 A_32_P167 592 2.2 7.4 1.1 1.2 4.6 3.4 1.3 1.3 5.6 4.1 1 1 0.9 0.9 A_24_P704 0 2.8 10.4 1 1 6.5 5.4 1.2 1.4 8.5 6.6 1 0.9 0.9 1.2 A_23_P202 245 2.2 13.4 1 1.2 8.8 8.6 1.1 1.1 6.3 10.2 1 0.6 0.7 1.4 A_32_P156 746 8.1 6.2 1.1 2.3 16. 2 4.8 1.3 2.9 15.9 3.1 1.3 1 2.5 3.5 A_24_P236 680 3.8 3.1 1.1 1.5 7.4 3.3 1.3 1.9 5.4 2.8 1 1.2 1.5 1.7 A_24_P157 02 2 4.3 0.5 0.9 3.8 1.8 0.5 1 1.8 2.3 0.5 0.9 1.1 1.4 A_32_P562 49 10 18.8 1.1 0.8 27. 3 21. 7 0.7 0.8 28.2 16.1 0.9 1 6.2 32 A_32_P171 793 6.2 12.8 0.9 1.7 18. 1 7.8 0.8 1.2 12.6 8.1 0.9 1.3 4.2 10.5 A_23_P203 629 1.5 4.8 1.6 1.1 2.9 3.8 1.7 1.2 2.8 3.7 1.4 1.1 1.3 2.9 A_23_P354 547 11.5 8.7 2.1 7.1 11. 7 10. 1 3.8 7.5 16.2 5.1 1.5 3.1 5.1 5 A_32_P154 321 1.9 4.4 1.3 1.1 2.6 1.5 1.3 1.3 2.9 2.3 1.3 1 1.8 1.7 A_24_P155 02 1.6 4.4 1 1 2.6 1.8 1.2 1.2 3.1 2.3 1 1 0.9 0.8 A_24_P254 933 2 8.1 1 1 3.6 2.6 1.2 1.3 4.4 3.8 1 1 0.9 0.9 A_24_P306 810 3.1 3.2 1.8 2.1 8.1 2.2 1.9 2.1 7.3 1.6 1.5 1 1.7 1.8 G AVIL advillin A_24_P419 087 1.3 1.6 1.8 2.7 3.1 5.2 2.4 3 5.2 3.1 2.1 0.9 1 1.2 CDH15 cadherin 15 preproprotein A_32_P253 57 1.7 1.1 1 1.3 6.8 3.3 1.2 1.1 1.6 2.5 1.2 1.3 3.2 1.6 CEBPD CCAAT/enhancer binding protein delta A_23_P318 10 2.7 1.3 2 1.5 3.7 1.9 1.6 1.5 5.2 1.4 1.7 1 0.8 1 CHRNA10 cholinergic receptor, nicotinic, alpha polypeptide 10 A_23_P411 188 0.8 1 1.8 1.6 1.7 7.5 1.8 1.5 4.9 4.3 1.5 1.4 1.1 1.2 DDEF1IT1 HSPC054 A_23_P146 325 1.3 1.5 4.5 2.3 2.8 7.6 3.5 2.3 7.9 2.9 3.2 1.5 1.3 1.6 FEV FEV (ETS oncogene family) A_23_P546 0 1.2 1.2 1.6 1.5 2.4 10. 9 1.7 1.2 1 1 1.2 2 1.5 2.5 IL1B interleukin 1, beta proprotein A_23_P795 18 1.2 1 0.8 1 1.8 4.3 0.8 1 1.6 0.8 0.9 1.1 1 3.5 ITGB1BP 2 integrin beta 1 binding protein 2 A_23_P454 24 0.9 1 2.8 1.8 2.1 3.8 1.9 1.7 4.7 2 1.8 1.2 1 1.3 MLL3 myeloid/lymphoid or mixed- lineage leukemia 3 isoform 2 A_32_P209 472 0.9 1.1 2.5 2.6 2.1 11. 4 2.5 2 7.6 4.4 1.5 1.5 1.2 1.8 SATB2 A_24_P928 408 1.7 1.7 3.9 3.1 2.4 7.9 3.2 2 7.1 2.6 2.4 1.4 1.5 1.2 SOCS3 suppressor of cytokine signaling 3 A_23_P207 058 2.4 1.2 1.4 1.4 4.8 1.3 1.6 1.8 4.1 0.9 1.2 1 0.8 0.7 SSH2 hSSH-2 A_23_P395 954 1.2 2 2.8 1.7 1.7 4.5 1.4 2.1 2.7 1.4 1.8 1.3 1.3 1.1 SYNPO2 A_23_P310 094 1.2 1.7 0.6 1.2 2.7 9.7 0.5 0.9 2.1 1.6 0.8 1.6 2.3 2.7 TTC18 tetratricopeptide repeat domain 18 A_23_P326 931 2.2 1 3 1.5 1.6 7.9 1.3 2.2 4.4 4.4 2.6 1.1 0.9 1.1 ZBTB20 A_32_P179 526 1.2 1.6 4.2 2.4 2.8 2.6 3.8 2.5 4.8 1.3 3.2 1.2 1.2 1.2 hypothetical protein A_24_P273 647 0.8 1.1 1.6 1.4 1 5.2 1.3 1 2.2 2.4 1.5 1.4 0.9 0.9 interferon epsilon 1 A_23_P302 060 1.1 1.2 1.6 2.2 2 6 2.2 1.8 2.6 2 1.5 1 1.5 1.1 KIAA1541 protein A_24_P940 218 1.1 1.1 2.6 2.1 1.9 6.4 1.9 1.7 3.7 3.9 2.2 1.8 1.5 1.5 A_32_P989 40 1.1 1.4 4.7 2.7 4.9 30. 3 3.3 2.2 15.5 11.5 3.9 1.8 1.6 3.3 A_32_P208 039 1 1.2 3.9 2.2 3.3 20. 3 2.7 1.8 10.3 8 2.9 1.6 1.3 2.4 A_32_P559 87 1.2 1.6 5.3 3 4.1 25. 3 4.4 2.5 13.6 8.9 4.1 1.8 1.7 2.9 A_32_P220 567 1 1.1 2.4 1.7 2.2 9.4 2.1 1.9 6.2 4.1 2 1.2 1.1 1.8 A_32_P216 122 0.8 1.6 4.2 2.5 3.1 16. 4 3.2 1.8 8 10.6 3.2 1.5 1.4 2.3 A_32_P214 054 0.8 1.4 3.8 2.8 2.7 11. 8 3.3 2.1 5.7 6.1 3 1.7 1.3 2.1 A_32_P158 74 1 1.1 2.2 1.9 1.6 6.3 2.4 2.1 4 3.8 1.7 1.3 1.3 1.4 A_24_P145 333 0.9 1.1 2.2 1.9 1.7 4.8 1.7 1.5 4.2 2.5 2.5 1.1 1 1.3 A_23_P242 75 1.2 1.1 2.4 2.2 1.4 6.7 2.1 1.7 3.4 3.2 1.8 1.8 1.4 1.1 A_32_P449 32 1 1.2 1.9 1.6 1.3 4.8 1.9 1.6 2.4 4.7 1.6 1 1.1 1.2 A_32_P101 33 0.8 2.2 3.9 2.6 2 10. 5 3.4 2.4 4.2 6 3.1 1.3 1.3 2 A_32_P406 73 1 1.5 2.1 2.3 1.5 5.1 2.5 2.1 4.1 4.4 2.5 1.2 1.1 1 A_32_P921 01 1 1.5 2.4 1.8 2 3.2 2.5 2.1 5.1 2.8 1.9 1.9 0.7 1.8 191 A_32_P839 73 1 1.5 4.3 2.1 2.2 6.3 3.3 2.2 5.6 2.2 2.3 1.4 1.3 1.6 A_32_P191 074 1.2 1.6 4.1 2.8 3.9 10. 9 3.1 2.8 11 3.9 3.1 1.9 1.6 2 A_24_P162 128 1 1.5 4 2.1 2.8 5 2.7 2.3 6.1 2.6 2.5 1.4 1.3 1.4 A_32_P751 6 1.1 1.6 2.5 1.9 2.8 4.3 2.5 2 5.2 2 1.9 1.2 1.2 1.2 A_23_P399 292 1.1 1.6 2.6 2 3 5 2.7 2 6 2.3 1.9 1.2 1.2 1.2 A_32_P198 282 2.4 1.7 4.4 3.1 2 9.7 3.9 3 7.9 3.5 2.9 1.4 1.4 2.1 A_32_P118 220 1.1 1.3 5.2 2.5 2 9.3 4.3 2.1 5.2 4.1 2.3 1.6 1.4 1.6 A_24_P932 388 1 1.8 5.9 1.6 4 10. 8 2.4 2.7 8 3.8 2.4 1.4 1.4 3.5 A_32_P110 485 1.1 1.6 2.6 2.7 3.5 8.9 1.7 3.7 4.5 2.8 1.7 1.5 1.2 1 A_24_P204 374 0.7 1 1 1.3 1.7 5.6 1.4 1.4 3 2.2 1.2 1.1 1.1 0.8 A_32_P211 048 1 3.1 5.5 2.6 1.9 9 4.7 2.5 4.1 2.8 4.7 1.7 1.6 1.3 A_32_P129 310 1.2 1.7 3.4 2 1.5 5.2 2.7 2.3 3.2 1.4 2.6 1.5 1.4 1.1 A_32_P539 76 1.5 1.7 6.3 3.3 3.3 5.4 6.8 3.3 6.4 2.1 3.1 1.7 1.5 1.7 A_32_P198 791 1.1 1.8 4.2 2.4 6.5 5.2 5.1 2.8 7.2 2.3 2.7 1.7 1.6 3 A_23_P156 408 0.8 1.3 1.5 0.8 0.8 4.3 1.2 0.9 1.1 3 2.4 0.6 0.6 1 A_32_P158 181 1.4 1.6 1.1 1.2 3.8 1.4 1.6 1.6 1.9 1.1 1.2 0.9 1.2 5.7 A_32_P331 7 1 0.8 0.6 0.7 2 2 0.5 0.8 0.7 0.6 0.6 0.8 3.8 5.2 H ABCB6 ATP-binding cassette, sub-family B, member 6 A_24_P197 196 1 0.9 1.3 1.1 1 0.2 1.4 1.2 0.7 0.4 1.1 1.1 1.2 1 ABCB6 ATP-binding cassette, sub-family B, member 6 A_23_P544 1 0.9 0.8 1.2 1.1 0.9 0.2 1.3 1.2 0.7 0.3 1.1 1 1.1 0.8 ABCC4 ATP-binding cassette, sub-family C, member 4 A_24_P169 13 1.1 0.9 0.8 0.8 0.8 0.1 0.8 0.8 0.5 0.3 1 0.9 0.8 0.7 ABCC6 ATP-binding cassette, sub-family C (CFTR/MRP), member 6 A_23_P152 72 1.3 1.4 1.6 1.3 0.9 0.2 1.3 1.1 0.7 0.3 1.5 1.4 1.1 0.8 ABHD6 abhydrolase domain containing 6 A_23_P211 850 1.1 1.3 1.4 1.2 0.9 0.2 1.2 1.1 0.7 0.5 1.4 1.4 1.4 1.1 ACSM3 SA hypertension-associated homolog isoform 2 A_23_P317 756 1.1 0.9 0.8 0.9 0.8 0.1 0.7 0.8 0.4 0.2 0.9 0.9 1 1 ADORA2 B adenosine A2b receptor A_23_P554 77 0.9 0.9 0.9 1 0.6 0.1 0.8 0.9 0.4 0.3 0.9 1.2 0.9 0.7 AGR2 anterior gradient 2 homolog A_23_P314 07 1.2 1.2 1.2 1.2 1.6 0.2 0.8 0.8 0.5 0.3 1.2 1.3 1.9 1.9 AGT angiotensinogen preproprotein A_23_P115 261 7.5 2.7 0.8 0.8 1.7 0.4 1 0.8 0.8 0.3 0.9 1 1 0.7 AKR1C1 aldo-keto reductase family 1, member C1 A_24_P152 968 1 0.8 1.1 1.2 1 0.1 1 1 0.7 0.2 1.1 1.2 1.2 0.8 AKR1C1 aldo-keto reductase family 1, member C1 A_24_P220 947 1.1 0.8 0.9 0.9 1 0.1 0.8 0.8 0.6 0.3 0.9 1.1 1.2 0.7 AKR1C1 aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha (3- alpha)-hydroxysteroid dehydrogenase) A_24_P312 578 1 0.9 0.9 1 0.8 0.2 0.8 0.8 0.6 0.2 1 1.1 1 0.8 AKR1C3 aldo-keto reductase family 1, member C3 A_23_P138 541 1.1 0.7 0.8 0.9 0.7 0.1 0.7 0.8 0.5 0.2 0.8 1.1 1 0.7 ALDH3A1 aldehyde dehydrogenase 3 family, member A1 A_23_P207 213 0.7 0.5 0.5 0.5 0.6 0.1 0.6 0.6 0.5 0.2 0.5 0.7 0.7 0.6 ALDH3A2 aldehyde dehydrogenase 3A2 isoform 2 A_23_P129 896 0.9 0.7 0.9 1 0.8 0.2 0.8 1 0.6 0.2 0.9 1.2 1.1 0.9 ANXA13 annexin A13 isoform b A_23_P600 09 1.1 0.6 0.7 0.7 0.6 0.1 0.8 0.7 0.3 0.1 0.6 0.8 1.1 0.7 ANXA13 annexin A13 isoform b A_23_P600 06 1.3 0.7 0.8 0.8 0.6 0.1 0.7 0.7 0.2 0.1 0.8 0.7 1 0.6 APBA2 amyloid beta A4 precursor protein-binding, family A, member 2 A_23_P146 849 1 0.7 1.1 0.9 0.6 0.1 0.9 0.9 0.6 0.2 1 1.1 0.8 0.6 APOBEC3 C apolipoprotein B mRNA editing enzyme, catalytic polypeptide- like 3C A_23_P120 931 0.9 0.9 0.7 0.8 0.8 0.2 0.7 0.7 0.6 0.3 0.7 1 0.9 0.8 APOH apolipoprotein H precursor A_23_P382 44 1.1 0.8 0.8 0.8 0.5 0 1 0.9 0.4 0.1 0.9 0.8 0.9 0.7 ARL6IP5 ADP-ribosylation-like factor 6 interacting protein 5 A_23_P166 640 1.3 1 0.8 0.8 0.7 0.2 0.6 0.7 0.4 0.4 0.8 1 1.1 1.2 ATAD4 ATPase family, AAA domain containing 4 A_23_P118 894 1.3 0.8 0.9 0.6 0.7 0 1 0.7 0.1 0 1 0.9 1.1 0.4 AYTL1 hypothetical protein A_24_P364 807 1.4 1 0.8 0.8 0.9 0.2 0.9 0.9 0.4 0.3 1 1.4 1.1 1.2 B3GNT1 UDP-GlcNAc:betaGal beta-1,3- N-acetylglucosaminyltransferase 1 A_23_P869 00 1 1 1 0.8 0.6 0.1 0.8 1 0.4 0.3 1.1 1 0.8 0.6 B3GNT3 UDP-GlcNAc:betaGal beta-1,3- N-acetylglucosaminyltransferase 3 A_23_P789 80 1.1 1.1 1.3 1 1 0.2 1.1 1.1 0.6 0.3 1.3 1 1.2 1 192 C12orf27 A_24_P314 515 0.9 1.2 1.5 1.3 1 0.1 1.7 1.5 0.8 0.2 1.4 1.1 1.2 0.8 C17orf58 hypothetical protein LOC284018 isoform a A_24_P491 90 1.2 1.1 0.8 0.9 0.7 0.2 0.8 0.8 0.5 0.4 0.9 1 1 1 C18orf56 hypothetical protein LOC494514 A_24_P238 499 1.1 1 0.6 0.7 0.6 0.1 0.6 0.6 0.4 0.3 0.7 0.7 0.8 0.8 C2orf32 chromosome 2 open reading frame 32 A_23_P329 353 1.3 0.9 0.8 0.8 0.7 0.1 0.7 0.8 0.5 0.3 0.8 0.9 0.9 0.8 C4BPA complement component 4 binding protein, alpha chain precursor A_23_P975 41 1.3 1.2 0.7 0.8 0.6 0.2 0.9 0.8 0.5 0.4 0.8 0.9 0.9 0.9 CA12 A_23_P163 338 1.3 1.1 2 1.8 0.8 0.2 1.5 1 0.4 0.2 1.6 1.5 1.6 1.1 CA12 A_23_P163 336 1.1 1.1 2.8 1.9 1.1 0.2 2.2 1.5 0.9 0.3 2.3 1.7 1.6 1.1 CALB2 calbindin 2 full length protein isoform A_23_P210 92 1 0.8 0.9 1 0.7 0.2 0.8 0.8 0.4 0.3 0.9 1.1 1 0.7 CD302 CD302 antigen A_24_P396 702 1.2 0.9 0.9 0.9 0.9 0.2 0.9 0.9 0.5 0.2 0.9 1 1.1 1 CDH1 cadherin 1, type 1 preproprotein A_23_P206 359 1.2 1.7 1.2 1 0.9 0.2 0.9 0.8 0.7 0.3 1.3 1 0.8 0.8 CDON surface glycoprotein, Ig superfamily member A_32_P172 141 0.9 0.7 1 0.9 0.6 0.2 0.9 0.9 0.3 0.2 1 1 1 0.8 CES1 carboxylesterase 1 isoform c precursor A_23_P206 733 0.9 0.8 0.7 0.7 0.7 0.2 0.7 0.8 0.4 0.2 0.7 0.9 0.9 0.7 CIT citron A_23_P420 551 0.9 0.8 0.9 0.9 0.7 0.2 1.1 1.2 0.8 0.4 0.9 0.8 0.8 0.9 CKMT1B creatine kinase, mitochondrial 1B precursor A_23_P163 235 0.9 0.7 0.6 0.8 0.7 0.3 0.5 0.8 0.3 0.2 0.9 0.9 1 0.8 CMBL carboxymethylenebutenolidase- like (Pseudomonas) A_23_P144 668 1 0.9 0.8 0.8 0.7 0.1 0.7 0.7 0.4 0.2 1 1 0.9 0.8 CNTN1 contactin 1 isoform 1 precursor A_23_P390 700 1.1 0.7 0.6 0.7 0.9 0.2 0.5 0.7 0.4 0.1 0.6 1 1.4 1 COL21A1 collagen, type XXI, alpha 1 precursor A_23_P311 24 2.4 0.8 0.7 0.9 0.8 0.2 0.7 0.9 0.8 0.1 1 0.7 1.5 0.8 CORO2A coronin, actin binding protein, 2A A_23_P135 061 1.1 1 0.9 0.9 1.1 0.2 1 1 0.8 0.3 0.9 0.9 0.9 0.9 CPLX2 complexin 2 A_24_P391 868 0.7 0.6 0.9 0.9 1 0.2 1 1 0.9 0.2 0.8 1 1 0.8 CRYZ crystallin, zeta A_23_P114 662 0.9 0.8 0.7 0.7 0.5 0.1 0.6 0.6 0.4 0.2 0.7 0.7 0.7 0.6 CRYZ A_24_P548 453 0.9 0.7 0.5 0.6 0.5 0.1 0.5 0.6 0.3 0.2 0.6 0.7 0.7 0.5 CTTNBP2 cortactin binding protein 2 A_23_P215 744 1 0.8 1.5 0.9 0.3 0 1.1 0.8 0.2 0.2 1 0.7 0.4 0.4 CXADR coxsackie virus and adenovirus receptor precursor A_32_P296 32 0.9 1 0.6 0.7 0.7 0.2 0.5 0.7 0.4 0.4 0.7 0.7 1 0.9 CXADR coxsackie virus and adenovirus receptor precursor A_24_P374 943 1 1.1 0.5 0.7 0.8 0.2 0.4 0.6 0.4 0.1 0.5 0.7 1 0.6 CYBRD1 cytochrome b reductase 1 A_23_P209 564 1.1 1.1 0.8 0.8 0.7 0.2 0.6 0.7 0.5 0.3 0.9 0.8 0.9 1 CYP24A1 cytochrome P450, family 24 precursor A_24_P356 930 0.9 1 1.5 0.9 0.8 0.2 1.2 1.1 0.9 0.4 1.4 0.7 0.8 0.9 CYP24A1 cytochrome P450, family 24 precursor A_23_P288 15 1 1 1.6 1 0.8 0.2 1.3 1.1 1 0.4 1.5 0.7 0.9 0.9 DHCR24 24-dehydrocholesterol reductase precursor A_23_P379 475 0.9 0.8 1.1 0.8 0.8 0.1 0.8 0.8 0.3 0.3 1 1.2 1.2 0.6 DPYSL2 dihydropyrimidinase-like 2 A_24_P383 47 1.1 0.9 0.8 0.8 0.8 0.2 0.7 0.7 0.5 0.3 0.9 0.9 1 1 DSCR1L2 Down syndrome critical region gene 1-like 2 A_23_P391 275 1.1 1.1 1.2 1 0.8 0.2 1.2 1 0.6 0.3 1.1 1 0.9 0.9 ECH1 peroxisomal enoyl-coenzyme A hydratase-like protein A_23_P153 853 0.9 0.8 0.7 0.8 0.8 0.2 0.7 0.8 0.5 0.3 0.8 1 1 0.7 ELL3 elongation factor RNA polymerase II-like 3 A_24_P479 88 1.2 1 1.2 0.8 0.7 0.2 1.2 0.9 0.5 0.4 1.1 0.9 1 1.1 EPHX1 epoxide hydrolase 1, microsomal (xenobiotic) A_23_P345 37 1 0.8 0.9 0.9 0.8 0.2 0.8 0.9 0.6 0.2 0.9 1.1 1 0.7 F2R coagulation factor II receptor precursor A_23_P213 562 0.8 1 0.5 0.5 0.7 0.2 0.4 0.6 0.3 0.3 0.5 0.6 1 1.2 FAM64A hypothetical protein LOC54478 A_23_P498 78 0.8 0.7 0.6 0.6 0.7 0.1 0.7 0.8 0.5 0.3 0.6 0.6 0.7 0.7 FGL1 fibrinogen-like 1 precursor A_23_P204 84 1.4 1.2 0.6 0.7 0.7 0.1 0.6 0.6 0.5 0.2 0.7 0.7 0.8 0.7 FMO5 flavin containing monooxygenase 5 A_24_P713 41 1.1 0.8 0.7 0.9 0.7 0.1 0.9 1.1 0.4 0.2 0.8 0.9 0.9 0.8 FOXQ1 forkhead box Q1 A_32_P164 246 0.8 1.2 1.3 1.1 0.7 0.1 1.2 1 0.6 0.3 1.2 1.1 0.6 0.5 GALNT5 UDP-N-acetyl-alpha-D- galactosamine:polypeptide N- acetylgalactosaminyltransferase 5 (GalNAc-T5) A_24_P408 736 1.2 1.2 0.8 0.8 0.7 0.2 0.6 0.8 0.3 0.3 1.1 0.9 1.1 1.3 GPX2 gastrointestinal glutathione peroxidase 2 A_23_P303 8 1.1 0.8 0.9 0.9 0.9 0.2 1 0.8 0.9 0.3 1 1 1.1 1 GRB14 growth factor receptor-bound protein 14 A_23_P154 526 1 0.9 1.6 1.3 0.9 0.2 1.4 1.3 0.8 0.3 1.4 1.2 1.3 0.9 GSTM3 glutathione S-transferase M3 A_24_P914 434 1 0.8 0.8 0.9 0.7 0.2 0.8 0.8 0.5 0.3 0.9 1 0.9 0.7 193 GSTO1 glutathione-S-transferase omega 1 A_24_P304 051 1.1 1.2 0.8 0.9 1 0.2 0.8 0.8 0.6 0.3 0.8 0.9 1 0.8 GSTO1 glutathione-S-transferase omega 1 A_23_P125 4 1 1.2 0.7 0.8 1 0.2 0.7 0.8 0.5 0.4 0.8 0.9 1 0.8 HHEX hematopoietically expressed homeobox A_23_P470 34 1 1.1 0.8 0.7 0.8 0.2 0.7 0.7 0.6 0.4 0.8 0.8 0.9 0.8 HLA-DMB major histocompatibility complex, class II, DM beta precursor A_32_P351 968 1 0.5 0.6 0.7 0.6 0.1 0.7 0.8 0.4 0.1 0.6 0.7 0.8 0.6 HPGD hydroxyprostaglandin dehydrogenase 15-(NAD) A_23_P213 050 1.1 1.2 0.9 0.9 0.7 0.2 0.9 1 0.5 0.3 1 0.9 0.9 0.9 HTRA1 HtrA serine peptidase 1 A_23_P979 90 0.9 0.6 1.1 0.9 0.7 0.1 0.9 0.9 0.6 0.2 1.1 0.9 0.8 0.7 IL6R interleukin 6 receptor isoform 1 precursor A_24_P379 413 1.2 1 1 1 0.7 0.1 1.2 1.1 0.5 0.2 0.9 0.9 1.1 0.8 INHBB inhibin beta B subunit precursor A_23_P153 964 0.8 0.8 1.3 1.1 0.8 0.2 1 1 0.7 0.4 1.3 1.1 1 0.9 KCNK3 potassium channel, subfamily K, member 3 A_23_P357 724 1.4 1.3 0.9 0.9 1.3 0.2 0.7 1 0.4 0.4 1 1.2 1.3 1 KCTD1 potassium channel tetramerisation domain containing 1 A_23_P130 352 0.9 1 0.6 0.8 0.6 0.2 0.7 0.8 0.5 0.3 0.8 1 0.8 0.7 KIAA1815 hypothetical protein LOC79956 A_23_P169 154 1 0.8 0.6 0.8 0.8 0.2 0.6 0.7 0.4 0.3 0.8 0.8 1.1 0.8 KIAA1822 L KIAA1822-like A_23_P860 59 1.1 1 1.6 1.3 1.4 0.2 1.2 1.2 0.8 0.2 1.5 1.3 1.4 0.8 MAGEH1 melanoma antigen, family H, 1 protein A_23_P341 44 1.1 0.8 0.9 0.9 0.6 0.2 0.6 0.7 0.4 0.3 1 0.9 0.8 0.8 MALAT1 A_24_P497 244 0.9 1.2 2.1 1.5 0.9 0.2 1.8 1.6 0.9 0.3 1.8 1.3 1.3 0.9 MALL mal, T-cell differentiation protein- like A_23_P102 551 0.7 0.5 0.5 0.6 0.6 0.2 0.5 0.6 0.3 0.2 0.5 0.7 0.9 0.8 MANEA mannosidase, endo-alpha A_23_P255 663 1.2 1 0.7 0.7 0.8 0.2 0.6 0.7 0.6 0.4 0.8 0.8 1.1 1 MANSC1 MANSC domain containing 1 A_23_P162 211 1 1 0.7 0.7 0.6 0.2 0.6 0.5 0.4 0.3 0.8 0.9 1 1 MATN2 matrilin 2 isoform b precursor A_23_P713 28 1 0.7 0.8 0.8 0.8 0.1 0.7 0.8 0.5 0.2 0.8 0.9 1 0.9 MATN3 matrilin 3 precursor A_23_P102 058 1.1 1.2 0.6 0.7 0.7 0.2 0.6 0.6 0.4 0.4 0.7 0.8 0.9 0.9 METTL7A methyltransferase like 7A A_23_P415 021 1.4 0.7 1.5 1.2 0.8 0.1 1.1 0.9 0.5 0.1 1.8 0.9 0.8 0.6 METTL7B methyltransferase like 7B A_24_P646 53 0.9 0.8 0.6 0.7 0.6 0.2 0.7 0.7 0.4 0.3 0.8 0.8 0.9 0.7 MGAT3 A_24_P245 838 1.2 1 1.5 1.2 0.8 0.1 1 1.2 0.7 0.3 1.3 1.2 0.9 0.6 MMP7 matrix metalloproteinase 7 preproprotein A_23_P527 61 1.3 1.3 1.1 1 0.8 0.2 0.9 0.9 0.6 0.4 1.1 0.9 0.8 0.7 MOSC1 MOCO sulphurase C-terminal domain containing 1 A_24_P233 995 0.9 0.8 1.3 1.2 0.6 0.1 1 1.2 0.6 0.3 1.2 1.1 0.9 0.6 NMU neuromedin U A_23_P695 37 1.2 0.9 0.8 0.8 0.8 0.1 0.9 0.8 0.5 0.2 0.9 1 0.8 0.8 NQO1 NAD(P)H menadione oxidoreductase 1, dioxin- inducible isoform a A_23_P206 661 1 0.9 0.7 0.8 0.8 0.2 0.7 0.8 0.5 0.3 0.8 0.9 1 0.8 NT5E 5' nucleotidase, ecto A_24_P316 430 1.2 1.7 0.4 0.6 0.9 0.2 0.7 0.8 0.9 0.7 0.5 0.6 0.7 1.1 NTS neurotensin/neuromedin N preproprotein A_23_P368 82 1.2 1.1 1.1 0.9 0.8 0.1 0.9 0.9 0.4 0.2 1.2 1.1 1 0.8 OLFML2A olfactomedin-like 2A A_24_P220 485 0.9 0.6 1.1 1 0.8 0.2 1.1 1 0.7 0.3 1.1 1 0.9 0.9 PCYOX1L prenylcysteine oxidase 1 like A_23_P302 75 1.2 1 1.2 1.1 0.8 0.2 1.2 1.1 0.8 0.4 1.2 1.1 0.9 1 PDGFD platelet derived growth factor D isoform 1 precursor A_24_P124 349 1.2 1.1 0.8 1 0.7 0.1 0.7 0.8 0.5 0.2 0.9 1 1 0.6 PDK3 pyruvate dehydrogenase kinase, isoenzyme 3 A_23_P250 478 1 0.8 0.7 0.8 0.7 0.2 0.8 0.9 0.4 0.3 0.8 0.8 1 0.9 PDK4 pyruvate dehydrogenase kinase 4 A_24_P243 749 1.4 1.3 1.1 1 0.8 0.1 1 1 0.5 0.3 1.2 0.8 1.1 1 PDK4 pyruvate dehydrogenase kinase 4 A_23_P257 087 1.2 1 0.7 0.9 0.6 0.2 0.9 1 0.4 0.2 0.9 1 1 0.7 PF4 platelet factor 4 (chemokine (C- X-C motif) ligand 4) A_24_P794 03 1.4 0.9 1.1 1.1 0.7 0.1 1 1 0.6 0.3 1.1 1.1 1.1 0.9 PLD1 phospholipase D1, phophatidylcholine-specific A_24_P123 190 1.1 1 1.4 1.3 0.9 0.3 0.8 1.1 0.5 0.2 1.2 0.7 2.1 1.1 PRDM13 PR domain containing 13 A_23_P256 581 1 1 1 1 0.7 0.2 0.7 0.7 0.5 0.3 1.1 1.1 0.9 0.9 PRKACB cAMP-dependent protein kinase catalytic subunit beta isoform 3 A_23_P371 410 1.3 1.1 0.7 0.9 1 0.2 1 0.9 0.5 0.2 0.8 1 1.1 0.9 PRKX protein kinase, X-linked A_23_P217 339 1 0.8 1 0.9 0.7 0.2 1 0.8 0.6 0.3 1 0.9 0.9 0.9 PRPS2 phosphoribosyl pyrophosphate synthetase 2 isoform 2 A_23_P966 41 1.1 0.9 0.6 0.6 0.6 0.2 0.6 0.6 0.4 0.4 0.6 0.7 0.9 0.7 RAB11FIP 4 RAB11 family interacting protein 4 (class II) A_24_P164 549 1.2 0.8 2 1.1 0.9 0.1 1.3 1.1 0.4 0.2 1.8 1.1 1 0.6 RAB37 RAB37, member RAS oncogene family isoform 3 A_23_P414 654 1.1 0.8 1.5 1.1 1 0.2 1 1 0.6 0.2 1.5 1.1 0.9 1.1 RALGPS1 Ral GEF with PH domain and SH3 binding motif 1 A_23_P390 148 1.3 0.9 1.3 1.7 1.2 0.2 1 1.1 0.8 0.2 1.5 1.5 1.6 1.2 194 RHOBTB1 Rho-related BTB domain containing 1 A_24_P158 536 0.9 1 1.2 1.1 0.9 0.1 1.1 1 0.6 0.2 1.1 0.9 1 0.9 RNASE4 ribonuclease, RNase A family, 4 precursor A_23_P205 531 1.5 0.9 0.8 0.9 1.1 0.2 0.6 0.8 0.5 0.2 1 1 1.1 0.9 RNF128 ring finger protein 128 isoform 1 A_23_P148 345 1.1 1 0.5 0.6 0.8 0.2 0.3 0.4 0.3 0.2 0.7 0.6 1.3 0.8 RNF157 ring finger protein 157 A_32_P578 10 0.9 0.8 0.9 0.9 0.7 0.2 0.8 0.8 0.5 0.3 0.9 1 0.8 0.6 SAMD13 dnaj-like protein A_32_P130 788 0.9 0.8 0.6 0.6 0.3 0.2 0.5 0.5 0.2 0.2 0.7 0.6 0.6 0.8 SCARA5 scavenger receptor class A, member 5 (putative) A_23_P941 03 1.1 0.9 1.3 1 1 0.2 0.9 1 0.5 0.3 1.4 1.3 1.2 1.2 SCD5 acyl-CoA-desaturase A_24_P385 134 1.3 1 1.2 1 0.6 0.2 0.9 0.8 0.4 0.3 1.4 0.9 1 0.8 SGCD delta-sarcoglycan isoform 1 A_23_P147 647 1.2 1.3 1 0.9 1.2 0.2 1.2 1 0.8 0.7 1.1 0.9 1.1 0.8 SLC22A3 solute carrier family 22 member 3 A_23_P197 33 1 1 0.9 0.9 0.9 0.1 0.8 0.9 0.6 0.2 1 0.9 1 0.8 SLC22A3 solute carrier family 22 member 3 A_32_P309 404 1.1 0.8 0.8 1 0.7 0.2 0.8 1 0.6 0.2 0.9 0.9 1.2 0.8 SLC23A1 solute carrier family 23 (nucleobase transporters), member 1 isoform b A_23_P219 90 1.2 1 0.4 0.5 0.9 0.1 0.6 0.6 0.6 0.2 0.5 0.6 1 0.7 SLC24A1 solute carrier family 24 (sodium/potassium/calcium exchanger), member 1 A_23_P205 913 1.1 1 0.7 0.7 0.7 0.2 0.4 0.7 0.5 0.4 0.7 0.7 0.7 1 SLC40A1 solute carrier family 40 (iron- regulated transporter), member 1 A_23_P102 391 1.2 1.2 1.1 1.1 0.4 0.1 0.8 0.8 0.3 0.2 1.2 1.1 0.8 0.6 SOX2 sex-determining region Y-box 2 A_23_P401 055 1.1 1.2 1 0.9 0.8 0.2 1 0.9 0.5 0.3 1.1 1.1 1 1.1 SPC25 spindle pole body component 25 A_23_P510 85 1.1 0.9 0.8 0.7 0.6 0.2 1.2 0.9 0.5 0.4 0.8 0.7 0.9 0.7 SPP1 secreted phosphoprotein 1 isoform b A_23_P731 3 1.2 1.1 1.1 1 0.9 0.2 0.7 0.8 0.5 0.2 1.1 1.1 1.3 0.9 SRI sorcin isoform a A_23_P597 18 1.2 0.9 0.7 0.9 0.6 0.2 0.7 0.8 0.5 0.4 0.7 0.9 0.8 0.8 ST3GAL5 ST3 beta-galactoside alpha-2,3- sialyltransferase 5 isoform 1 A_23_P311 869 0.8 0.9 0.8 0.6 0.5 0.2 0.8 0.8 0.5 0.5 0.7 0.7 0.7 1.1 ST6GAL2 KIAA1877 protein A_32_P126 157 0.9 0.7 0.8 0.7 0.7 0.2 0.7 0.7 0.4 0.2 0.8 0.6 1.3 1.3 ST8SIA4 ST8 alpha-N-acetyl- neuraminide alpha-2,8- sialyltransferase 4 isoform a A_23_P361 984 4.1 1.6 0.9 1.2 3 1.4 0.9 1 1.3 0.6 1.7 0.8 2.6 1.6 SULF2 sulfatase 2 isoform a precursor A_23_P154 605 1.2 0.9 1.8 1.6 1.1 0.2 1.3 1.1 0.7 0.2 1.8 1.6 1.3 0.9 TACSTD1 tumor-associated calcium signal transducer 1 precursor A_23_P910 81 1.3 0.9 0.9 0.9 0.8 0.1 0.8 0.8 0.5 0.2 0.9 0.9 1 0.8 TFAP2A transcription factor AP-2 alpha isoform a A_32_P141 87 1.4 1 1.1 0.9 0.9 0.1 0.9 0.9 0.8 0.4 1.1 1.1 1 0.7 THRA thyroid hormone receptor, alpha isoform 2 A_23_P207 742 1.2 1.1 1.2 1.2 1 0.2 1.3 1.2 0.9 0.3 1.2 1.1 0.8 0.9 TM4SF18 transmembrane 4 L six family member 18 A_24_P120 251 1 0.9 0.7 0.7 0.7 0.2 0.7 0.8 0.5 0.3 0.7 0.7 0.8 0.7 TM4SF4 transmembrane 4 superfamily member 4 A_23_P252 432 1.1 0.7 0.6 0.7 0.5 0.1 0.7 0.8 0.4 0.2 0.7 0.6 0.7 0.5 TRIM2 tripartite motif-containing 2 A_23_P213 141 1.3 0.8 1.3 1.2 1.1 0.2 1.2 1.1 0.7 0.4 1.4 1.2 1.2 1.2 TSC22D1 TSC22 domain family 1 isoform 1 A_23_P162 739 0.9 1.1 0.7 0.8 0.7 0.2 0.6 0.7 0.5 0.3 0.7 0.8 0.8 0.8 UGT1A6 UDP glycosyltransferase 1 family, polypeptide A6 isoform 1 precursor A_24_P222 872 1 0.7 0.7 0.7 0.5 0.1 0.5 0.7 0.3 0.2 0.7 1 1.1 0.6 UGT1A6 UDP glycosyltransferase 1 family, polypeptide A6 isoform 1 precursor A_23_P605 99 1.2 1.1 1.2 1.2 0.7 0.2 1 1 0.6 0.4 1.3 1.2 1.2 0.9 UGT2B11 UDP glycosyltransferase 2 family, polypeptide B11 A_23_P212 968 1.1 0.5 0.6 0.8 0.8 0.2 0.7 0.8 0.9 0.2 0.7 1.1 1.1 0.7 UPK1B uroplakin 1B A_24_P200 219 1 0.7 0.3 0.6 0.5 0.3 0.5 0.6 0.5 0.2 0.6 0.7 0.9 0.8 USH1C A_23_P127 818 0.9 1.2 1.7 1.1 1.6 0.2 1.9 1.8 1 0.3 1 0.8 1.1 0.9 VAV3 vav 3 oncogene isoform 1 A_23_P201 551 1.1 0.9 1.2 1 1 0.1 1 1 0.5 0.1 1.4 0.9 1.4 0.7 hypothetical protein LOC134145 A_23_P133 279 1.1 0.5 0.8 0.8 0.6 0.2 0.8 0.8 0.4 0.3 0.9 0.9 0.9 0.9 hypothetical protein LOC25854 A_23_P416 965 1.2 0.8 0.9 0.9 0.9 0.2 0.9 0.8 0.6 0.3 0.9 1 0.9 0.8 hypothetical protein LOC54842 A_23_P285 30 1.1 1 0.7 0.7 0.7 0.2 0.6 0.6 0.4 0.3 0.8 0.8 0.8 1.1 OK/SW-CL.92 A_24_P919 683 1.2 0.9 1.8 1.5 0.8 0.1 1.4 1.3 0.6 0.3 1.3 1 1.1 0.8 phospholysine phosphohistidine inorganic pyrophosphate phosphatase A_24_P355 493 0.9 0.8 0.6 0.8 0.8 0.2 0.7 0.8 0.6 0.3 0.8 0.9 0.9 0.7 A_32_P992 4 1 1 1 1.1 0.9 0.2 0.9 1 0.6 0.3 1 1 1.1 0.9 A_23_P464 47 1 0.8 1 1 0.8 0.2 0.8 1 0.6 0.2 1 1.1 1 0.9 A_32_P218 707 1.1 0.9 1.3 1.1 1.1 0.2 1.2 1 0.8 0.4 1.2 1.2 1.3 1.3 A_24_P451 992 0.9 0.7 0.7 0.7 0.7 0.1 0.6 0.8 0.5 0.2 0.7 0.9 0.8 0.6 195 A_24_P918 762 1.1 0.9 1 0.9 0.9 0.2 0.8 0.8 0.5 0.3 1 1 1.1 0.9 A_24_P354 488 1.1 0.9 0.9 0.9 0.7 0.2 0.7 0.8 0.5 0.3 0.9 1 1 0.9 A_24_P307 306 1 0.8 0.7 0.8 0.7 0.2 0.7 0.8 0.4 0.3 0.8 1 0.8 0.8 A_24_P161 725 0.9 0.9 0.8 0.9 0.9 0.2 0.7 0.9 0.5 0.3 0.8 0.8 1.2 0.9 A_24_P152 845 1.1 0.8 0.6 0.7 0.9 0.2 0.6 0.7 0.5 0.3 0.7 0.9 1.2 0.8 A_32_P959 73 1.1 1 1 1.1 0.9 0.2 0.9 1.1 0.5 0.3 1.1 1.3 1.3 1 A_32_P145 502 1.1 1.2 1.1 1.2 1.1 0.2 1.1 1 0.8 0.5 1.4 1.4 1.2 1 A_32_P161 755 0.8 0.7 0.9 1 0.8 0.2 0.8 0.9 0.5 0.3 0.9 1 1 0.7 A_24_P920 388 0.9 0.9 1.1 1.5 0.9 0.2 0.9 0.9 0.5 0.3 1 1 1 0.8 A_24_P128 524 1.2 1 1.1 1.1 0.8 0.1 0.9 0.9 0.7 0.4 1 1.2 1 0.9 A_23_P796 22 1.3 1.1 1.1 1 0.8 0.2 1 0.9 0.6 0.3 1.1 1.1 0.9 0.9 A_24_P680 548 0.9 0.8 0.8 0.8 0.6 0.1 0.8 0.8 0.6 0.3 0.8 0.8 0.8 0.7 A_24_P647 163 1.1 0.9 0.8 0.8 0.6 0.1 0.8 0.8 0.5 0.2 0.9 0.8 0.8 0.7 A_24_P159 06 1.2 1.1 0.9 0.9 0.6 0.1 1 1 0.5 0.3 0.9 0.9 0.9 0.7 A_24_P108 281 1.1 0.7 0.9 0.9 0.6 0.2 1 0.8 0.5 0.4 0.8 1 0.9 0.8 A_32_P925 36 1.3 1 1.4 1.2 0.8 0.2 1.2 0.9 0.6 0.3 1.3 1 1.2 1 A_32_P130 522 1.2 1 1.1 1.1 0.9 0.2 1 0.9 0.6 0.4 1.2 1 1 1.3 A_32_P717 44 1.1 0.9 1.2 1 0.9 0.2 1 1 0.6 0.3 1.2 0.9 1.2 1.1 A_24_P706 752 1.2 0.8 1 0.9 0.8 0.2 0.9 0.9 0.6 0.3 1 1.1 1.1 1.1 A_24_P303 145 1.1 0.7 1.3 1 0.7 0.2 0.9 0.9 0.5 0.2 1.1 1.1 1 0.9 A_23_P259 561 1 0.6 0.8 0.8 0.8 0.1 0.8 1 0.4 0.2 1 0.9 1 0.7 A_24_P118 938 1 1.1 0.8 1 0.7 0.2 1 0.8 0.6 0.3 1.4 1.1 1.1 0.8 A_32_P914 91 1.2 1.2 0.8 0.8 0.8 0.1 0.9 1 0.6 0.3 0.8 0.9 1 1 A_32_P662 97 1 0.9 0.8 1 0.7 0.2 0.8 0.9 0.5 0.4 0.7 0.9 1 0.8 A_32_P228 254 1.1 1 0.7 0.7 0.5 0.2 0.7 0.7 0.4 0.4 0.9 0.7 0.8 0.7 A_23_P753 62 1.1 0.9 0.9 0.8 0.9 0.2 0.8 0.9 0.6 0.4 0.8 1 1 0.9 A_24_P625 898 0.9 1 0.5 0.6 0.5 0.2 0.5 0.6 0.4 0.4 0.6 0.7 0.7 0.8 A_23_P213 527 1.3 1.3 1.2 1 1.3 0.2 0.8 0.8 0.9 0.4 1.2 1 0.9 0.9 A_32_P710 32 0.9 0.5 0.7 0.8 0.4 0.1 0.7 0.9 0.4 0.1 0.8 0.8 0.9 0.7 A_32_P697 83 1.1 0.6 1.4 1.2 0.8 0.2 1.1 1.1 0.5 0.1 1.2 1.2 1.3 1 A_24_P340 112 1.3 0.4 1 0.9 0.6 0.2 0.9 0.7 0.3 0.1 0.9 1 0.9 1.1 A_24_P132 703 1.3 1 1.1 1.1 0.8 0.2 1.1 0.7 0.4 0.3 1.5 1.1 1 1 A_32_P640 04 0.8 1.1 0.9 1 0.8 0.1 1.1 1 0.6 0.3 0.9 0.8 0.9 0.9 A_32_P231 25 1.1 1.2 1.5 1.4 1.2 0.2 1.4 1.1 0.8 0.3 1.5 1.1 0.9 1 A_24_P878 419 1 1 1.3 1.1 0.8 0.1 1.2 1.1 0.7 0.3 1.3 1 0.8 0.7 A_24_P213 325 1 1 1.3 1.2 0.9 0.1 1.3 1.2 0.8 0.3 1.4 1.1 0.9 0.8 A_24_P670 63 1 0.9 1.3 1.2 0.9 0.1 1.2 1.2 0.8 0.3 1.2 1 0.8 0.7 A_24_P676 81 1 1 1.2 1.1 0.9 0.1 1.2 1 0.8 0.3 1.2 1 0.8 0.7 A_23_P373 119 1 1 1.3 1.1 0.9 0.1 1.2 1.1 0.7 0.2 1.3 1.1 0.9 0.8 A_24_P871 208 1.5 1.3 1.6 1.5 1 0.2 1.5 1.3 0.7 0.3 1.6 1.4 1.1 1 A_24_P341 106 1.1 1.1 1 1.1 0.8 0.1 1.1 1 0.6 0.2 1.3 1 0.8 0.7 A_24_P246 85 1.1 1.1 1.2 1.2 0.8 0.1 1.2 1.1 0.6 0.3 1.2 1 0.8 0.7 A_24_P827 794 1.3 1.1 1.2 1.2 0.8 0.2 1.1 1.2 0.7 0.4 1.3 1.1 0.8 0.7 A_24_P475 556 1.1 1 1.1 1.1 0.8 0.2 1.3 1.1 0.8 0.3 1.1 1 1 0.9 A_24_P761 490 1.1 1.2 1.4 1.1 0.8 0.2 1.5 1.3 0.9 0.4 1.3 1.1 0.9 0.8 A_24_P109 962 1.1 1.1 1.4 1.2 0.8 0.2 1.5 1.3 0.9 0.4 1.2 1 0.9 0.8 A_23_P316 81 1.1 1.1 1.2 1.3 0.8 0.2 1.3 1.3 1 0.4 1.1 0.9 0.9 0.9 A_24_P324 084 1.2 1.1 1.3 1.3 0.9 0.2 1.5 1.4 1 0.5 1.2 1.4 1.2 1 A_24_P249 82 1 1 1.1 1.2 0.8 0.2 1.3 1.2 0.9 0.5 1 1.3 1.1 0.8 196 A_32_P233 049 1.1 1 1.8 1.5 1 0.2 1.9 1.4 0.6 0.3 1.6 1.4 1.2 1.2 A_23_P327 370 0.8 0.8 1.2 1 0.8 0.2 1.2 1.1 0.6 0.3 1.1 0.9 0.8 0.9 A_32_P198 731 1 0.9 1.3 0.9 0.7 0.2 1.2 1.1 0.6 0.3 1.4 0.9 0.8 0.8 A_24_P150 466 1 0.9 1.4 1.3 0.8 0.2 1.1 1 0.6 0.4 1.4 1.3 0.9 1 A_23_P153 958 0.9 0.9 1.8 1.4 0.9 0.2 1.3 1.3 0.7 0.4 1.6 1.4 1.1 1 A_32_P223 504 2.3 1.7 4.7 3.9 1.6 0.1 3.4 1.9 1 0.2 2.9 2 1.4 1 A_24_P419 017 1.7 1.3 3.4 2.3 1.6 0.1 3.1 2.2 0.7 0.2 2.9 1.9 1.1 0.6 A_32_P213 509 1 1.1 3.8 2.4 0.9 0.1 3.4 2.2 1.1 0.4 3 1.6 1.2 0.8 A_24_P268 474 1 1 2.5 1.4 0.9 0.2 2.2 1.3 0.9 0.4 1.8 1.4 1 0.8 A_32_P133 37 1.2 1.2 2.1 1.6 1 0.1 2.2 1.6 1 0.2 1.7 1.2 1 0.7 A_24_P163 009 0.8 0.8 1.7 1.2 0.8 0.1 1.4 1.2 0.9 0.2 1.4 0.9 0.9 0.7 A_23_P480 80 0.8 0.9 1.5 1.3 0.9 0.2 1.4 1.3 0.7 0.3 1.2 1.2 1 0.9 A_32_P195 346 1.2 1.7 4 1.8 1.1 0.2 4.4 2.3 1 0.3 2.3 1.3 1.2 0.9 A_24_P163 78 0.9 1 1.6 1.4 0.8 0.2 1.9 1.4 0.8 0.4 1.3 1 0.9 1.1 A_32_P546 28 1.5 1.2 0.7 0.9 1.3 0.1 0.7 1.1 1.2 0.2 0.8 0.9 0.9 0.6 A_32_P174 874 1 1.1 1.3 1 1.1 0.2 1 1.4 1 0.3 1.1 1 1.2 0.9 A_32_P378 3 0.9 1.9 3.5 2.4 0.8 0.2 3.8 2.4 1 0.6 2.7 1.3 1.1 1.2 A_24_P665 185 1.3 2 4.1 2.3 1.1 0.2 3.4 2.4 1.3 0.6 3.2 1.4 1.2 1.1 A_32_P977 39 0.9 0.7 0.4 0.5 0.6 0.2 0.3 0.6 0.4 0.2 0.4 0.6 1.1 1 A_23_P316 487 0.9 0.7 0.4 0.4 0.8 0.3 0.4 0.5 0.5 0.2 0.4 0.6 1.2 0.9 A_23_P305 245 1.2 1.2 0.5 0.6 0.8 0.2 0.6 0.6 0.4 0.2 0.6 1 1.3 1.1 A_32_P442 10 1.3 0.9 1.1 0.7 1.3 0.4 0.6 0.8 0.5 0.2 1.1 0.9 1.4 1.2 A_32_P873 23 1.2 0.9 0.9 0.8 1 0.2 0.7 0.8 0.6 0.2 0.7 1.1 1.1 1 A_32_P131 998 1 0.8 0.7 0.9 1 0.2 0.8 0.8 0.5 0.3 0.7 1 1.5 1.4 A_32_P452 29 1.3 1.1 0.8 0.8 0.6 0.2 0.8 0.7 0.3 0.3 0.8 0.7 1 0.9 A_32_P448 31 1.1 0.9 0.8 0.8 0.7 0.2 0.6 0.6 0.5 0.4 0.8 1 1.1 1.2 A_32_P116 70 1.4 1 0.5 1.2 0.9 0.2 0.5 1.2 0.8 0.2 0.6 1 1 0.9 A_23_P156 996 1.3 1.4 0.9 1.2 2.3 0.2 1.1 0.9 1.8 0.4 0.8 1.3 1.4 1.2 A_32_P203 688 1.1 0.5 1 0.7 0.5 0.3 0.8 0.8 0.3 0.2 1.3 0.9 1.1 1.2 A_23_P416 305 1 0.2 1.2 1 0.4 0.2 1.2 1.1 0.3 0.3 1 1.1 0.9 0.7 A_32_P214 503 0.9 0.4 0.8 0.8 0.8 0.3 0.8 0.7 0.4 0.2 0.9 1 1.5 1.4 A_23_P204 541 1 0.6 0.6 0.8 1 0.3 0.5 0.7 0.5 0.2 0.7 0.9 1.6 1.7 ?Genes listed in each cluster correspond to the same genes hierarchically clustered in Figure 4. *Rows in bold correspond to 76 additional interferon induced genes from Table S1. ?Rows in blue correspond to the 17 predominantly IFN-responsive but wt HPIV1 unresponsive genes from cluster C of figure 1. 14 of those genes are found in cluster F, 1 gene in cluster C, 1 gene in cluster H, and 1 gene (RET) was not significantly differentially expressed above 4-fold between virus infections or with IFN? treatment. 197 Table S3. Table S3. Comparison of IFNB mRNA message by qPCR IFNB Expression (Van Cleve et al. 2006) IFNB Expression with Taqman Assay Virus Time vs Mock Ratio CF170S:WT vs Mock Ratio CF170S:WT WT 5.9 261.7 CF170S 24 h 240.5 40.8 27007.7 103.2 WT 12.2 1227.4 CF170S 48 h 5595.3 458.3 853061.5 695.0 198 Table S4. Symbol Product Agilent 6 h 24 h 6 h 12 h 24 h 48 h 6 h 12 h 24 h 48 h 6 h 12 h 24 h 48 h AKT1 v-akt murine thymoma viral oncogene homolog 1 A_23_P296 0 0.6 0.6 0.7 0.7 0.8 0.5 0.7 0.8 0.7 0.5 0.7 0.9 0.8 0.6 BAG1 BCL2-associated athanogene isoform 1L A_23_P146 654 0.9 0.9 0.8 0.8 1.1 1.1 0.8 0.9 1 0.9 0.8 0.8 0.9 1.1 BAG3 BCL2-associated athanogene 3 A_23_P525 52 1.2 1.1 0.8 0.9 0.9 0.6 0.7 0.7 0.6 0.8 0.8 1 1 0.8 BAG3 BCL2-associated athanogene 3 A_23_P470 77 1.1 1 0.8 0.8 0.7 0.6 0.7 0.8 0.6 0.7 0.8 0.9 0.9 0.7 BAG4 BCL2-associated athanogene 4 A_23_P146 217 1.2 1.1 0.8 0.9 1 0.8 0.7 0.7 0.5 0.7 0.7 0.9 1 0.7 BCL2 B-cell lymphoma protein 2 alpha isoform A_23_P352 266 1.2 1.3 0.9 0.9 0.9 0.6 0.8 0.8 0.5 0.9 1.1 1.2 0.9 1.1 BCL2 B-cell lymphoma protein 2 beta isoform A_23_P208 132 1 1.1 1.7 1.4 1.3 2.1 1.6 1.6 1.7 1.5 1.6 1.4 1.1 1.1 BCL2A 1 BCL2-related protein A1 A_23_P321 703 1 1 0.8 0.9 0.9 1.5 0.9 0.9 1.1 1.3 0.8 0.9 0.8 1 BCL2A 1 BCL2-related protein A1 A_23_P152 002 1.2 1.4 0.9 0.8 1.8 29.3 1 0.9 3.9 13.9 0.9 1.1 1.3 5 BCL2L 1 BCL2-like 1 isoform 1 A_23_P210 886 0.6 0.6 0.5 0.7 0.8 0.5 0.6 0.7 0.6 0.6 0.6 0.8 0.8 0.7 BCL2L 1 BCL2-like 1 isoform 2 A_24_P100 130 0.8 0.9 0.5 0.8 0.7 0.5 0.7 0.8 0.7 0.9 0.6 0.8 0.7 0.7 BCL2L 10 BCL2-like 10 (apoptosis facilitator) A_23_P163 209 1.5 1.1 1.9 1.4 1.1 1.2 1.4 1.2 0.9 1.4 1.6 1.5 1.4 0.8 BCL2L 12 BCL2-like 12 isoform 1 A_23_P504 77 0.9 0.9 1 1 1.1 1.5 1 1.1 1.4 1.6 1 1 1 1 BCL2L 13 BCL2-like 13 (apoptosis facilitator) A_24_P410 389 1.4 1.4 0.8 0.6 2.6 0.8 0.6 0.7 1.3 1.4 0.5 0.9 1.2 1.2 BCL2L 13 BCL2-like 13 (apoptosis facilitator) A_24_P409 494 0.9 0.9 0.7 0.9 0.9 1.4 0.9 0.9 0.8 1.2 0.7 0.9 1 1.1 BCL2L 13 BCL2-like 13 (apoptosis facilitator) A_23_P102 965 1.7 1.4 1 1 2.7 2.1 1 1 1.8 1.9 1 0.8 1 1.4 BCL2L 2 BCL2-like 2 protein A_23_P418 373 1 1.1 1.1 1.1 1.2 1.6 1.1 1 1.1 1.4 1.1 1 1.1 1.2 BFAR apoptosis regulator A_24_P184 931 0.8 0.9 0.5 0.6 0.7 0.6 0.6 0.6 0.6 0.8 0.7 0.7 0.7 0.8 BFAR apoptosis regulator A_23_P659 63 0.9 0.9 0.8 0.9 0.9 0.9 0.8 0.9 0.8 0.9 0.8 0.9 0.9 1.1 BIRC2 baculoviral IAP repeat-containing protein 2 A_24_P115 774 1 1.1 0.7 0.8 1.3 3 0.7 0.8 1.4 2.5 0.8 0.9 1.1 1.6 BIRC3 baculoviral IAP repeat-containing protein 3 A_23_P983 50 1.1 1.9 1 0.9 6.5 40.8 0.7 0.9 11.6 34.9 1 1 1.7 9.2 BIRC4 baculoviral IAP repeat-containing protein 4 A_23_P224 60 1.2 1.4 0.8 0.9 1.4 2.4 0.9 0.9 1.3 2.4 0.9 0.9 1 1.4 BIRC5 baculoviral IAP repeat-containing protein 5 isoform 3 A_23_P118 815 0.9 0.8 0.7 0.7 0.9 0.5 0.9 0.9 0.8 0.8 0.7 0.7 0.9 1 BIRC6 baculoviral IAP repeat-containing 6 A_23_P142 616 1.2 1 1 0.9 1 1.2 0.9 0.9 1.1 1 0.9 0.9 1.2 1.1 BIRC7 livin inhibitor of apoptosis isoform beta A_23_P797 69 0.8 0.7 1.3 1.1 0.9 0.6 1.3 1.3 1.3 0.6 1.1 0.9 0.7 0.7 BIRC8 baculoviral IAP repeat-containing 8 A_23_P900 58 0.8 0.8 0.9 1 1 1.2 1 0.9 1.2 1 1 0.9 1 1 BNIP1 BCL2/adenovirus E1B 19kD interacting protein 1 isoform BNIP1-a A_23_P765 5 0.9 0.9 1 1 1.2 1.9 1 1 1.2 2.2 1 1.2 1.1 1.2 BNIP2 BCL2/adenovirus E1B 19kD interacting protein 2 A_23_P317 001 1.2 1.1 1 1.1 1.1 1.8 0.9 1 1.2 1.7 1 1 1.1 1.4 BRAF v-raf murine sarcoma viral oncogene homolog B1 A_23_P429 35 1.1 1.1 0.9 1.1 1.1 3.6 1 1.1 1.7 2.8 0.9 1 1 1.3 CFLAR CASP8 and FADD- like apoptosis regulator A_24_P120 115 1 1.1 0.9 0.8 1.4 1.6 0.8 0.8 1.8 1.3 0.8 0.8 0.8 1.3 CFLAR FLAME-1-delta A_23_P209 394 0.9 1.1 0.6 0.7 1.2 3.2 0.7 0.8 1.4 2.9 0.6 0.8 0.8 1.5 199 GDNF glial cell derived neurotrophic factor isoform 1 preproprotein A_24_P255 44 0.8 0.7 0.8 1.1 1 1.1 1 1 1 1 1 0.8 1 0.9 GDNF glial cell derived neurotrophic factor isoform 1 preproprotein A_23_P167 683 0.8 1 5.8 1.1 1 1.2 1 0.9 1.2 0.9 1 0.9 0.9 1 GDNF HFK4-GDNF A_24_P376 451 1.1 1.3 1.9 1.4 1.4 2.6 1.7 1.5 2.4 1.7 1.7 1.3 1 1.3 IGF1R IGF1R protein A_23_P417 282 1.2 0.9 1.4 1 0.9 0.4 1.2 1 0.8 0.5 1.3 1 0.9 0.8 IGF1R insulin-like growth factor 1 receptor precursor A_23_P205 986 1 0.9 0.8 0.8 0.8 0.9 0.8 0.8 0.7 0.9 0.8 0.8 0.7 0.7 IGF1R unknown protein A_23_P305 680 1.2 0.7 1.3 0.7 1.1 1.3 1.1 0.8 1.5 1 1 1.1 0.8 0.8 MCL1 myeloid cell leukemia sequence 1 isoform 1 A_24_P336 759 1.1 1.1 0.8 0.9 2.3 2.3 0.8 1 2.9 1.7 0.8 0.9 1 1.2 MCL1 myeloid cell leukemia sequence 1 isoform 1 A_24_P336 754 1.2 1.2 1.1 1.1 3.3 4.6 1.1 1.3 4.6 3.7 1 1 1.2 1.5 MCL1 myeloid cell leukemia sequence 1 isoform 1 A_24_P319 635 1.4 1.4 0.6 0.9 3.8 9.2 0.7 1 5.1 8.4 0.7 0.9 1.1 1.6 NOL3 nucleolar protein 3 A_23_P206 371 1 1 1.2 1.2 1.3 0.6 1.2 1.2 1.2 0.6 1.1 1.3 1.2 0.9 200 Table S5. Table S5. Expression of known intrinsic apoptosis mediators after HPIV1 infection or IFN treatment Avo nex Avo nex F17 0S F17 0S F17 0S F17 0S P(C- ) P(C- ) P(C- ) P(C- ) WT WT WT WT Symbol Product Agilent 6 h 24 h 6 h 12 h 24 h 48 h 6 h 12 h 24 h 48 h 6 h 12 h 24 h 48 h APAF1 apoptotic peptidase activating factor 1 isoform c A_23_P3661 1 1 1 0.9 1 0.9 1.1 1.2 0.9 0.8 1.3 1 0.8 1 1.1 BAK1 BCL2- antagonist/killer 1 A_23_P1453 57 1.2 0.9 0.8 0.9 1.2 0.8 1 1 0.9 0.9 0.9 1 1 1 BAX BCL2-associated X protein isoform epsilon A_23_P2087 06 0.8 0.7 0.9 1 0.9 0.5 0.9 1 0.9 0.6 0.9 1 0.9 0.9 BID BH3 interacting domain death agonist isoform 1 A_24_P1879 48 1 1 0.9 1 0.9 0.6 0.9 1 0.8 0.8 0.9 0.9 1 1.2 BID BH3 interacting domain death agonist isoform 1 A_23_P1549 29 1 1 0.8 0.9 0.7 0.5 0.8 0.9 0.6 0.7 0.9 0.9 1 1.2 CASP9 caspase 9 isoform alpha preproprotein A_24_P1113 42 1 1 1.1 1 1 0.9 1.1 1.1 0.9 0.9 1 1 0.9 1 CASP9 caspase 9 isoform alpha preproprotein A_23_P9730 9 1.1 1.2 1.3 1.2 1.2 1 1.3 1.2 1.1 1.1 1.3 1.2 1.1 1.2 DIABL O diablo isoform 1 precursor A_23_P4780 0 0.9 0.8 1 1 1 1.2 1 1 0.9 1.1 0.9 1 0.9 1 PMAIP 1 phorbol-12- myristate-13- acetate-induced protein 1 A_23_P2079 99 2 1.4 0.6 1.1 12.6 34.2 0.7 2 24.5 21.1 0.7 1.1 1.8 5.3 201 Table S6. Table S6. Genes Differentially Expressed between CF170S and P(C-) infection by 2-Fold followed by P < 0.01 with MTC F170S P(C-) Ratio F170S : P(C-)a Symbol Product Agilent 6 12 24 48 6 12 24 48 6 12 24 48 ABHD6 abhydrolase domain containing 6 A_23_P211850 1.4 1.2 0.9 0.2 1.2 1.1 0.7 0.5 1.2 1 1.4 0.5 ASMTL acetylserotonin O- methyltransferase- like A_23_P159539 1 1 1.4 4.9 1.3 1.1 3.4 3.2 0.8 0.9 0.4 1.5 BTN2A2 butyrophilin, subfamily 2, member A2 isoform b A_24_P337592 1.4 1.3 3.3 27.3 1.4 1.4 9 19 1 1 0.4 1.4 CASP3 caspase 3 preproprotein A_23_P92410 0.9 1 2.3 6.3 1 1 6.5 4.1 1 1 0.3 1.6 DUSP8b dual specificity phosphatase 8 A_24_P322867 1.8 2 2.7 10 1.7 1.8 7.4 5.2 1.1 1.1 0.4 1.9 FAM64A hypothetical protein LOC54478 A_23_P49878 0.6 0.6 0.7 0.1 0.7 0.8 0.5 0.3 0.8 0.8 1.3 0.5 HIVEP2 human immunodeficiency virus type I enhancer binding protein 2 A_23_P214766 1.5 1.3 2.1 14.2 1.5 1.2 5.1 9.1 1 1 0.4 1.6 IFIT3 interferon-induced protein with tetratricopeptide repeats 3 A_23_P35412 0.9 7.3 226.3 421.9 2.2 21.2 353.2 224.7 0.4 0.3 0.6 1.9 MAP3K8 mitogen-activated protein kinase kinase kinase 8 A_23_P23947 1.4 1.4 4.1 6.6 1.2 1.8 4.7 2.9 1.1 0.8 0.9 2.3 NAV2 steerin2 protein A_24_P933458 1.9 1.5 2.9 14.3 2.1 1.8 8.3 7.3 0.9 0.8 0.3 2 NCOA7 nuclear receptor coactivator 7 A_23_P156957 1 1.2 7.7 15.9 1 3.2 12.2 7.2 1 0.4 0.6 2.2 PRIM1 DNA primase small subunit, 49kDa A_23_P25019 0.6 0.7 0.7 0.2 0.8 0.8 0.5 0.4 0.9 0.9 1.2 0.5 PTGS2 prostaglandin- endoperoxide synthase 2 precursor A_24_P250922 0.8 0.9 5 12.3 0.6 1.2 7.6 4.4 1.4 0.8 0.7 2.8 PTPRG PTPRG protein A_32_P93493 0.9 2.6 1.3 0.2 1.1 2.3 1.8 0.4 0.8 1.1 0.7 0.4 RBM35A RNA binding motif protein 35A isoform 1 A_23_P259127 1 1.1 0.8 1.2 0.7 1.1 1.2 5.6 1.4 1 0.6 0.2 RELB reticuloendotheliosis viral oncogene homolog B A_23_P55706 1.2 1.1 2.4 25.7 1.1 1 6.2 22.2 1.1 1 0.4 1.2 SIPA1L2 signal-induced proliferation- associated 1 like 2 A_23_P137470 1.4 1.2 1.7 7.7 1.1 1.2 3.8 3.7 1.2 1.1 0.5 2.1 SLC27A2 solute carrier family 27 (fatty acid transporter), member 2 A_23_P140450 0.8 0.9 0.7 0.2 0.7 0.8 0.5 0.5 1 1.1 1.3 0.4 SLC2A13 solute carrier family 2 (facilitated glucose transporter), member 13 A_23_P10211 1.1 1 1.5 14 0.9 0.9 3.5 11.3 1.2 1.1 0.4 1.2 SLCO5A1 organic anion transporter polypeptide-related protein 4 A_23_P135669 0.7 0.9 2.3 38.3 0.9 0.8 8.4 17 0.8 1.1 0.3 2.3 202 SPANXA1 sperm protein associated with the nucleus, X chromosome, family member A1 A_23_P96302 0.8 0.9 0.7 0.2 1.1 1 0.7 0.5 0.7 0.9 1 0.4 STK19 serine/threonine kinase 19 isoform 2 A_23_P19479 1.1 1.1 2.8 10.6 1.1 1.2 6.4 4.2 1 0.9 0.4 2.5 TNFRSF10B tumor necrosis factor receptor superfamily, member 10b isoform 1 precursor A_23_P169030 0.9 1 2.3 11.5 1.1 1 4.8 8.9 0.9 1 0.5 1.3 ZEB2b zinc finger homeobox 1b A_23_P142560 1.1 1.1 6.1 90.6 1 1.1 19.2 62.2 1.1 1 0.3 1.5 A_24_P127641 1.4 1.8 16.3 36.6 1.3 2 20 16.6 1.1 0.9 0.8 2.2 A_24_P696924 2.3 1.7 3.8 4 2 2.5 5.2 1.8 1.2 0.7 0.7 2.3 A_32_P207802 3.1 2.4 4.8 17.4 2.6 2 14 5.5 1.2 1.2 0.3 3.1 A_32_P51855 1 1 0.9 9.3 1 1.2 2.2 5.7 1.1 0.9 0.4 1.6 aBoxes indicate times post-infection at which gene expression between CF170S and P(C-) infection differed by greater than 2=fold bGenes shaded in gray were also identified as significantly differentially expressed between CF170S and P(C-) infection by 2-fold with P < 0.01 simultaneously 203 Table S7. Table S7. Genes Differentially Expressed by 4-fold and an ANOVA P < 0.01 with MTCa between CF170S and P(C-) Infection F170S F170S F170S F170S P(C- )b P(C- ) P(C-) P(C-) WT WT WT WT Symbol Product Agilent 6 hr 12 hr 24 hr 48 hr 6 hr 12 hr 24 hr 48 hr 6 hr 12 hr 24 hr 48 hr CCL5 small inducible cytokine A5 precursor A_23_P152838 0.9 19.6 33.6 125.7 5.3 21.8 55.5 90.3 1.8 17.5 23 29.9 IFIT2 interferon-induced protein with tetratricopeptide repeats 2 A_23_P24004 2.4 20.5 926.4 6100.4 11.9 81.2 2569.2 3777.9 2.9 14.5 42.9 531 OASL 2'-5'-oligoadenylate synthetase-like isoform a A_23_P139786 2.1 29.7 77 187.9 11.5 41.5 114.8 135.5 2.4 24.5 33.3 58.9 C20orf141 A_32_P133182 0.8 1.5 48.5 166.7 0.8 3.4 237.7 99.4 1.1 1.1 4.1 13.9 CXXC4 CXXC finger 4 A_23_P121676 1.2 0.7 2.2 35.4 1.1 1 11.2 30.5 1.8 1.1 1.1 2.8 DICER1 dicer1 A_24_P22163 1.5 1.1 2.2 25.7 1.4 1.2 9.5 13.4 1.4 1 1.1 2.7 OTUD1 A_32_P60459 0.8 0.9 7.2 150.9 0.7 1.1 29.3 102.4 0.9 0.6 1.1 7.1 PLCB4 phospholipase C beta 4 isoform a A_23_P28898 1.3 1.7 3.6 22.5 1.5 1.7 16 23.9 1.1 1.2 1.2 1.9 SLC12A7 solute carrier family 12 (potassium/chloride transporters), member 7 A_24_P60634 1 0.8 2.4 20.4 0.8 1.1 11.8 17.7 0.9 1.2 1.3 3.1 SLC12A7 solute carrier family 12 (potassium/chloride transporters), member 7 A_23_P251855 0.7 0.9 2.4 23.8 0.7 0.9 14.6 16 0.7 0.8 1 3.4 SLC12A7 solute carrier family 12 (potassium/chloride transporters), member 7 A_23_P61688 1.7 1.3 3.6 50.1 1.5 1.5 26.5 45.1 1.5 1.3 1.5 5.1 SNPH syntaphilin A_23_P102706 1.4 1.2 5.2 34.9 1.3 1.5 24.9 24.5 1.5 1.2 1.2 4.7 TNFAIP6 tumor necrosis factor, alpha- induced protein 6 precursor A_23_P165624 0.5 0.5 1 50.9 0.9 1 4.3 26.5 0.5 0.6 0.7 7 A_24_P255384 0.9 1 1.8 52.3 0.8 1.3 13.5 31 0.9 0.9 0.9 7.3 A_32_P95766 0.9 1.1 16 125.9 1 1 84.6 88.1 1 1 1 25.5 A_32_P226205 2.1 1.3 3.2 31.6 1.3 1.3 15.7 24.2 1.6 1.1 1 2 FEV FEV (ETS oncogene family) A_23_P5460 1.6 1.5 2.4 10.9 1.7 1.2 1 1 1.2 2 1.5 2.5 SYNPO2 A_23_P310094 0.6 1.2 2.7 9.7 0.5 0.9 2.1 1.6 0.8 1.6 2.3 2.7 aBenjamini-Hochberg multiple testing correction (MTC) bBoxes indicate time at which gene expression differed by greater than 4-fold between CF170S and P(C-) infections. All 18 genes identified as significantly differentially expressed between CF170S and P(C-) infection by two-way ANOVA are also present in the 43 genes identified by a paired t-test without MTC. 204 Table S8. Table S8. RT-qPCR Primers Gene Symbol Assay ID* TaqMan Probe Context Sequence IRF7 Hs00242190_g1 ACCGCGGTGCAAGAGCCCAGCCCAG IFNB1 Hs01077958_s1 TCTTCAACCAGCAGATGCTGTTTAA MX1 Hs00182073_m1 CCTATCACCAGGAGGCCAGCAAGCG NFKB1 Hs00231653_m1 CAAAGCAGCAGGAGCAGATCCCCTG TRAF1 Hs01090170_m1 GCCCTTCCGGAACAAGGTCACCTTC GAPDH Hs99999905_m1 TTGGGCGCCTGGTCACCAGGGCTGC *Applied Biosystems TaqMan Gene Expression Assay ID 205 Supplementary Information for Chapter 4 Supplementary Figure 1. Activation of components of the IFN? enhanceosome by HPIV1 infection. Phosphorylated ATF2 (Thr 71), total ATF2, phosphorylated P38 (Thr 180 / Tyr 182), and total P38 were detected by Western blot in A549 cells (A) infected with WT, F170S, or P(C-) HPIV1 at a MOI of 5 for 48 h or (B) treated with 10 ?g/mL anisomycin for 30 or 60 minutes. n.s. indicates a non-specific band. 206 Supplementary Figure 2. Examination of C protein interaction with RIG-I pathway components. 293T cells were transfected with a Myc-tagged C expression plasmid along with (A) V5-tagged TRIM25 and/or Flag-tagged Riplet, (B) RIGI and/or MDA5, (C) IPS1 and/or TBK1, (D) TRAF3 and/or TRAF6, (E) BIRC2 and/or BIRC4, or (F) TRADD and/or FADD. Myc-tagged TRADD and Flag-tagged TRAF3 was transfected together as a positive control. Myc-tagged proteins were immuno-precipitated using immobilized anti-Myc antibodies. 207 Supplementary Figure 3. Levels of viral genomic versus anti-genomic RNA levels over time during infection with WT and mutant HPIV1. HPIV1 genomic and anti-genomic RNA was distinguished using genome- and anti-genome-specific primers respectively for reverse transcription (RT) followed by real-time PCR. (A) A549 cells were infected with WT, F170S, and P(C-) HPIV1 using a MOI of 5 for 12, 24, or 48 h and genomic (left axis, intact lines) and anti-genomic RNA (right axis, dotted lines) were measured by real- time PCR, normalized to the level of WT HPIV1 anti-genome at 12 h. (B) The ratio of genomic to anti-genomic RNA from (A). 208 Supplementary Figure 4. Relative levels of (A) IRF3 phosphorylation, (B) I?B? degradation, (C), P38 phosphorylation, and (D) PKR knockdown were measured by densitometry of Figure 8a and normalized to mock-infected shCON levels and to GAPDH protein expression. (E) Relative levels of C? protein expression normalized to WT HPIV1-infected shCON levels and to GAPDH. 209 References 1. Ahmed, M., and D. S. Lyles. 1998. 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