ABSTRACT
Title of Document:REGULATION OF INFECTED CEL FUSION
BY THE VACINIA VIRUS A56 AND K2
PROTEINS.
Timothy Robert Wagenaar, Ph.D., 2008
Directed By:Dr. Bernard Moss, Adjunct Professor,
Department of Cel Biology and Molecular
Genetics
Poxviruses are a group of large double-stranded DNA virus that replicate in
the cytoplasm of the cel. The Orthopoxvirus genus includes variola virus, the
etiological agent of smalpox, and vacinia virus (VACV), the prototypical member
of the genus.  Cels infected with VACV display very litle cel-cel fusion, however
VACV mutants deleted for either the A56R or K2L gene display extensive cel-cel
fusion. A56 and K2 interact with one another (A56/K2) and expresion of both
proteins is important for preventing cel fusion. VACV entry and fusion requires a
multiprotein entry fusion complex (EFC) composed of at least eight proteins. In the
absence of a functional EFC infected cell fusion does not occur even when the viruses
lack either A56 or K2. A panel of recombinant VACVs was used to define protein
interaction important for regulation of cel fusion. Afinity purification of A56, K2
and the EFC revealed an interaction betwen A56/K2 and the EFC. This interaction
required expresion of both A56 and K2 as A56 did not bind the EFC in the absence
of K2 and vice versa.  Interestingly, the ability to bind the EFC corelated with the
inhibition of infected cel fusion by A56 and K2. Although the EFC contains eight
proteins, only two entry proteins, A16 and G9, were important for binding A56/K2.
Individualy, A16 and G9 did not bind A56/K2; instead both A16 and G9 were
neded for eficient interaction with A56/K2. A16 and G9 copurified with one
another when expresed by transfection in uninfected cels, confirming that the two
proteins bind to one another sugesting they directly interact within the EFC. To
suport a biological role for A56/K2 binding the EFC, cels expresing A56 and K2
were tested for infectivity as wel as their ability to undergo cel-cel fusion. In both
cases, cels expresing A56 and K2, but not individual expresion of A56 or K2,
showed reduced cel-cel fusion and virus entry. Colectively, these data suport a
model by which A56/K2 regulate infected cel fusion through an interaction with the
viral EFC.
? Copyright by
Timothy Robert Wagenaar
2008
REGULATION OF INFECTED CELL FUSION BY THE VACCINIA VIRUS A56
AND K2 PROTEINS.
By
Timothy Robert Wagenaar
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
2008
Advisory Committee:
Dr. Jonathan Dinman, Chair
Dr. Bernard Moss
Dr. James Culver
Dr. Anne Simon
Dr. Siba Samal
ii
Dedication
I would like to dedicate this work to my parents Fred and Diane Wagenar. Without
their suport and emphasis on the importance of a god education I would never have
reached this point. This work is a tribute to the sacrifices they have made over the
years.
iii
Acknowledgements
There are a number of people that have ben instrumental in completion of this work.
My mentor Dr. Bernard Mos, whose pasion for science is infectious and from his
helpful sugestions I have learned so much. Dr. Charles Grose, who first introduced
me into the wonderful world of viruses and whose lab was a playground for a na?ve,
inexperienced undergraduate. Finaly, Kristie Grebe, who during this proces learned
more about vaccinia virus then she probably cared to, but whose help and support was
critical to my completion.
iv
Table of Contents
Dedication............................................................ii
Acknowledgements.................................................iii
Table of Contents...................................................iv
List of Tables......................................................vi
List of Figures.....................................................vii
List of Abbreviations...............................................viii
Chapter 1:  Review of literature.......................................1
1.1The Poxviridae..............................................1
1.1.1Classification.............................................1
1.1.2Smallpox, Edward Jenner and Vaccinia virus.....................2
1.1.3Genome organization.......................................3
1.1.4Virion Morphology and Infectious particles......................4
1.1.5Viral Proteins.............................................6
1.1.6Recombinant techniques for the investigation of VACV.............9
1.2Virus Entry and Replication...................................12
1.2.1Virus Attachment.........................................12
1.2.2Virus Entry.............................................13
1.2.3Virus gene expression.....................................16
1.2.4Genome replication.......................................19
1.2.5MV morphogenesis.......................................21
1.2.6Formation of Wrapped and Extracellular virus...................23
1.3Virus Fusion and Regulation of Infected Cell Fusion................24
1.3.1Low pH induced cell-cell fusion..............................24
1.3.2A56R and K2L...........................................26
Chapter 2: The VACV A56R and K2L associate with proteins of the
Multicomponent Entry/Fusion Complex.....................29
2.1Introduction...............................................29
2.2Materials and Methods.......................................30
2.2.1Cell and virus propagation..................................30
2.2.2TAP and mass spectrometry.................................31
2.2.3Recombinant virus construction..............................32
2.2.4Western blotting..........................................35
2.2.5Antibodies..............................................37
2.2.6Synthesis and purification of soluble A56/K2....................37
2.3Results...................................................38
2.3.1Neutral pH Cell-Cell Fusion Requires the Entry/Fusion Complex....38
2.3.2The anti-fusion proteins A56 and K2 interact with the viral EFC.....39
2.3.3A56 does not bind the EFC in the absence of K2.................43
2.3.4Both K2 and A56 are needed for association with the EFC..........44
2.3.5Association of A56 and K2 with a TAP-tagged EFC..............48
2.3.6Interaction of soluble A56/K2 with the EFC.....................50
2.4Discussion................................................53
v
Chapter 3: The VACV fusion regulatory proteins A56 and K2 interact with a
subcomplex of A16 and G9................................56
3.1Introduction...............................................56
3.2Material and Methods........................................57
3.2.1Cells and virus...........................................57
3.2.2Plasmid and recombinant VACV construction...................57
3.2.3A16 and G9 Codon Optimization.............................60
3.2.3Affinity purification.......................................61
3.2.5Transfection and coimmunoprecipitation.......................62
3.2.6Western blotting and antibodies..............................62
3.3Results...................................................63
3.3.1A56/K2 interacts with a subset of the proteins within the EFC.......63
3.3.2A16 and G9 selectively copurify with A56/K2...................66
3.3.3A56/K2 copurify with A16/G9...............................70
3.3.3Both A16 and G9 are required for  their association with A56/K2....71
3.3.4Association of A56/K2 with G9 requires A16...................73
3.3.5A16 and G9 stably associate with each other in uninfected cells......73
3.4Discussion................................................76
Chapter 4: Cells expressing A56 and K2 show reduced virus entry and fusion
with VACV infected cells.................................80
4.1Introduction...............................................80
4.2Material and Methods........................................81
4.2.1Cells and virus...........................................81
4.2.2Purification of VACV.....................................81
4.2.3Codon optimization of A56 and K2...........................81
4.2.4Transfection.............................................82
4.2.5Antibody staining and flow cytometry.........................82
4.2.6Quantification of cell-cell fusion.............................83
4.2.7Measuring virus entry.....................................83
4.3Results...................................................84
4.3.1Expression of A56 and K2 in transfected cells...................84
4.3.2A56/K2 expression correlates with reduced fusion with virus induced
syncytia...............................................86
4.3.3A56 and K2 expression reduce virus infection...................89
4.4Discussion................................................91
Chapter 5:  Conclusions and Future Direction..........................93
5.1Conclusions...............................................93
5.2Future Directions...........................................98
Bibliography.....................................................102
vi
List of Tables
Table 2-1 Recombinant viruses utilized for experiments in chapter 2.
Table 3-1 Recombinant viruses utilized for experiments in chapter 3.
vii
List of Figures
Figure 1-1:Vaccinia virus genome organization.
Figure 1-2:Lifecycle of vaccinia virus.
Figure 1-3:Diagram of inducible gene expression in vaccinia virus.
Figure 1-4:Model of VACV induced low pH cell-cell fusion.
Figure 2-1:The EFC is required for neutral pH cell fusion.
Figure 2-2:Tandem affinity purification of A56.
Figure 2-3:Western blot analysis of A56TAP.
Figure 2-4:Entry proteins co-purify with K2TAP only in the presence
of A56.
Figure 2-5:A TAP tagged A28 protein associates with A56 and K2.
Figure 2-6:Soluble A56/K2 interacts with the EFC.
Figure 3-1:A56/K2 physically associate with A16.
Figure 3-2:A16 and G9 selectively co-purify with A56/K2.
Figure 3-3:A56/K2 binds to affinity purified A16 and G9.
Figure 3-4:Both A16 and G9 are required for binding A56/K2.
Figure 3-5:A16 is required for G9 to bind A56/K2.
Figure 3-6:A16 and G9 interact in uninfected cells.
Figure 3-7:Model by which A56/K2 inhibit infected cell fusion.
Figure 4-1:Cell surface expression of A56 and K2.
Figure 4-2:Efect of A56 and K2 expresion on fusion betwen
transfected cells and VACV induced syncytia.
Figure 4-3:Effect of A56 and K2 expression on VACV entry.
viii
List of Abbreviations
A Adenine
bps Base pairs
C Cytosine
o
C Degrees Celsius
CBBCalmodulin binding buffer
DMEMDulbecco?s modified Eagle medium
DNADeoxyribonucleic acid
dsDNADouble-stranded DNA
dsRNADouble-stranded RNA
DsRedDiscosoma sp. red fluorescent protein
E. coliEscherichia coli
EFCEntry fusion complex
EGFPEnhanced green fluorescent protein
EGTA
2-[2-[2-[2-[bis(carboxymethyl)amino]ethoxy]ethoxy]ethyl-(carboxymethyl)amino]acetic acid
EM Electron microscopy
EMEMEarle?s modified Eagle medium
EV Extracellular virus
FBSFetal bovine serum
g Gravity
G Guanine
GAGsGlycosaminoglycans
GAPDHGlyceraldehyde-3-phosphate dehydrogenase
GFPGreen fluorescence protein
HcRedHeteractis crispa red fluorescent protein
h Hours
IPTGIsopropyl-?-D-thiogalactopyranoside
kbpsKilobase pairs
kDa Kilodaltons
ix
LDSLithium dodecyl-sulfate
Luc Luciferase
minsMinutes
ml Milliliters
mM Millimolar
MOIMultiplicity of infection
mRNAMessenger ribonucleic acid
MV Mature virus
nm Nanometers
ORFOpen reading frame
PCRPolymerase chain reaction
PFUPlaque forming unit
RFPRed fluorescent protein
SBBStreptavidin binding buffer
SERPINSerine protease inhibitor
SDS-PAGESodium dodecyl sulfate-polyacrylamide gel electrophoresis
T Thymidine
TAPTandem affinity purification
TBSTTris-buffered saline with Tween-20
ts Temperature sensitive
!l Microliter
?M Micromolar
UTRUntranslated region
WR Western Reserve
WV Wrapped virus
VACVVaccinia virus
VARVVariola virus
1
Chapter 1:  Review of literature
1.1The Poxviridae
1.1.1 Classification.
Poxviruses are among the largest animal viruses. They have a characteristic
brick-shaped virus particle that contains the virus genome along with al of the viral
enzymes and factors required for early RNA synthesis. Poxviruses are unusual
among DNA viruses, replicating entirely within the cytoplasm. The family
Poxviridae is divided into two subfamilies: Chordopoxvirinae and the
Entomopoxvirinae based on their respective vertebrate and insect host range. The
chordopoxviruses have ben more intensely studied than entomopoxvirues due in part
to their ability to infect humans and domesticated animals. There are eight genera
within the Chorodopoxvirinae: Orthopoxvirus, Parapoxvirus, Avipoxvirus,
Capripoxvirus, Leporipoxvirus, Suipoxvirus, Moluscipoxvirus, and Yatapoxvirus.
Viruses within the Orthopoxvirus genus are primarily responsible for infection of
humans, with the most notable members of the genus including variola virus
(VARV), the etiological agent of smalpox, and vacinia virus (VACV), which was
utilized sucesfuly as a vacine in the eradication of smalpox. VACV is the
prototype of the Orthopoxvirus genus and has ben intensively studied in the
laboratory. Many of the poxviruses within the Chordopoxvirinae exhibit a narow
host range and infect only a single species, although ocasionaly the viruses wil
infect humans by zonosis. VARV and moluscum contagiosum virus
2
(Moluscipoxvirus genus) are obligate human pathogens with no known animal
reservoir.
1.1.2 Smallpox, Edward Jenner and Vaccinia virus.
Smalpox is estimated to have evolved around 100 BCE, about the same
time as the first agriculture setlement [1]. The disease ravaged the human population
and spared no clas from its efects as evident from the skin lesions found on
mummified bodies of Egyptian pharaohs.
Prior to the advent of vacination, the practice of ?variolation? was introduced
in India and China around 100 years ago as a strategy to prevent infection with the
smalpox virus [1]. Variolation involved the use of a smal amount of infectious
material obtained from the smallpox lesion or scab.  This infectious material was used
to inoculate an individual in the hope of developing a milder form of the disease.
Variolation was born from the observation that once individuals infected with
smalpox had recovered they were protected from subsequent infection with the virus.
Variolation, posesed a significant risk to the individual by modern standards,
asociated with a low mortality rate which may have in part ben due to an inability
to control the inoculating dose.
It was the English physician Edward Jener in the late 18 
th
 century who
introduced the concept of vacination [2]. Edward Jener observed that local
milkmaids infected with cowpox virus failed to be infected by VARV. Jener?s
observation led him to inoculate James Phips with cowpox virus and later chalenge
the boy with VARV. The prior infection with cowpox virus efectively prevented the
3
child from developing smalpox and changed the course of modern medicine leading
to the eventual eradication of the disease nearly 200 years later.
1.1.3 Genome organization.
The poxvirus family is divided into two subfamilies: viruses that infect
vertebrate hosts (Chordopoxvirinae) and viruses that infect insect hosts
(Entomopoxvirinae) [3]. The chordopoxviruses have a dsDNA genome of variable
length that ranges from 260 kbps for fowlpoxvirus to 140 kbps for Orf virus. The
nucleotide composition of the chordopoxvirus genomes is quite diverse and ranges
from 64% G/C in Parapoxvirus to 3% G/C for Yatapoxvirus [3]. Despite the
variation in size and nucleotide composition, poxviruses share a comon genome
organization. The central region of the genome is conserved with respect to gene
content and arangement. Many of the genes located in the central region are
esential for virus replication in cel culture and have important roles in virus
transcription, replication and asembly of nascent particles. The genome termini are
more divergent and the genes located in this region are generaly not required for
virus replication in cel culture but instead function in host range and imune
evasion.  There are 90 genes conserved among the Chordopoxvirinae with most of the
conserved genes being located in the central portion of the virus genome. Inclusion
of the entomopoxviruses reduces the number of conserved gene to 49 [4].
Vacinia is the prototypical member of the poxvirus family. Many of the
conserved poxvirus genes have ben investigated in the laboratory by examination of
VACV. VACV has a linear dsDNA genome of 190 kbps with covalently closed
hairpin termini [5]. Prior to sequencing of the genome of VACV it was characterized
4
by digestion with restriction endonuclease HindII. The comon VACV laboratory
strain Western Reserve (WR) contains 15 HindII fragments designated A to O based
on their mobility in an agarose gel, with the A fragment being the largest and O the
smalest [6, 7]. VACV genes are named acording to their location, position, and
direction of transcription within the HindII fragments. For example, A56R is the
56
th
 gene within the A HindII fragment and is transcribed to the right as indicated by
the R. Meanwhile, K2L is the second gene of the K HindII fragment and transcribed
to the left. It is comon to refer to the gene with the L and R designation, while the
L and R are omitted when referring to the protein (Figure 1-1).
The ends of the VACV genome are located within the B and C HindII
fragments and consist of 10 kbps of inverted repeat DNA [8, 9]. The sequence of the
inverted repeats consists of a total of 30 DNA repeats, with each repeat being 70 bps
in length [10]. There is significant diversity in the number of repeats betwen strains
of VACV. The virus genome terminates with a conserved A/T rich region of 104 bps
to form a covalently closed hairpin [1]. The terminal portion of the virus genome,
encompasing the viral hairpin, which have ben sugested to have several important
functions including initiation of DNA replication and resolution of the viral genome
concatemers [12-15].
1.1.4 Virion Morphology and Infectious particles.
Cels infected with VACV produce thre morphologicaly distinct virus
particles: mature virus (MV), wraped virus (WV) and extracelular virus (EV). The
MV particle is the simplest and most abundant.  Most MV particles remain
5
Figure 1-1: Vacinia virus genome organization. Diagram representing the DNA
fragments generated by digestion of the VACV genome with the HindII restriction
endonuclease. The DNA fragments are letered acording to their size; A is the
largest fragment, while P is the smalest. Viral genes are named acording to their
location and position within the HindII fragment with L or R indicates the direction
of transcription. Shown are two examples: K2 is the second gene of the K fragment
and transcribed to the left. A56 is the 56
th
 gene of the A fragment and transcribed to
the right.
6
intracelular until cel lysis but a portion acquires two aditional membranes of the
trans-Golgi or endosomal origin to form WV [16].  The WV is transported to the cel
periphery on microtubles and is released from the cel by exocytosis [17]. During the
release of WV the outer of the two wraping membranes is lost to form the EV. The
EV particle is surounded by one aditional membrane with respect to MV. EV is
important for virus spread in cel culture and is thought to mediate disemination of
the virus in vivo.  The different stages of VACV lifecycle are indicated in figure 1-2.
The MV particle has ben visualized by a combination of cryo-electron
tomography and atomic force microscopy [18-20]. These studies define the MV
particle as a brick shaped structure with an average dimension of 360nm X 270nm X
250 nm. Transmision electron microscopy of thin-sectioned MV reveal the virus is
surounded by a single lipid membrane [21, 2], although others interpret the images
diferently, sugesting the MV particle is enveloped by two tightly aposed
membranes [23-25]. The virus particle contains a dumbel shaped electron dense
core housing the viral genome with two protein lateral bodies paralel to the
biconcave core.
1.1.5 Viral Proteins.
The 190 kbps genome of VACV is predicted to encode nearly 20 proteins.
Many of these proteins are incorporated into the virus particle. Early analysis of the
MV virus particle by 2D electrophoresis sugested the particle was comprised of
nearly 10 proteins [26, 27]. More recently thre groups have revaluated the protein
composition of the MV particle by mass spectrometry.  The number of viral proteins
7
Figure 1-2: Lifecycle of Vaccinia virus.
Virus particles (extracelular virus (EV) or mature virus (MV) enter the cel by direct fusion at the
plasma membrane or an endocytotic pathway to release the virus core into the cytoplasm. The virus
core contains the DNA template, viral enzymes and transcription factors required for transcription of
early viral mRNA. The early gene products encode imunomodulatory proteins, transcription factors
and enzymes for viral DNA replication. Disasembly of virus core ocurs during early gene
expresion and leads to uncoating of viral DNA and DNA replication. Intermediate mRNA is
transcribed after DNA replication and includes transcription factors for late gene expresion. Late
mRNA encodes many of the structural proteins for nascent particle formation as wel as the early
transcription factors and enzymes to be packaged into the virus core.  Virus asembly begins with the
formation of membrane crescents. The virus crescents are filed with the viroplasm, a mixture of
viral proteins, which evolves into spherical imature virions (IV). Genome concateers are resolved
during late gene expresion and the unit length genome is packaged into the IV. Virus maturation is
acompanied by proteolytic procesing of viral membrane and core proteins.  A double membrane of
trans-Golgi or endosomal origin leads to wraping of a portion of the MV to form wraped virus
(WV). Egres of WV ocurs folowing transport of the WV on microtubles to the plasma membrane
and the outer membrane fuses with the plasma membrane to release extracelular virus (EV). Viral
proteins present in the WV and EV membranes are important for recruitment of celular proteins to
form virus tipped actin tails that aid in virus spread.
8
within the MV particle varied betwen the studies and ranged from 63 to 80 viral
proteins [28-30]. A consensus of 73 viral proteins was identified within two of the
thre studies [30]. Nearly 50% of the viral proteins within the MV particle have a
role in forming the structure or asembling the MV, while the remaining proteins are
enzymes and factors important for synthesis of viral RNA. Most of the viral proteins
packaged into the MV particle are encoded by genes located in the central conserved
region of the genome with few proteins from the more divergent genome termini [30].
Poxvirus replication ocurs entirely in the cytoplasm and very few host
proteins are required for virus gene expresion. Consequentialy, poxviruses have
evolved an aray of proteins for synthesis, regulation and procesing of its mRNA
[31]. The virus encodes a multisubunit DNA dependent RNA polymerase [32, 3], a
heterodimeric caping enzyme [34-36], and a poly (A) polymerase [37]. Viral gene
expresion is cordinated by early [38, 39], intermediate [40-43] and late [4]
transcription factors, while two viral decaping enzymes: D9 [45] and D10 [46, 47]
are thought to regulate turnover of host and viral mRNA. Formation of protein
disulfide bonds typicaly ocurs within the oxidizing environment of the host ER, as
the reducing environment of the cytoplasm is generaly thought to prevent their
formation. Interestingly, multiple viral membrane proteins (L1, F9, A28, H2, A16,
G9 and A21) have ben identified to contain intramolecular disulfide bonds. Thre
viral proteins: E10 [48], A2.5 [49] and G4 [50, 51] are required for the formation of
these intramolecular disulfide bonds. The E10 viral protein contains a domain with a
conserved thiol active site motif C-X-X-C comon to the ERV1 (Esential for
Respiration and Vegetative Growth)/ARL (Augmenter of Liver Regeneration) family
9
of celular thiol oxidoreductases [52]. Colectively, E10, A2.5 and G4 form a
cytoplasmic pathway essential for the catalysis of intramolecular disulfide bonds [53].
VACV encodes numerous imunomodulatory proteins that help the virus
evade both the inate and adaptive imune response of the host [54, 5]. During
viral gene expresion dsRNA is produced, a potent triger of the inate imune
response. However, the virus encodes a dsRNA binding protein, E3, which is
important for the inhibition of dsRNA dependent protein kinase R [56, 57]. The
VACV C3L gene encodes a soluble complement control protein that inhibits both the
clasical and alternative pathways of complement by binding C4B and C3B [58, 59].
Poxviruses also expres a number of proteins that function as defective receptors for
cytokines and chemokines [60]. The terms virokines and viroceptor was coined to
describe these decoy molecules, which are important virulence factors as viruses
lacking these proteins are attenuated in vivo.
1.1.6 Recombinant techniques for the investigation of VACV.
The roles of many VACV genes have ben elucidated through targeted
deletion of viral genes and examination of the resulting phenotype. Targeted deletion
is useful for the study of virus genes that are not esential for the virus life cycle,
however alternative strategies are used to investigate the function of esential virus
genes. Early investigation of esential viral genes relied on random mutagenesis to
generation conditional lethal viruses in which virus growth was temperature sensitive
(ts). VACV is normaly grown at 37 
O
C, however the permisive temperature for ts
viruses is typicaly 31
 O
C with viruses containing a ts legion being unable to grow at
an elevated temperature of 40 
O
C. Temperature sensitive viruses were characterized
10
biochemicaly as wel as by electron microscopy and placed into complementation
groups. Ultimately, characterization of a temperature sensitive virus relied on
identification of the defective virus gene through complementation [61]. A more
targeted aproach for the development of ts viruses employs alteration of charged
amino acids to the nonpolar amino acid alanine [62, 63].
A second aproach developed to characterize genes esential for the virus
lifecycle utilizes components of the E. coli lac [64] and tet [65] operons. Control of
viral gene expresion relies on integration of the lac or tet represor into the virus
genome and constitutive expresion of the represor protein throughout the virus
lifecycle. Virus gene expresion is controled at the level of transcription by inserting
the DNA sequence of the lac or tet operator betwen the virus promoter and the
initiating methionine of the protein. An inducer, isopropyl-?-D-thiogalactopyranoside
(IPTG) for the lac operon or Doxycycline for tet operon, is aded to the medium to
alow gene expresion. The inducer prevents the represor protein from binding to its
cognate operator thereby alowing viral gene expresion. In the absence of the
inducer the represor protein binds to the operator and stringently repres
transcription, most likely through steric hindrance. Basicaly, in the presence of the
inducer, the gene is expresed, alowing the virus to grow, while in the absence of the
inducer gene expresion is represed and the virus is unable to grow. An alternative
aproach for control of virus gene expresion utilizes the phage T7 RNA polymerase
along with components of the lac operon. Instead of relying of the virus promoter for
gene expresion the gene is expresed from a T7 promoter. Expresion of T7
polymerase is dependent on the addition of IPTG [66] (See Figure 1-3).
11
Figure 1-3:  Diagram of inducible gene expression in vaccinia virus.
Gene expresion is controled at the level of transcription. The lac represor (lacI) is
expresed constitutively from a viral early/late promoter (P7.5). Expresion of the
phage T7 polymerase is regulated by the lac operator (lacO) located betwen the late
viral promoter (P1) and the initiating codon of the protein. GFP is expresed from
the T7 promoter (PT7), with the lac operator located betwen the T7 promoter and
initiating codon of GFP. In the absence of IPTG the lac represor binds to the
operator preventing expresion of the T7 polymerase. A second lac operator
downstream of the T7 promoter further represes transcription. In the presence of
IPTG the lac represor does not bind the lac operators, alow expresion of T7
polymerase, which catalyzes transcription of the GFP gene.
12
1.2Virus Entry and Replication
1.2.1 Virus Attachment.
Entry and fusion of poxviruses is complicated by the existence of multiple
forms of the virus. Most of the studies of VACV entry have focused on the MV
particle, since it is stable and easily purified. VACV has a broad tropism in cel
culture and is able to infect many diferent types of cels making isolation of a
celular receptor dificult. VACV fails to infect human resting T cels, but does infect
activated T cels, sugesting na?ve T cels lack specific celular factors required for
virus atachment or entry that are expresed upon activation [67]. A monoclonal
antibody reactive to a cel surface antigen was isolated and shown to prevent virus
attachment, although the specificity of the antibody remains to be identified [68].
Initial atachment of the MV particle to the cel is facilitated by interactions
betwen the virus and cel surface glycosaminoglycans (GAGs). Thre VACV
proteins have ben reported to mediate virus atachment: D8, H3 and A27.
Individualy, D8 [69], H3 [70] and A27 [71, 72] are not required for virus growth in
cell culture.  This may not be all that surprising as A27, H3 and D8 proteins may have
slightly redundant function with respect to virus atachment, which would be
consistent with individual deletions of the proteins not having a more severe efect on
virus growth [73-75].  However, virus attachment is not solely dependent on GAGs as
VACV stil binds and infects Sog9 cels, which lack the GAGs heparin sulfate and
chondroitin sulfate [76]. A recent report sugested that celular laminin is an
important celular atachment factor since soluble inhibited virus atachment in a dose
dependant maner [76]. Cholesterol within the cel membrane has also ben
13
demonstrated to be important for virus entry. Depletion of cholesterol from the cel
membrane by treatment with methyl-!-cyclodextrin reduced cel infectivity by 90%.
Infectivity was restored by adition of exogenous cholesterol, indicating an important
role for cholesterol in VAV atachment/entry [7]. The celular factors important for
virus atachment and entry remain to be fuly characterized and further investigations
will likely reveal other cellular factors that play a role in this process.
1.2.2 Virus Entry.
Entry and fusion of VACV requires a conserved multiprotein complex of at
least eight proteins: A16 [78], A21 [79], A28 [80], L5 [81], G3 [82], J5, G9 [83], and
H2 [71]. The proteins of the EFC are integral membrane proteins located within the
MV membrane and critical for infectivity of the virus, although it remains to be
determined if the EFC directly mediates virus fusion. Al of the entry proteins
contain a single transmembrane anchor and the proteins fal into two groups based on
their predicted topology within the virus particle. A28, A21, H2, G3 and L5 poses
an N terminal transmembrane domain and have 0-2 intramolecular disulfide bonds,
while A16, G9 and J5 have a C terminal transmembrane domain with 4-10
intramolecular disulfide bonds. The cysteine rich domains of A16, G9 and J5 are
homologous, sugesting the proteins arose through gene duplication and diverged to
have independent roles in virus entry.  There is no detectable homology between A28,
A21, H2, G3 and L5. The entry proteins have no role in asembly of the MV, WV or
EV particle. Viruses that lack a single entry protein have ben isolated and bind
normally to cells, but fail to enter indicating a block in virus entry or fusion.
14
The EFC is critical for virus entry and al proteins of the complex must be
expresed for the virus to enter. The absence of a single entry protein is suficient to
prevent the complex from asembling, although if the complex does not form the
remaining entry proteins are stil incorporated into the MV membrane [84]. This
potentialy indicates the entry proteins localize to the virus membrane prior to
forming the EFC. In fact, the EFC does not form in the absence of the virus
membrane, which would be consistent with the entry proteins localizing to the viral
membrane prior to assembling into the complex.
Intramolecular disulfide bonds have ben shown to form in the cytoplasmic
domains of several of the entry proteins: A16 [78], G9 [83], A21 [79], L5 [81] and
A28 [85]. Interestingly, represing the E10 protein, an esential component of the
VACV cytoplasmic disulfide bond pathway, had no efect on asembly of the EFC.
Therefore disulfide bonds do not apear to be important for asembly of the complex,
however because E10 is essential for virus morphogenesis the role of disulfides bonds
in the entry of the virus has not ben asesed. With eight proteins forming the EFC
it is likely that a number of protein interaction are required for formation of the entry
complex, however these interactions remained to be defined. In the absence of A16,
the H2 and A28 entry proteins were observe to remain asociated, sugesting the two
proteins may interact within the EFC [84].
In adition to the proteins of the EFC, several other viral proteins have ben
sugested to be important for MV entry. The ability to isolate neutralizing antibodies
to the A27 and L1 proteins sugested that these two proteins might be important for
virus entry [86-8]. Analysis of the A27 protein indicated a role in the wraping of
15
MV to form WV. Viruses lacking A27 remained infectious, indicating the A27
antibody may inhibit virus entry through steric hindrance [71, 72]. The role of the L1
protein in virus entry has not ben investigated because the protein is required for
virus morphogenesis preventing isolation of virus particle deficient in L1 [89].
The L1 protein shares 20% amino acid identity with F9, sugesting these two
proteins may have had a comon gene ancestor (i.e. one of the proteins arose from
gene duplication of the other). Unlike L1, there is no block in morphogenesis in the
absence of F9 and al of the forms of the virus are produced. Virus particles lacking
F9 bind to the cell, however their cores do not penetrate into the cytoplasm [90].  This
phenotype is identical to that observed for the proteins of the EFC. The F9 protein
was shown to interact with proteins of the EFC, although the interaction was dificult
to detect sugested the protein may only asociate with a portion of the entry
complexes. The functional role of F9 is consistent with its interaction with the EFC,
although the significance of the interaction remains to be determined.
The MV particle of VACV has ben shown by EM to fuse at the cel surface
[91-93]. Virus particles are also observed in intracelular vesicles sugesting entry
through an endocytic route [94, 95]. Several recent studies investigating MV entry
have used a recombinant VACV that expreses the firefly luciferase under the control
of an early viral promoter [94]. Luciferase (Luc) expresion, which can be detected
as early as 30 minutes postinfection, is measured and used to quantify virus entry.
MV bound to the cel surface and exposed to low pH display a 10-fold increase in
Luc activity compared to an identical sample treated with neutral pH [94]. The
ability of low pH to influence virus entry is consistent with entry through an
16
endocytic route, with low pH treatment mimicking the pH reduction that ocurs
within the endosome. Inhibitors of endosomal acidification decrease entry of MV
particles by 60-80% depending upon the cel type [94]. The efect of inhibiting
endosomal acidification can be particularly rescued by treating the virus bound to the
cel surface with low pH. The low pH presumably trigers fusion of the particle with
the cel surface thereby bypasing the requirement of the low pH within the
endosome.
While most of the studies on the entry of VACV have focused on the MV
particle, few studies have loked at EV. Entry of the EV, which contains one
additional membrane with respect to the MV particle, is difficult as the EV membrane
is fragile and prone to damage during purification. EV was proposed to enter by
endocytosis, with the low pH environment of the endosome disrupting the EV
membrane [96].  Recently EV has been shown to enter the cell by directly fusion with
the plasma membrane. The EV membrane is disrupted by soluble GAGs, to release
the MV particle [97]. The EV membrane serves as an acesory during entry of EV
with virus-cell fusion depending on the EFC in the MV membrane.
1.2.3 Virus gene expression.
Virus entry is folowed by release of the virus core into the cytoplasm. The
core is transported on microtubules to a perinuclear site within the cytoplasm [98].
Disassembly of the core, which releases the virus genome into the cytoplasm, requires
early gene products and is prevented by protein synthesis inhibitors [9]. The virus
core contains al of the enzymes and factors required for viral transcription and early
viral mRNA synthesis ocurs independent of de novo protein synthesis. The
17
transcriptional aparatus of VACV has ben extensively characterized by isolating
the enzymes present within the virus core. An extract from the virus core is able to
synthesize RNA in vitro and the resulting mRNA is caped at its 5? end and contains
a 3? poly(A) tail [100].
Virus genes are divided into prereplicative and postreplicative clases
depending on whether they are expressed before or after viral DNA replication.  Early
viral gene expresion ocurs prior to DNA replication, while intermediate and late
gene expresion ocur after viral DNA replication. Clas specific promoters and
transcription factors cordinate expresion from each clas. Early transcription
factors are the products of late viral genes that are packaged into the virus particle
during the previous infectious cycle. Early viral gene expresion ocurs shortly after
the virus enters into the cytoplasm with RNA/DNA hybridization studies sugesting
nearly 50% of the genome is transcribed during this early phase [101, 102].
Intermediate transcription factors are the products of early genes, however
intermediate transcription is delayed until after viral DNA replication. Inhibitors of
viral DNA replication block intermediate transcription [4]. Interestingly, an
intermediate promoter transfected into viraly infected cels is transcribed even under
conditions in which viral DNA replication is inhibited. This sugests the template for
intermediate gene expresion is not acesible until after DNA replication, likely
indicating the newly replicated DNA serves as the template [4]. Intermediate
transcription in vitro requires three viral factors: viral intermediate transcription factor
(VITF) VITF-1, VITF-2 [41], and VITF-3. The E4L gene encodes VITF-1 [40],
while VITF-3 is a heterodimer of A8R and A23R [43]. In contrast, VITF-2 is a
18
heterodimer of two celular proteins: Ras-GTPase-activating protein SH3 domain-
binding protein (G3BP) and p137 [103]. Both G3BP and p137 are found in distinct
regions of the viral factory along with the intermediate transcription factor A23R.
G3BP, p137 and A23R localized to similar regions of the viral factories as viral
mRNA and two translation initiation factors: eIF4E and eIF4G. These findings
sugest virus transcription and translation is cordinated within the virus factory
[104].
The viral late transcription factors are the products of intermediate genes.
Thre viral proteins are required for the transition from intermediate to late gene
expresion: A1 [105], A2 [106] and G8 [107]. In vitro, the H5 protein has ben
shown to enhance late viral gene transcription [108]. Late gene expresion continues
until the cellular resources are depleted or cell lysis occurs.
VACV early, intermediate and late gene promoters are located imediately
upstream of viral genes. The poxvirus promoters are conserved, with the promoter
from one type of poxvirus functioning within cels infected with a diferent poxvirus.
The features of both early and late VACV promoters have ben characterized by
single nucleotide substitution. Early promoters contain an A+T rich core sequence
betwen ?13 and ?28, folowed by a spacer region of 12 nucleotides and initiation of
transcription starting with an A or G nucleotide (Note: +1 is the transcription start
site) [109]. Late promoters contain a highly conserved TAAT sequence with
transcription initiating within the A triplet of TAAT [10]. The TAAT is
comonly folowed by a G nucleotide to form TAATG, with ATG serving as the
initiating codon for protein synthesis. Intermediate promoters contain an important
19
core region from ?26 to ?13 and have a conserved sequence TAA, with
transcription initiating within the A triplet [11]. The 5? of intermediate and late
mRNA contains a poly(A) leader of 30-40 bps [12, 13]. The poly(A) leader may
arise as a consequence of the polymerase stutering within the AA found in both
intermediate (TAA) and late promoters (TAAT). Although the poly(A) leader is
generaly absent from early mRNA, several early promoters contain a TAAT
sequence and analysis of the 5? end revealed a poly A tract, although the length of the
leader was reduced to only 15-20 bps [114].  The significance of the 5? poly(A) leader
is unknown, but it may have a role in enhancing translation of the mRNA.
The length of the mRNA transcript varies depending on the transcription
clas. Early transcripts have a defined length resulting from transcription termination
downstream of the sequence TTTTTNT, where N is any nucleotide including T [115].
The termination sequence is recognized within the nascent mRNA transcript [116]
and transcription ends 20-50 bps after the sequence. Intermediate and late mRNA
lack defined termination signals resulting in mRNA of heterogeneous length [17,
18]. Several late viral mRNAs undergo cleavage at their 3? UTR [19]. The activity
responsible for in vitro cleavage of the 3? UTR copurified with the H5 protein.
However, a recombinant H5 protein expresed in E. coli was unable to cleave the
3?UTR suggesting there may be additional factors important for cleavage [120].
1.2.4 Genome replication.
VACV encodes most, if not al, of the proteins required for replication of its
genome. Enucleated cels suport replication of the virus, albeit at a reduced rate
[121, 12]. VACV infected cels form discrete cytoplasmic bodies known as ?viral
20
factories? that localize adjacent to the nucleus and stain densely with fluorescent
DNA dyes. The viral proteins important for DNA replication are synthesized from
early genes and viral replication begins within 1 to 2 h postinfection. The viral
proteins important for DNA replication include, but are not limited to, the DNA
polymerase (E9) [123], uracil DNA glycosylase (D4) [124], the protein kinase (B1)
[125, 126], nucleic acid-independent nucleoside triphosphatase (D5) [127, 128], and a
viral DNA procesivity factor (A20) [62, 129]. The B1 kinase phosphorylates the
celular protein barier to auto integration factor (BAF) [130]. BAF is sugested to
bind to the viral DNA and inhibit its replication. B1 phosphorylation of BAF is
thought to prevent the protein from binding the viral DNA, although the exact
mechanism by which BAF inhibits DNA replication is unknown [131]. The viral D5
protein shares limited homology with the archaeoeukaryotic primase superfamily
[132] and purified recombinant D5 protein was shown to catalyze oligoribonucleotide
synthesis consistent with a role as a primase [13]. The D4 and A20 proteins interact
and are important for processivity of the viral DNA polymerase in vitro [134].
The mechanism of VACV DNA replication is porly characterized. Atempts
to identify a viral origin of replication were unsucesful and instead showed the
virus is able to replicate plasmid DNA devoid of any viral genomic DNA [135].
Plasmid DNA is thought to replicate by a roling circle mechanism within the viral
factory and was shown to depend on the same viral proteins esential for genome
replication [136]. The curent model of VACV DNA replication hypothesizes that
the viral DNA replicates by a roling hairpin mechanism, with nicking of the virus
genome within hairpin termini serving as a primer for DNA replication. The
21
replication of both the viral genome and plasmid DNA results in long DNA
concatemers (i.e. direct repeats of the genomic DNA). An A/T rich DNA sequence
located near the hairpin termini of the viral genome is important for resolution of the
plasmid DNA concatemers [137]. This sequence is conserved amongst poxvirus,
sugesting an important role in resolution of viral genome concatemers. Resolution
of the viral concatemers requires the action of a late viral gene product encoding a
holiday junction resolvase (A2) [138]. The purified A2 protein resolves synthetic
holiday junctions in vitro and represion of the A2 gene prevents resolution of
genome concatemers [139, 140]. Folowing DNA replication, the virus transition to
intermediate and late gene expresion in which the replicate viral genome is packaged
into the nascent virus particle.
1.2.5 MV morphogenesis.
Asembly of VACV ocurs within cytoplasmic viral factories. The various
stages of MV morphogenesis have ben defined by electron microscopy. The first
structures of virus morphogenesis observed by electron microscopy are membrane
crescents, although the celular organele from which the membrane crescents are
formed remains controversial. Some studies propose the membrane is synthesized de
novo [2] or originates from the ER/Golgi intermediate compartment [25], while
more recent evidence favors the idea that the membrane crescents are derived from
the ER membrane [141]. Formation of membrane crescents does not ocur in the
absence of the viral proteins F10 [142], A1 [143], H5 [14] or G5 [63]. Membrane
crescents asociate with an electron dense granular viroplasm containing viral
proteins destined for the viral core. Asociation of membrane crescents with the
22
viroplasm requires a complex of seven proteins: F10, A30, G7, J1, D2, D3 and A15
[145].
Membrane crescents evolve into spherical imature virus (IV) particles. The
transition from membrane crescent to IV is inhibited by the antibiotic rifampicin.
Viral mutants resistant to rifampicin have ben isolated and the mutations map to the
D13L gene [146]. D13 forms a honeycomb latice around the IV, but is not packaged
into the MV particle. Instead, D13 may serve as a scafold for the asembly of IV
[147]. The viral genome is packaged into the virus core at the IV stage. Two viral
proteins, A32 [148] and I6 [149], have ben shown to be important for encapsidation
of the viral genome.
The transition from IV to MV is porly defined, but is asociated with the
proteolytic cleavage of viral membrane and core proteins. VACV has two predicted
proteases encoded by G1L and I7L. G1L is a predicted metaloprotease with an
esential role in the transition from IV to infectious MV [150], however G1 is not
required for cleavage of the viral core proteins. The I7 protein shares homology with
other known cysteine proteases and is responsible for cleavage of the viral core
proteins A3, A10 and L4 as wel as the viral membrane protein A17 [151]. There are
several other proteins, including A12 and G7, which also undergo proteolysis. The I7
protease recognizes a consensus AG/X motif [(/) denotes site of cleavage], however
other undefined factors influence substrate specificity. For example, the F10 protein
contains an AG/X motif, yet does not apear to be cleaved. When synthesis of I7 is
represed, cleavage of the viral core and membrane proteins does not ocur and
noninfectious iregular virus particles containing an aberant core structure are
formed.
23
1.2.6 Formation of Wrapped and Extracellular virus.
There are at least eight viral proteins that localize to either the EV or the WV
membrane: A3, B5, A34, A56, K2, and F13 are found in the EV membrane, while
A36 and F12 specificaly localize to the WV membrane. Wraping of MV is
severely compromised in the absence of the EV proteins F13 [152] and B5 [153, 154]
as wel as the MV protein A27 [15]. WV are transported to the periphery of the cel
on microtubles [156]. In the absence of A36, WV transport is impaired, sugesting a
role for this protein in WV transport [157]. This was suported by yeast two hybrid
screning which identified an interaction betwen A36 and the light chain of the
microtubule motor protein kinesin [158]. A more severe defect in WV transport was
noted in the absence of F12, indicating A36 is not solely responsible for transport of
WV to the cel surface [159]. Upon reaching the cel surface the WV particle
undergoes exocytosis and loses one of the two wrapping membranes to form EV.
The virus recruits celular machinery to form virus tiped actin tails important
for spread of the virus [160]. In the absence of actin tails, the virus forms smal
plaques, indicating an important role for actin tails in virus-cel spread. Actin tail
formation requires multiple WV and EV proteins including A36 [161], A33 [162] and
A34 [163]. Phosphorylation of two tyrosine residues within A36 is critical for the
recruitment of the cellular proteins required for actin tail formation [164].
24
1.3Virus Fusion and Regulation of Infected Cell Fusion
1.3.1 Low pH induced cell-cell fusion.
During a typical VACV infection there is very litle fusion betwen infected
cels, however a brief exposure to a pH below 6 induces extensive cel-cel fusion.
There are two forms of low pH cel fusion: i) fusion from without and i) fusion from
within. Fusion from without requires adsorption of a large amounts of purified virus
(MOI 30-50) to the cel surface [165, 16]. Fusion from within ocurs at late times
during infection and requires cel surface EV [72, 152]. Folowing exposure to low
pH, cel fusion develops over several hours, while a similar treatment with neutral pH
fails to triger cel fusion. The ability of VACV to induce cel fusion folowing low
pH is characteristic of viruses that enter through a low pH endocytic route, with low
pH mimicking the pH drop in the endosome and stimulating virus fusion at the
plasma membrane instead of the endosome. It is thought that low pH activates the
virus entry machinery and trigers fusion of the virus membrane with the plasma
membrane. Consequentialy, the viral proteins formaly in the viral membrane are
relocated to the cel membrane positioning the EFC to mediate fusion with adjacent
cels, eventualy forming large multinucleated syncytia (Figure 1-4). Neutralizing
antibodies are able to inhibit the development of low pH cel-cel fusion consistent
with the proces of cel-cel fusion being closely related to virus cel fusion [86-8].
More recently VACV entry has been shown to require a multiprotein EFC, although it
is uncertain
25
Figure 1-4: Model of VACV induced low pH cell-cell fusion.
(Fusion from without.) A large number of mature virus (MV) particles is bound to
the surface of an uninfected cel and then treated briefly with low pH (5.5 or below)
medium. Low pH synchronizes MV fusion with the plasma membrane and deposits
the viral membrane with the entry fusion complex (EFC) into the cel membrane.
The EFC within the cel membrane mediates fusion betwen adjacent cel leading to
formation of multinucleated syncytia. (Fusion from within.) Cels infected with
VACV poses surface extracelular virus (EV). Folowing treatment with low pH
medium the EV membrane ruptures to expose the MV particle, which fuses with the
cell membrane.
26
whether the EFC directly mediates fusion or instead is important for asembly of the
viral fusion apparatus.  Never the less, the EFC is required to mediate all forms of low
pH cell fusion.
1.3.2 A56R and K2L.
In adition to low pH cel fusion described above, VACV trigers neutral pH
cell fusion when either the VACV A56R [167] or K2L [168-170] gene is absent.  A56
is known as the viral hemaglutinin for its ability to aglutinate red blod cels of
certain species. K2 is one of thre viral proteins with homology to serine protease
inhibitors (SERPINS) and has ben refered to as serine protease inhibitor 3 (SPI-3),
[171]. Tisue culture cels infected with closely related cowpox virus with deletions
of the cowpoxvirus HA (VACV A56R homologue) or SPI-3 of cow pox (VACV K2L
homologue) also develop extensive cel-cel fusion. Neutral pH cel fusion is induced
by specific monoclonal antibodies that react with A56 [172] or K2 [173]. The
mechanism by which the A56 and K2 antibodies triger cel fusion is unknown,
although the antibodies may perturb protein interactions of A56 or K2 required for
inhibition of cel fusion. Neutralizing antibodies (which normaly prevent virus
infection) aded to cultures folowing infection with VACV lacking A56 or K2
inhibit neutral pH cel fusion [169]. Neutral pH cel fusion depends on EV, which is
also required for low pH fusion from within, suggesting the two forms of fusion occur
by a similar mechanism [152].
Both A56 and K2 localize to the plasma membrane of infected cels and are
incorporated into the membrane of EV [174]. K2 binds to A56 and this interaction is
required for proper localization of K2 [173]. K2 lacks a membrane anchor and is
27
secreted from infected cels in the absence of A56. The K2 protein is glycosylated,
however point mutations that abolish the four putative N linked glycosylation sites
have no efect on the ability of K2 to inhibit cel-cel fusion [174]. A56 poseses
both N and O-linked glycosylation [175], but the role of glycosylation in the anti-
fusion activity of A56 has not ben studied. Although A56 is an integral membrane
protein in the EV particle, the protein has no role in formation of EV and the virus is
able to form virus tiped actin tails in the absence of A56 or K2. Immunoelectron
microscopy reveals that A56 is inconsistently incorporated into the EV membranes
with aproximately one third of EV particles lacking A56, although the significance
of this is unknown [176]. Deletion of A56 caused a slight atenuation of disease in an
intranasal mouse model [17], while no efect on viral virulence was observed when
K2 was deleted [168]. Recombinant K2 was shown to function as a SERPIN in vitro
[178]. The cowpox SPI-3 and myoxoma SERP1 both exhibit similar proteinase
inhibition profiles in vitro, however the myoxoma SERP1 is unable to complement
the anti-fusion activity of SPI-3 [179]. This result may have ben predicted as it was
shown earlier that point mutations that abolish the SERPIN activity of the cowpox
SPI-3 (VACV K2L homologue) in vitro have no efect on the anti-fusion activity of
the protein [180].
The mechanism by which A56 and K2 regulate cel-cel fusion is porly
understod. Curiously, cel-cel fusion only ocurs among infected cels that are
deficient for A56. This was established by infecting cels with an A56 deletion virus
and a separate cel population with wild-type virus that expresing A56. The infected
28
cels were mixed and cel fusion was noted to ocur only among hemaglutination
negative cells [167].
The deletion of A56 and K2 causes neutral pH cel fusion which is thought to
develop as a result of reinfection by cel surface EV. There are several stages at
which A56 and K2 could prevent EV reinfection.  Entry of EV depends on expression
of the EFC within the MV membrane and is only exposed upon rupturing of the EV
membrane. Since both A56 and K2 are found in the EV membrane the proteins could
stabilize the membrane to prevent premature rupturing. However, since the EV
membrane apears to be quite fragile even in the presence of A56 and presumably K2
[181] inhibition of fusion is probably due to an alternative mechanism.  Both A56 and
K2 are abundant in the plasma membrane of infected cels and there is evidence to
sugest this localization is important as cel fusion develops with (i) monoclonal
antibodies to A56 [172] or SPI-3 [173] or when (i) the membrane anchor of HA is
removed [173] or (ii) the signal sequence of SPI-3 is removed [174]. This thesis wil
define the protein interactions of VACV A56R and K2L with the aim of elucidating a
mechanism by which these proteins regulate infected cell fusion.
29
Chapter 2: The VACV A56R and K2L asociate with proteins of the
Multicomponent Entry/Fusion Complex
2.1 Introduction
VACV is the prototypical member of the Orthopoxvirus genus. Poxviruses
are large DNA viruses with a brick shaped virus particle [3]. VACV replicates in the
cytoplasm of the cel and produces several types of infectious particles, the simplest
of which is MV. The MV particle is surounded by a lipid membrane that contains
nearly 20 viral membrane proteins [182]. The virus core houses the viral genome
along with al of the enzymes and factors necesary for early RNA synthesis. The
MV particle remains intracelular until lysis, however a portion of MV acquires a
double membrane derived from odified trans-Golgi or endosomal cisternae to form
WV [16], which is transported on microtubles to the cel surface and released by
exocytosis [183]. The resulting EV particle is esentialy an MV with one aditional
membrane.
Entry of the MV particle has ben shown to ocur by fusion with the plasma
membrane [91], while entry is also enhanced by briefly lowering the pH of the
medium below 6, characteristics of entry through a low pH endocytic route [94].
Chemical inhibitors of endosome acidification reduce MV entry by 80%, but can be
partialy rescued by treatment with low pH [94]. Low pH treatment of infected cels
trigers cel-cel fusion [152, 165, 16]. Cel fusion also ocurs spontaneously at
neutral pH when cels are infected with VACV or the closely related cowpox virus in
which the A56R gene encoding the viral hemaglutinin (HA) [167] or K2L encoding
a serine protease inhibitor (SPI-3) [168, 169, 170] is mutated or deleted. The A56R
30
gene of VACV is a type-I membrane protein that localizes to the EV and plasma
membrane [172, 184]. The K2L gene (SPI-3) does not contain a membrane anchor
and asociates with the EV and plasma membrane through an interaction with A56R
[173, 174]. The anti-fusion activity requires both proteins to localize to the cel
membrane. This is suported by the folowing evidence as syncytia form when (i)
poxvirus-infected cels are incubated with antibodies to HA [172] or SPI-3 [173], (i)
the membrane anchor is removed from HA [173] or (ii) the signal sequence of SPI-3
is removed [174]. A56 poses no putative catalytic motifs and the serine protease
inhibitory activity of SPI-3 is not required for fusion inhibition [180].
Virus-cel fusion and low pH-induced cel-cel fusion requires the same EFC
found in the MV membrane, which consists of at least the folowing eight viral
proteins: A16, A21, A28, G3, G9, H2, J5 and L5 [84, 94]. The study described in this
chapter investigates the role of the EFC in neutral pH cel fusion asociated with
deletion of A56R or K2L gene. Furthermore, tandem affinity purification is utilized to
determine the protein interaction of A56 and K2 which may be important for their
anti-fusion activity.  This study has been previously described in reference [185]
2.2Materials and Methods
2.2.1 Cell and virus propagation.
BS-C-1 (ATC CL-26) and RK13 (ATC CL-37) cels were grown in
Minimum Esential Medium with Earle?s balanced salt suplement (EMEM; Quality
Biologicals, Gaithersburg, MD) containing 2 mM L-glutamine, 10% fetal bovine
serum (FBS), penicilin and streptomycin. HeLa S3 (ATC CL-2.2) suspension
31
cels were cultured in Minimum Esential Medium, Spiner modification (Quality
Biologicals) with 5% equine serum and L-glutamine.  Unles specified, al
recombinant viruses were derived from the Western Reserve (WR) strain (ATCC VR-
1354; acesion number AY24312). Virus stocks were prepared as described [186].
Viral titers were determined by plaque asay using a confluent monolayer of BS-C-1
grown in six-well cluster plate.   A 10-fold serial dilution of virus stocks was prepared
in EMEM containing 2.5% FBS, glutamine and antibiotics (2.5% EMEM). Medium
was removed from the six-wel plate and 0.5ml of serialy-diluted virus inoculum was
incubated 1 h at 37 
o
C and 5% CO
2
. The cels were then overlaid with 2.5% EMEM
containing 0.5% methylcelulose and incubated 48 h. The plaques were visualized by
staining with 0.1% crystal violet (w/v) in a solution of 20% ethanol and deionized
water.
2.2.2 TAP and mass spectrometry.
HeLa S3 cels (1.5 x10
9
) were infected at a multiplicity of 5 plaque-forming
units (PFU) and after infection for 24 h the cels were colected, washed once with
ice-cold bufer (150 mM NaCl and 50 mM Tris-HCl pH 7.4), and lysed by incubating
for 1 h at 4
o
C in streptavidin binding bufer (SB) (1% Triton X-10, 150 mM NaCl,
50 mM Tris-HCl pH 7.4) with complete protease inhibitor (Roche, Indianapolis, IN).
The lysate was centrifuged for 15 min at 300 X g and the clarified supernatant was
colected. The later, except for 0.3 ml reserved for later analysis, was aded to 0.5 ml
to 1 ml of streptavidin sepharose (GE Healthcare, Piscataway, NJ) that had ben
washed with SB and the mixture rotated overnight at 4oC. The beads were washed
3 times with 10 ml of ice cold SB and the bound proteins eluted by 3 washes with 1
32
ml of SB containing 1mg/ml of D-Biotin (USB corporation; Cleveland, OH). The 3
ml of streptavidin eluate was suplemented with Mg acetate, imidazole, and CaCl
2
 to
final concentrations of 1 mM, 1 mM and 2 mM, respectively. Calmodulin Sepharose
(0.5 ml to 1 ml of packed resin; GE Healthcare) was washed with calmodulin binding
bufer (CB; which consists of SB suplemented with Mg acetate, imidazole and
CaCl2 at final concentrations of 1 mM, 1 mM and, 2 mM, respectively). Calmodulin
Sepharose was aded to the suplemented streptavidin eluate along with an aditional
2 ml of CB and the mixture rotated overnight at 4
o
C. The beads were washed 3
times with 10 ml of CB and 3 times with 0.75ml of SB containing 25 mM 2-[2-[2-
[2-[bis(carboxymethyl)amino]ethoxy]ethoxy]ethyl-(carboxymethyl)amino]acetic acid
(EGTA) to elute proteins.  The proteins in the calmodulin eluate were concentrated by
trichloroacetic acid precipitation and then resuspended in lithium-dodecyl sulfate
(LDS) sample bufer (Invitrogen, Carlsbad, CA) containing NuPage sample reducing
agent (Invitrogen) and separated on a 4-12% NuPage gel (Invitrogen) with 2(N-
morpholino)ethanesulfonic acid bufer. Gels were stained with Comasie blue
(GelCode blue stain Reagent, Pierce, Rockford, IL) and bands of interest were
excised from the polyacrylamide gel and subsequently digested with trypsin.  Tandem
mas spectrometry and database searching were performed at the National Institute of
Allergy and Infectious Diseases core facility.
2.2.3 Recombinant virus construction.
The folowing recombinant viruses were constructed for this study (Table 2-
1): vK2TAP, vA28TAP, and vA56TAP vA28i"A56 vA28i"K2 v"A56 and
33
vK2TAP"A56 v"A56"K2, vA56TAP"K2 and vA28TAP"K2, vsA56TAPi,
vK2i"A56, vsA56TAPi"C3, vT7lacOI"F13
vK2TAP, vA28TAP, and vA56TAP were constructed using DNA encoding
(i) K2L, A28L or A56R genes with 30 bps of downstream flanking region, (i) the
TAP tag derived from pCTAP (Stratagene, La Jola, CA), and (ii) a strong VACV
promoter adjacent to the gene for a fluorescent protein namely Heteractis crispa red
fluorescent protein 1 (HcRed) from Clontech (Mountain View, CA) for K2TAP and
enhanced gren fluorescent protein (EGFP) from Clontech for A28TAP. The
constructs were prepared by overlaping PCR (Acuprime Pfx, Invitrogen, Carlsbad,
CA) so that the TAP tag sequence was apended imediately before the stop codon
of the modified gene. The gene encoding the fluorescent reporter was inserted
betwen the stop codon and the 30 bps flanking region. The PCR product was
cloned into pCR-BluntI-TOPO (Invitrogen) and the TOPO plasmids encoding
K2TAP and A28TAP were transfected with Lipofectamine 200 (Invitrogen) into
BS-C-1 cels that had ben infected 1 h earlier with 1 PFU per cel of VACV.
Parental and recombinant viruses were distinguished by fluorescence microscopy and
thre rounds of plaque isolation clonaly purified the later. vA56TAP was
constructed as above except that v"A56 was used as the parental virus and the EGFP
gene in the A56R locus was replaced with A56TAP; recombinant viruses were
distinguished from the parental virus by the absence of green fluorescence.
Deletion of the A56R and K2L genes was achieved by replacing the open
reading frames with the DNA encoding EGFP or HcRed, respectively. Briefly, 30
bps of DNA coresponding to the left and right flanks of A56R and K2L were fused
34
by recombinant PCR to the fluorescent protein gene. To construct vA28i"A56 and
vA28i"K2, BS-C-1 cels were infected in the presence of 10 ?M IPTG with vA28i
[85] at 1 PFU per cel and then transfected with the respective A56 or K2 deletion
plasmid. Recombinant viruses were distinguished from parental virus by
fluorescence microscopy and clonaly purified by thre rounds of plaque isolation.
v"A56 and vK2TAP"A56 were constructed by deletion of the A56R gene from
VACV strain WR and vK2TAP, respectively, utilizing an aproach analogous to that
described for vA28i"A56. The K2L gene was deleted from v"A56, vA56TAP and
vA28TAP as described for vA28i"K2 and the resulting viruses were designated
v"A56"K2, vA56TAP"K2 and vA28TAP"K2, respectively
vK2i"A56 was designed to inducibly over expres an influenza HA epitope-
taged inducible K2. The coresponding transfer plasmid was asembled by
recombinant PCR from: (i) 20 bps of DNA upstream of the A56R gene, (i)
bacteriophage T7 promoter and encephalomyocarditis virus leader sequence
containing an internal ribosome entry site from pVote 1 [187], (ii) K2L with a C
terminal influenza HA epitope tag sequence, (iv) HcRed gene regulated by a strong
VACV promoter, and (v) 20 bps of DNA downstream of A56R. The final PCR
product was cloned into PCR-BluntI-TOPO and sequenced. The K2L expresion
plasmid was transfected into cels infected with vT7lacOI [6]and the vK2i"A56
plaques were detected by fluorescence microscopy. vK2i"A56 was clonaly purified
by three rounds of plaque isolation.
vsA56TAPi, a virus encoding an inducible A56 that is secreted from cels
because of deletion of its transmembrane segment and contains TAP, V5 and 10-
35
histidine tags, was constructed. The virus vT7lacOI"F13 was created by first deleting
the F13L gene from vT7lacOI in order to provide subsequent plaque selection [18].
A DNA segment was asembled by overlaping PCR using DNA encoding (i) the T7
promoter, encephalomyocarditis leader sequence, and E. coli lac operator from pVote
1 to provide inducible expresion and cap-independent translation, (i) A56R gene
with a V5 tag inserted betwen codons 18 and 19 and replacement of codons 280 to
315 with a TAP tag sequence folowed by 10 tandem copies of a histidine codon, and
(ii) T7 termination sequences from pVote 1. This DNA was then cloned into pRB21
[18] and the resulting plasmid was used to transfect BS-C-1 cels that had ben
infected with vT7lacOI"F13. The new recombinant virus vsA56TAPi formed large
plaques and was clonaly purified. The C3L gene was deleted from vsA56TAPi and
vK2i"A56 in a similar fashion as described for deletion of A56 to construct
vsA56TAPi"C3 and vK2i"A56"C3, respectively.
2.2.4 Western blotting.
Samples subjected to TAP from 2-3 X 10
8 
HeLa S3 cels were separated by
loading onto a 10% or 4-12% NuPage Bis-Tris gel (Invitrogen). Folowing
electrophoresis, the proteins were transfered to nitrocelulose membranes and
blocked with Tris-buffered saline supplemented with 5% nonfat dried milk and 0.05%
Twen-20 (TBST) for 1 h at rom temperature. The membranes were then incubated
with the apropriate primary antibody, washed, incubated with horseradish
peroxidase-conjugated secondary antibodies (GE healthcare, Piscataway, NJ), and
analyzed with the SuperSignal West Dura or Femto Maximum Sensitivity Substrate
chemiluminescence reagents (Pierce, Rockford, IL).
36
Table 2-1. Recombinant VACV
a 
TAP-tag at C-terminus
b 
C-terminal HA tag
c 
Transmembrane and cytoplasmic tail of A56 removed and replaced with TAP-tag
Recombinant virusParent   [Reference]Description
v"A56 VACV WRDeletion of A56R
v"A56"K2 v"A56 Deletion of A56R and K2L
vA28TAPVACV WRA28-TAP
a
vA28i"A56vA28i              [80]Inducible A28-HA
b
; deletion of A56R
vA28i"K2 vA28i              [80]Inducible A28-HA
b
; deletion of K2L
vA28TAP"K2vA28TAPA28-TAP
a
 tag; Deletion of K2L
vA56TAPv"A56 A56-TAP
a
vA56TAP"K2vA56TAPA56-TAP
a
; deletion of K2L
vsA56TAPivT7lacOI"F13
vsA56TAPi"C3vsA56TAPiInducible and secreted A56-TAP
c
;
deletion of C3L
vK2i"A56vT7lacOI        [66]Inducible K2-HA
b
; deletion of A56R
vK2TAPVACV WRK2-TAP
a
vK2TAP"A56vK2TAPK2-TAP
a
; Deletion of A56R
vT7lacOI"F13vT7lacOI        [66]Inducible T7 polymerase; constitutive
expresion of lac represion; deletion
of F13L
37
Primary and secondary antibodies were removed from the membrane by
incubating with Restore Western Blot Stripping Buffer (Pierce) for 30 min at 55
o
C.
2.2.5 Antibodies.
Rabit polyclonal antisera used to detect VACV proteins were: anti-A21 [79],
anti-L5 [81], anti-A16 [78], and anti-p4b/4b (R. Doms and B. Mos, unpublished).
Antibody to the A28 was prepared by imunizing rabits with purified recombinant
protein provided by Gretchen Nelson, NIAID. K2 and A56 rabit antisera were raised
against a synthetic peptide PFDITKTRNASFTNKYGTKT derived from K2 amino
acids 176-195 and SEKPDYIDNSNCSVF derived from A56 amino acids 151-16
with the adition of a C-terminal cysteine for conjugation to keyhole limpet
hemocyanin (Covance Research Products, Denver, PA). A monoclonal antibody
against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from
Covance.
2.2.6 Synthesis and purification of soluble A56/K2.
Ten roler botles of RK13 cels were coinfected with 1 PFU per cel each of
vsA56TAPi"C3 and vK2i"A56"C3 for 2 h at 37
o
C. After virus adsorption the cels
were washed twice with Dulbecos?s phosphate bufered saline with calcium and
magnesium (Quality Biological Inc.). Then 50 ml of Opti-Mem (Invitrogen) with 2
mM IPTG was added and the infection was allowed to proceed for 48 h.  The medium
was clarified by centrifugation and any remaining debris removed by filtering through
a 0.45 !M membrane. The medium was suplemented with glycerol to 10% final
concentration and NaCl to 40 mM. The protein was purified by binding to wheat
38
germ aglutinin agarose (Vector laboratories, Burlingame, CA) and eluted with 50
mM N-acetyl-#-D-glucosamine (Calbiochem, La Jola, CA). The eluate was then
bound to streptavidin Sepharose, washed with phosphate bufered saline and eluted
with 1 mg/ml of biotin.
2.3Results
2.3.1 Neutral pH Cell-Cell Fusion Requires the Entry/Fusion Complex.
The first goal of this study was to beter understand neutral pH cel fusion that
ocurs folowing infection with VACV deleted for the A56R or K2L gene. Low pH
cel fusion of VACV requires a functional EFC, however this complex has not ben
demonstrated to be required for neutral pH cel-cel fusion. The function of the
proteins of the EFC in virus entry and fusion was initialy characterized by
constructing conditional lethal viruses in which gene expresion was regulated by
E.coli lac represor [71, 78, 79, 81-85]. Represing the synthesis of any of the entry
proteins produced viruses with a similar phenotype, mainly an inability of the virus
particle to penetrate the cel along with failure to triger low pH cel fusion. The
curent strategy was to determine the efect of deleting the A56R or K2L gene from
one of the inducible mutants of the entry complex. If spontaneous fusion of infected
cels ocured when EFC gene was represed, it would indicate an alternative
pathway of cell-cell fusion.
The vA28i was used as the parental inducible virus because regulation of A28
expression was stringently repressed in the absence of IPTG and assembly of the EFC
was prevented [85]. A56R and K2L genes were deleted individualy from vA28i by
39
replacing the viral gene with the coding sequence for EGFP to form vA28i"A56 and
vA28i"K2, respectively. DNA sequencing confirmed the deletion of the A56R or
K2L gene. vA28i"A56 and vA28i"K2 stocks were prepared in the presence of IPTG
so that virions posesed A28 and were therefore able to infect cels. After infection
in the absence of IPTG, however, the progeny virions would lack A28 and be unable
to spread to neighboring cels. Both recombinant viruses expresed EGFP regardles
of the presence or absence of IPTG but only formed plaques under the former
conditions. In the presence of IPTG, HeLa cels infected with vA28i"A56 or
vA28i"K2 formed large multinucleated syncytia visualized by fluorescence
microscopy (Figure 2-1). No cel fusion was observed in the absence of IPTG for
either vA28i"A56 or vA28i"K2 even though the cels were infected as shown by
expresion of EGFP (Figure 2-1). The results showed the A28 protein, in adition to
its role in virus entry and low pH trigered cel-cel fusion, was required for cel-cel
fusion ocuring at neutral pH in the absence of the A56R or K2L gene. It sems
likely that the other components of the EFC would also be required for neutral pH
cel fusion indicating the multicomponent EFC mediates both low pH and neutral pH
virus-induced membrane fusion.
2.3.2 The anti-fusion proteins A56 and K2 interact with the viral EFC.
A56 contains no putative enzymatic motifs and the serine protease inhibitor
active site of the K2 protein is not required to inhibit cel fusion. This sugested the
proteins may regulate cel fusion through protein-protein interactions. To investigate
possible protein interaction of the A56 protein, a recombinant VACV called
40
Figure 2-1: The EFC is required for neutral pH cel fusion. HeLa cels were
infected with vA28i"A56 or vA28i"K2 in the absence (-) or presence (+) of IPTG.
After 24 h, the cels were examined under an inverted fluorescence microscope to
visualize cels expresing GFP encoded by the recombinant VACVs. The percentage
of nuclei in syncytia, as defined by a cel containing 3 or more nuclei, is averaged
from two independent experiments.
41
Figure 2-2: Tandem affinity purification of A56.
HeLa cels were infected with VACV WR or vA56TAP and the cels were disrupted
with Triton X-10. The post-nuclear supernatants were purified sucesively on
streptavidin and calmodulin afinity columns. The bound proteins were eluted,
concentrated, resolved by SDS-PAGE and detected by staining with Comasie blue.
The protein bands were excised from the gel, digested with trypsin and analyzed by
mas spectrometry. The identities of the proteins are indicated to the right of the
stained bands. In one case, peptides coresponding to two proteins (G9 and I1) were
obtained from the same band. Marker proteins with masses in kDa are indicated at the
left.
42
vA56TAP was constructed in which codons for streptavidin and calmodulin binding
peptides were fused in frame to the end of the A56R open reading frame. The TAP
tag did not compromise the function of the A56 protein, as syncytia did not form
when cels were infected with vA56TAP. Infected cels were lysed with Triton X-10
detergent and the post-nuclear supernatant was incubated with Sepharose beads
linked to streptavidin. The resin was washed extensively and the bound proteins were
eluted from the streptavidin beads by incubation with D-biotin. The eluate was
subjected to a second afinity purification by incubating with Sepharose beads linked
to calmodulin. The calmodulin Sepharose was washed and the bound proteins were
eluted with EGTA. The purified proteins were concentrated, resuspended with a
solution of LDS, resolved by polyacrylamide gel electrophoresis, and then visualized
by staining with Comasie blue. Cels infected with VACV lacking a TAP tag were
purified in paralel to serve as a negative control. Multiple intensely stained bands
were observed in the A56TAP sample, but were absent from the control. The protein
bands were excised and then digested with trypsin folowed by mas spectrometry to
identify the peptides. The proteins coresponding to the observed peptides are
indicated next to the bands in figure 2-2. One of the bands coresponded to the K2
protein, confirming an asociation with A56. The intensity of the K2 band, however,
was much les than that of A56, even taking into acount the diference in their
mases. This sugests either a surplus of A56 relative to K2, not al of the K2 was
bound to HA, or the complex partly disociated during purification.  Interestingly
members of the EFC A16, G9 and J5 co-purified with A56. Several other proteins,
namely calmodulin and the VACV products C3, I1 and O2 also copurified with A56
43
and were identified by mas spectrometry (Figure 2-2). Calmodulin undoubtedly
came from the afinity beads. C3 is a secreted modulator of complement activation
[58, 59] I1 is a DNA telomere binding protein [189], [190] and O2 is a non-esential
glutaredoxin [191 {Rajagopal, 195 #54, 192]. Although these aditional bands
were not detected in the control lane (Figure 2-2), any biological significance of the
latter interactions with A56 remains to be determined.
Western bloting with specific antisera to A21, A28 and L5 was used to
identify thre aditional EFC proteins recovered after TAP of A56 (Figure 2-3). It is
likely that the other entry proteins G3 and H2 were also present, though antibodies to
these proteins were not available to confirm this. Cels infected with wild type
VACV lacking a tag on A56 did not asociate with A21, A28 and L5 when subjected
to TAP, even though these proteins were present in the starting material (Figure 2-3).
Furthermore, antisera to K2 and A16 were used as positive controls and antisera to
D8, an MV membrane protein not asociated with the EFC, and the celular protein
GAPDH as negative controls. In this experiment a smal amount of GAPDH was
detected; however this was non-specific as it was not dependent on K2 (se next
section). Colectively the mas spectrometry and Western bloting data indicated that
at least 6 of the 8 EFC proteins were associated with A56 either directly or indirectly.
2.3.3 A56 does not bind the EFC in the absence of K2.
Regulation of infected cel fusion requires K2 as wel as A56, yet the role of
the individual proteins in the interaction with the EFC was unknown. Therefore the
primary binding of the EFC may be mediated by either A56 or K2. To investigate if
A56 alone was able to asociate with the entry complex, the K2L gene of vA56TAP
44
was replaced with DNA encoding EGFP. As expected, cels infected with the K2L
deleted virus vA56TAP"K2 fused to neighboring cels. When extracts of cels
infected with vA56TAP"K2 were subjected to TAP in paralel with extracts of cels
infected with vA56TAP, EFC proteins were only detected in the later (Figure 2-3).
Cels infected with vA56TAP"K2 expresed A16, A21, A28 and L5 similar to those
infected with either WR or A56TAP (Figure 2-3). The results sugested K2 is
important for interaction with the EFC, possibly by direct association.
2.3.4 Both K2 and A56 are needed for association with the EFC.
The inability of A56 to asociate with the EFC in the absence of K2 sugested
the EFC interacts with A56/K2 through K2 and only indirectly with A56. To test this
hypothesis, a recombinant VACV was constructed in which K2 has a TAP tag and
A56 was deleted. The virus created in several steps, the first was to make vK2TAP, a
recombinant VACV encoding K2 with a TAP tag apended to the C-terminus. A
smal increase in syncytia formation was noted in cels infected with vK2TAP
sugesting the presence of the TAP tag slightly afected the function of K2, though
the amount of cel fusion was wel below the level of a K2 deletion virus. Western
blot analysis of affinity purified K2 showed the protein co-purified with both A56 and
components of the EFC and confirmed that the tag did not greatly compromise the
function of K2 (shown later). To determine whether K2 was suficient for interaction
with the EFC the A56 gene was removed to construct vK2TAP"A56. Cels infected
with vK2TAP"A56 formed large multinucleated syncytia consistent with the
phenotype observed upon infection with an A56 deletion mutant. Initial experiments
suggested the EFC did not associate with affinity purify
45
Figure 2-3: Western blot analysis of A56TAP.
Mock infected cels (M) or cels separately infected with VACV WR, vA56TAP or
vA56TAP"K2 were subjected to tandem afinity purification over sucesive
streptavidin and calmodulin afinity resins. The bound proteins were eluted, resolved
by SDS-PAGE, and detected by Western bloting with antibodies to the indicated
proteins. Components of the EFC are represented by: A16, A28, A21, and L5; D8 is
an MV protein and absent from the EFC; GAPDH is a celular protein used as a
control. Starting material is designated PreTAP, samples subjected to dual afinity
purification are labeled TAP.  Viruses are noted at the top.
46
Figure 2-4: Entry proteins co-purify with K2TAP only in the presence of A56.
Cels were mock infected or infected separately with VACV WR, vK2TAP or
vK2TAP"A56. The post-nuclear supernatent was afinity purified sequentialy over
streptavidin and calmodulin Sepharose.  The bound material was eluted, separated by
SDS-PAGE and analyzed by Western bloting with antibodies to the proteins A56,
K2, A16, A21, L5 and p4b as indicated. Starting material is labeled PreTAP.
Afinity purified samples refered to as TAP. The - and + signs indicate the absence
or presence of brefeldin A, respectively.
47
K2TAP in the absence of A56. However, in the absence of A56 the K2 protein was
partially secreted into the medium, since K2 relies on association with A56 for
membrane asociation. To prevent secretion and potentialy alow more oportunity
for K2 to interact with the EFC, the antibiotic brefeldin A was used to disrupt the
Golgi aparatus and prevent protein traficking along the secretory pathway [193,
194]. Brefeldin A has ben shown to have litle efect on the formation of MV, while
significantly reducing the amount of EV [195]
To determine whether A56 was required for K2 to bind the EFC, HeLa cels
were infected with VACV WR, vK2TAP or vK2TAP"A56 in the presence or
absence of 10 ?g/ml of brefeldin A. Cel lysates from the starting material confirmed
A56 was not synthesized in cels infected with vK2TAP"A56 (Figure 2-4). Brefeldin
A altered the mobility of glycosylated A56 expresed in cels infected with VACV
WR or vK2TAP which was expected due to a failure of the protein to transit through
the Golgi aparatus (Figure 2-4). There apeared to be more A56 in the presence of
brefeldin A, posibly because sheding of EVs was prevented. Similarly, brefeldin A
increased the amount of K2 and changed the mobility of the protein, particularly in
the absence of A56. There was no noticeable efect of brefeldin A on the non-
glycosylated EFC proteins or on the core protein p4b (Figure 2-4). K2 purified from
cels infected with vK2TAP demonstrating an asociation with A56 and the EFC
proteins A16, A21, A28 and L5 in the absence or presence of brefeldin A (Figure 2-
4). K2 did not interact with p4b indicating the interaction was specific. In the
absence of A56, the K2 protein was unable to asociate with A16, A21, A28 and L5
48
even though brefeldin A increased the amount of K2. These results show both A56
and K2 are needed to association with the EFC.
2.3.5 Association of A56 and K2 with a TAP-tagged EFC.
The EFC has ben shown to copurify with both a TAP taged A56 and a TAP
taged K2 protein. To confirm this interaction, the reciprocal experiment was
performed by constructing an aditional recombinant VACV with DNA encoding the
TAP tag at the 3? terminus of the A28L gene. The A28 protein was chosen as a
representative member of the EFC and the protein was previously shown to exhibit
normal function with the adition of the influenza HA epitope, sugesting the protein
may tolerate the adition of the TAP tag [85]. The recombinant VACV vA28TAP
grew to high titers indicating no defect in A28 function. The vA28TAP was used to
construct another recombinant VACV in which the K2L gene of vA28TAP was
replaced with the gene encoding EGFP. Cels infected with vA28TAP"K2 formed
syncytia at neutral pH.  Lysates from HeLa cells infected with VACV WR, vA28TAP
or vA28TAP"K2 were subjected to tandem afinity purification and then analyzed by
Western bloting. As expected, the mobility of the A28 protein was reduced in cels
infected with A28TAP due to the increase in mas caused by the TAP tag, while K2
was absent in cels infected with vA28TAP"K2 (Figure 2-5). Both A56 and K2 co-
purified with the A28TAP entry protein (Figure 2-5). The interaction betwen A28
and A56/K2 was specific as neither the core protein p4b nor the celular protein
GAPDH co-purified with A28TAP, in adition no proteins were detected from the
afinity-purified lysates of VACV WR (Figure 2-5). The major A56 band did not
associate with A28TAP when the protein was purified from the lysate of
49
Figure 2-5: A TAP-tagged A28 protein associates with A56 and K2.
Cels were infected individualy with VACV WR, vA28TAP or vA28TAP!K2 as
indicated and lysed. The A28 protein was purified sequentialy by streptavidin and
calmodulin chromatography. The co-purifying proteins were eluted, resolved by
SDS-PAGE, and detected by Western bloting with antibodies to the proteins: A56,
K2, A28, p4b, GAPDH as indicated. PreTAP, starting material; TAP, afinity purified
samples.
50
vA28TAP"K2 (Figure 2-5). A much les intense doublet migrating faster than the
major A56 bands was observed to copurified with A28TAP in the presence and
absence of K2 (Figure 2-5). Initialy our thought was that the doublet represented
cros-reactivity of the anti-A56 peptide antibody, however the same result was
obtained when the Western blot was caried out with a monoclonal antibody against
A56. This sugests a minor form of A56 is able to bind A28TAP even in the absence
of K2. In the absence of K2, the major bands of A56 fail to asociate with A28TAP
confirming the importance of K2 for binding the EFC.
2.3.6 Interaction of soluble A56/K2 with the EFC.
To further characterize the asociation betwen the fusion regulatory proteins
and the EFC, a soluble form of A56/K2 was prepared. The basic idea was to prepare
two recombinant VACVs that each inducibly over expresed either K2 or a secreted
form of A56 using vT7lacOI [6] as the starting virus. One of the recombinant
VACVs had a deleted A56 gene and a K2 gene regulated by the bacteriophage T7
promoter and E. coli lac operator. A second recombinant VACV posesed an
inducible A56 with the membrane anchor and cytoplasmic tail sequences substituted
for codons of the TAP tag. Pilot experiments confirmed secretion of a K2/A56TAP
complex from co-infected cels, yet the C3 protein also asociated with the complex.
(Note that asociation of C3 with the ful-length K2/A56 complex is shown in Figure
2-2). The C3 protein is a non-esential virus-encoded host defense protein and was
removed by replacing the gene with one encoding EGFP in both recombinant VACVs
to form vK2i"A56"C3 and vsA56TAPi"C3 which inducibly expresed K2 and a
soluble TAP-tagged A56 in the absence of C3.
51
To isolate a soluble complex of A56 and K2 RK13 cels were coinfected with
vK2i"A56"C3 and vsA56TAPi"C3 in low serum edium suplemented with IPTG.
The presence of IPTG was required to induce protein expression, while the low serum
reduced the level of contaminating proteins. Both A56 and K2 are glycosylated so an
imobilized lectin, wheat germ aglutinin, was used to concentrate the proteins from
the medium. Folowing this step the complex was isolated by an afinity step with
streptavidin Sepharose. This two-step purification isolated a soluble complex of A56
and K2 that was free of major contaminating proteins (Figure 2-6A).
To investigate an interaction betwen purified soluble A56TAP/K2 and the
EFC, the infected cel lysate neded to lack endogenous A56 and K2. To facilitate
this, a recombinant VACV, v"A56"K2 was constructed by sequential replacement of
the A56R and K2L genes with DNA encoding EGFP and HcRED, respectively.
Deletion of A56 and K2 was confirmed by Western blot analysis (Figure 2-6). The
soluble complex of A56TAP/K2 was incubated with a post-nuclear lysate isolated
from HeLa cels infected with v"A56"K2 or mock infected. Afinity purification of
the soluble A56TAP/K2 was determined by TAP, utilizing the tag on the recombinant
A56TAP protein. Western blot confirmed an asociation betwen soluble
A56TAP/K2 and A16, A21, A28 and L5, but not with the core protein p4b (Figure 2-
6B). The EFC only co-purified with the soluble A56TAP/K2, as none of the entry
proteins were detected in its absence. The secreted form of A56TAP apears to be a
homogenous, fuly glycosylated protein, whereas A56TAP from infected cel lysates
(shown on the right of Figure 2-6B) is heterogeneous reflecting varying stages of
glycosylation.
52
Figure 2-6: Soluble A56/K2 interacts with the EFC.
(A) Soluble A56/K2 was isolated by afinity purification from the medium of cels
co-infected with vK2i"A56"C3 and vsA56TAPi"C3 in the presence of IPTG. A
complex of A56/K2 was purified by a 2-step procedure using imobilized wheat
germ aglutinin and streptavidin. Folowing concentration, the purified proteins were
analyzed by SDS-PAGE and silver staining. (B) A post nuclear lysate of mock
infected HeLa cels or cels infected with v"A56"K2 was incubated with the purified
soluble A56/K2. The A56/K2 complex was tandem afinity purified, separated by
SDS-PAGE and analyzed by Western bloting by using antibodies to the proteins
indicated on the left.
53
2.4Discussion
Entry of VACV core is preceded by fusion of the MV membrane with the
plasma membrane at neutral pH [91] or with the endosomal membrane at low pH
[94]. Fusion of MV at the plasma membrane is greatly enhanced by lowering the pH
of the medium below 6, a proces that is thought to miic the decrease in pH within
the endosome [94]. At late times during an infection, briefly lowering the pH of the
medium trigers cel-cel fusion, a proces that depends on cel surface EV (caled
fusion from within) [152, 165, 16]. Cel fusion also ocurs when cels are
inoculated with large numbers of purified MV and subsequently treated with low pH
(caled fusion from without) [16]. Cels infected with VACV normaly do not fuse,
but deletion of the A56 or K2 protein leads to spontaneous cel fusion of infected
cells at neutral pH [167-170].
EV is required for infected cel fusion triger by low pH as wel as cel fusion
asociated with deletion of A56 (and presumably K2), indicating virus-cel fusion and
cel-cel fusion are closely related phenomena. Previous investigations have revealed
virus-cel fusion and low pH-induced cel fusion depended on a conserved
multiprotein EFC [84, 94]. The study described in this chapter demonstrated neutral
pH cel fusion requires the A28 protein. Represing synthesis of A28 prevents
formation of the EFC [84]. It is likely that other proteins of the complex wil also be
required for neutral pH cel fusion. The first stage in the development of cel fusion
is thought to be fusion betwen MV and cel membrane (Figure 1-4). The absence of
a functional EFC would prevent virus-cel fusion and as a result the later steps would
not occur.
54
The mechanism by which A56 and K2 regulate cel fusion is unknown. The
A56 protein has no putative catalytic motifs and mutations in K2 that disrupt the
SERPIN activity have no efect on syncytia formation. Given this information it was
suspected that A56/K2 might inhibit cel fusion through protein-protein interaction.
To investigate this hypothesis a number of recombinant VACV were constructed with
the TAP tag atached to A56, K2 or the A28 EFC protein. Both A56 and K2 were
observed by a combination of mas spectrometry and Western bloting to interact
with proteins of the EFC. A56 was unable to interact with the EFC in the absence of
K2, and eficient interaction of K2 with the EFC required A56. A minor form of A56
was observed to copurified with TAP-taged EFC in the absence of K2, although the
significance is not yet understod. The requirement of both A56 and K2 to eficiently
bind the EFC corelates with the ned of both proteins to eficiently prevent
spontaneous fusion of infected cels. The dynamics of the interaction betwen the
EFC and A56 and K2 remain to be studied. It is unknown whether a conformational
change ocurs within A56 or K2 upon asociating with one another. Alternatively,
the conformation of the complex of A56 and K2 may be required to bind the EFC.
Both A56 and K2 are present in the EV membrane [184] and are located
within the plasma membrane as wel [172]. It is suspected that plasma membrane
localization of A56/K2 is important for preventing re-infection of cels by progeny
extracelular virions. In order for A56/K2 to interact with the EFC within the MV
membrane, the outer EV membrane must first be disrupted. It was recently reported
that the EV membrane is ruptured by interaction with cell surface GAGs [97].
55
The interaction of A56/K2 with the EFC was observed to ocur post-lysis.
This was shown by the ability of soluble A56/K2 to interact with the EFC from an
infected cel lysate devoid of endogenous A56/K2. Therefore under the experimental
conditions the adition of the detergent lysis the cels and alows A56/K2 to interact
with the EFC. Aditional investigation wil be required to determine the proteins of
the EFC that mediate an interaction with A56/K2.
56
Chapter 3: The VACV fusion regulatory proteins A56 and K2
interact with a subcomplex of A16 and G9
3.1 Introduction
VACV induces cel fusion folowing low pH treatment of infected cels or
spontaneously at neutral pH when either the A56R or K2L gene is absent. VACV
entry and fusion requires a conserved multiprotein complex of at least eight proteins
that resides within the MV membrane. The EFC is esential for entry of both the MV
and EV forms of the virus as is also required for low pH cel-cel fusion, while in the
previous chapter the A28 entry protein was shown to be esential for neutral pH cel
fusion associated with deletion of A56 or K2.  Therefore virus-cell fusion, neutral cell
fusion and low pH cel fusion al depends on the viral EFC. Asembly of the EFC
does not ocur in the absences of a single entry protein, although the remaining entry
proteins are stable and localize to the MV membrane.
Litle is known about the mechanism by which A56 and K2 regulate cel
fusion. Tandem afinity purification of the A56 protein was utilized to identify a
novel protein interaction with the EFC, sugesting the anti-fusion activity of A56 and
K2 may be mediated through an interaction with the viral entry proteins. Both fusion
regulatory proteins were required to bind the EFC; neither A56 nor K2 alone was
suficient. The previous chapter did not determine the protein within the EFC
required for an interaction with A56/K2. To determine the minimal components of
the EFC required for binding to A56/K2 a series of conditional lethal viruses were
constructed in which various entry proteins were represed and interaction with
A56/K2 were asesed. Our analysis revealed both A16 and G9 were neded to bind
57
A56/K2 as neither A16 nor G9 alone bound eficiently to A56/K2. Furthermore A16
and G9 were shown to interact with one another in transfected cels sugesting the
two proteins directly interact within the EFC.
3.2Material and Methods
3.2.1Cells and virus.
BS-C-1 (ATC CL-26), HeLa S3 (ATC CL-2.2) suspension cels were
grown as described in chapter 2, section 2.2.1. 293T cels, stably expresing the
large T antigen, were provided by Chris Buck [196], and were grown in Dulbeco?s
minimum esential medium (Quality Biological) suplemented with 10% fetal bovine
serum, 2 mM L-glutamine and 40 !g/ml hygromycin (Invitrogen, Carlsbad, CA).
The Western Reserve (WR) strain of VACV was used in the construction of al
recombinant viruses unles noted otherwise. General procedures for preparing and
titrating stocks were done as previously described in chapter 2, section 2.2.1.
3.2.2 Plasmid and recombinant VACV construction.
The recombinant viruses constructed for this study (Table 3-1) were:
vA28iA56TAP, vA21iA56TAP, vA28iA56TAPJ5Flag, vA28iA56TAPG93XFlag,
vA28iA163XFlag, vA28iG93XFlag, vA16iA56TAPG93XFlag, and vG9iA56TAP (where i
indicates an inducible gene, TAP refers to a tandem afinity tag, 3XFlag indicates 3
copies of the Flag epitope). Recombinant viruses were screned by PCR to confirm
the absence of parental virus and the sequence of the inserted DNA was confirmed.
The vA28iA56TAP, vA21iA56TAP, vA16iA56TAP and vG9iHA56TAP were constructed
from vA28i [85], vA21i [79], vA16i [78] and vG9iHA [83], respectively by appending
58
Table 3-1. Recombinant VACV
Recombinant virus    Parent (Reference)Description
vA16ivT7lacOI      [66]Inducible A16
vA16iA56TAP vA16i           [78]Inducible A16; A56-TAP
a
vA16iA56TAPG93XFlagvA16iA56TAPInducible A16; A56-TAP
a
; G9-Flag
b
vA21ivT7lacOI      [66]Inducible A21
vA21iA56TAP vA21i           [79]Inducible A21; A56-TAP
a
vA28ivT7lacOI      [66]Inducible A28-HA
c
vA28iA163XFlagvA28i           [80]Inducible A28-HA
c
; A16-Flag
b
vA28iA56TAP vA28i           [80]Inducible A28-HA
c
; A56-TAP
a
vA28iA56TAPG93XFlagvA28iA56TAPInducible A28-HA
c
; A56-TAP
a
; G9-
Flag
b
vA28iA56TAPJ5FlagvA28iA56TAPInducible A28-HA
c
; A56-TAP
a
;
J5-Flag
d
vA28iG93XFlagvA28i            [80]Inducible A28-HA
c
; G9-Flag
b
vA56TAP v"A56A56-TAP
a
vG9iA56TAP vG9i                     [83]Inducible G9-HA
e
; A56-TAP
a
a 
TAP-tag at C-terminus
b 
3 copies of Flag tag at C-terminus
c 
1 copy of
 
C-terminal HA tag
d 
1 copy of Flag tag at N-terminus
e 
1 copy of N-terminal HA tag
59
the codons for a C-terminal TAP tag to the A56 gene. The DNA used to construct the
C-terminal TAP tag has been described in chapter 2, section 2.2.3.  vA28iA56TAP was
the parental virus for the construction of vA28iA56TAPJ5Flag.   Overlapping PCR
(Accuprime Pfx; Invitrogen) was used to assemble the DNA used for recombination.
The layout of the DNA sequence for the J5Flag construct from the 5? to 3? was as
follows: (i) 500 bps of DNA sequence upstream of the J5R gene, (ii) EGFP expressed
from the I1L promoter, (iii) 70 nucleotides containing the J5R promoter and (iv) the
initial methionine of J5R, followed by the DNA sequence for the Flag epitope
(DYKDDDK) and then the remaining DNA sequence of the J5R gene.  The
recombinant PCR product was cloned into pCR-BluntII-TOPO (Invitrogen) and
verified by DNA sequencing.  The J5Flag plasmid was linearized by cleavage with a
unique restriction endonuclease.  BS-C-1 cells were infected with vA28iA56TAP for 1
h, then transfected with the linearized plasmid using Lipofectamine 2000
(Invitrogen). IPTG was added to the medium at 100 ?M to allow expression of the
inducible A28 gene.  The parental and recombinant viruses were distinguished by the
GFP fluorescence of the latter.  Recombinant plaques were clonally isolated through
3 rounds of plaque purification.
vA28iA56TAPG93XFlag and vA28iG93XFlag were constructed from vA28iA56TAP
and vA28i, respectively. vA16iA56TAPG93XFlag was constructed sequentialy by first
generating vA16iA56TAP from vA16i. The 3XFlag was aded to the G9 protein to
form vA16iA56TAPG93XFlag, vA28iA56TAPG93XFlag, vA28iG93XFlag as folows. The
G9R gene was PCR amplified from genomic DNA of the Western Reserve (WR)
strain (ATC VR-1354, acesion number AY24312). The 3XFlag epitope was
60
apended to the C terminus of G9 prior to the stop codon. This was folowed by the
coding sequence of the Discosoma sp. red fluorescent protein (DsRed) expresed
from the I1L intermediate promoter for screning of recombinant viruses. The L1R
gene was PCR amplified along with 90 bps upstream of the gene to include the L1R
promoter. Recombinant PCR was utilized to ad L1R with the promoter after the
DsRed coding sequence. The promoter for the LIR gene is located within the C-
terminal codons of G9. To conserve expresion of the L1R gene, the C-terminus of
G9 was duplicated causing a direct repeat of DNA sequence before and after the
DsRed gene. Direct repeats are unstable in the virus genome [197]. Therefore to
prevent the eventual los of the DsRed gene the codons of the final 3 amino acids
from G9 were altered, while conserving the amino acid sequence. The recombinant
G93XFlag PCR was cloned into pCR-BluntI-TOPO and verified by DNA sequencing.
Recombinant viruses were generated by infecting BS-C-1 cels with the apropriate
parental virus and then transfecting linearized G93XFlag plasmid. Recombinant viruses
were distinguished from parental by DsRed fluorescence and were clonaly isolated
through 3 rounds of plaque purification.
3.2.3A16 and G9 Codon Optimization.
The DNA sequence for the VACV WR A16L and G9R genes was optimized
(Geneart, Regensburg, Germany) to alter codon usage and G-C content to improve
RNA procesing and translation. The optimized A16L and G9R genes were PCR
amplified with oligonucleotides that contained the sequence of the influenza virus HA
or 3XFlag epitope apended to the C-terminus of the respective ORFs. The PCR
61
products of A16HA and G93XFlag were cloned into the directional TOPO vector
pcDNA3.1 (Invitrogen) and sequenced to confirm proper insertion and sequence.
3.2.3 Affinity purification.
BS-C-1 cels (6 x10
6
) were infected at a multiplicity of 3 to 5 plaque forming
units per cel in EMEM with 2% FBS. After 24 h the cels were scraped into the
medium and subjected to low sped centrifugation. The cel pelet was washed once
by resuspending in 150 mM NaCl with 50 mM Tris-HCl (pH 7.5). The cels were
then lysed with ice-cold SB [1% Triton X-10 (Sigma, St. Louis, MO), 150 mM
NaCl, 50 mM Tris-HCl (pH 7.5)] suplemented with complete protease inhibitor
(Roche, Indianapolis, IN) and rotated at 4 
o
C for 30 min. The lysate was peleted at
4
o
C in a benchtop centrifuge at 20,00 x g for 10 min and the postnuclear supernatent
was colected and 50 ?l saved for analysis. Tandem afinity purification was caried
out as previously described in chapter 2, section 2.2. Single step purifications were as
folows. Packed beads (20-30 ?l) of either streptavidin Sepharose (Milipore,
Bilerica, MA) or anti-Flag conjugated agarose (Sigma) were washed once with 1 ml
of lysis bufer and the postnuclear supernatant was aded to the afinity resin and
rotated overnight at 4 
o
C. The afinity resin was washed 5 times with 1 ml of SB
prior to elution. The bound material was eluted from the anti-Flag agarose by aded
50 !l of 1X LDS sample bufer (Invitrogen) suplemented with reducing agent
(Invitrogen) and incubated at 10 
o
C for 5 min. The beads were peleted by
centrifugation and the supernatent was collected.  Bound material was eluted from the
streptavidin Sepharose by incubation with 30 ?l of lysis bufer suplement with 2
mM d-Biotin (US Biological, Swampscot, MA). The elution was repeated two times
62
and the eluates were combined prior to concentration by precipitation with
trichloroacetic acid. The precipitated material was resuspended in 40 ?l of 1x LDS
buffer with reducing agent.
3.2.5 Transfection and coimmunoprecipitation.
293T cels were plated at a density of 2x10
6
 per 9.2 cm
2
 the day before
transfection in 10% DMEM plus glutamine, but without hygromycin. Cels were
transfected with 2 ?g of total DNA using Lipofectamine 200 (Invitrogen). After 24
h fresh 10% DMEM was aded and the incubation continued for an aditional 24 h at
which time cell extracts were prepared and subjected to immunoprecipitation.
3.2.6 Western blotting and antibodies.
Samples were loaded onto a 4-12% Novex NuPAGE acrylamide gel
(Invitrogen) and separated by electrophoresis using 2(N-morpholino)ethanesulfonic
acid bufer (Invitrogen). The protein samples were transfered to a nitrocelulose
membrane and then blocked with 5% nonfat milk in TBST. Primary antibody was
incubated a minimum of 1 h prior to extensive washing with TBST. The secondary
antibodies (Pierce, Rockford, IL) were diluted in 5% nonfat milk TBST and incubated
for at least 1hr. The nitrocelulose membrane was washed and then developed with
Dura or Femto Chemilumenscent substrate (Pierce). The antibodies were striped
from the nitrocelulose by incubating 20 min at 5 
o
C with Restore (Pierce).
Antibodies to A56 (chapter 2, section 2.5), K2 (chapter 2, section 2.5), A21 [79], L5
[81], A16 [78], and p4a/p4b (R. Doms and B. Mos, unpublished data) were used in
Western blot analysis. A monoclonal antibody (conjugated to horseradish peroxidase)
63
against the influenza hemaglutinin epitope was acquired from Bethyl laboratories
(Montgomery, TX). The anti-Flag M2 monoclonal antibody was obtained from
Sigma (St. Louis, MO) and a monoclonal antibody to the celular glyceraldehyde-3-
phosphate dehydrogenase was purchased from Covance Research (Princeton, NJ).
The rabit antiserum to H2 and A28 was generated by imunizing rabits with
purified recombinant H2 or A28 proteins, respectively and provided by Gretchen
Nelson, NIAID. Peptide antibody was generated to the G3 protein by imunizing
rabits with the synthetic peptide [SLNGKHTFNLYDNDIRT] coupled to
keyhole limpet hemocyanin through an N terminal cysteine (Covance).
3.3Results
3.3.1A56/K2 interacts with a subset of the proteins within the EFC.
The interaction of A56/K2 with the EFC was described in chapter 2. Both
A56 and K2 polypeptides were required to interact with the EFC, although the entry
proteins required for the interaction were not determined. Our analysis was based on
previous observations that (i) the viral membrane is required for asembly of the EFC
and (i) that the EFC fails to form in the absence of A28 or A21 even though the
remaining EFC proteins are incorporated into the viral membrane [84]. It may be
posible by represing synthesis of A28 and A21 to identify either individual
polypeptides or previously uncharacterized subcomplexes of the EFC capable of
interacting with A56/K2. To implement this strategy two recombinant viruses were
constructed, vA28iA56TAP and vA21iA56TAP, in which A28 and A21 were
conditionaly expresed, respectively. Conditional expresion of A28 and A21 was
64
regulated by components of the E. coli lac operon in combination with the T7 phage
DNA-dependant RNA polymerase, such that viral gene expresion depended on the
adition of IPTG. Both recombinant viruses were constructed with a TAP tag
apended to the C-terminus of the A56R gene to alow purification of the A56/K2
heteromultimer along with any asociating polypeptides. In adition, A28 had a C-
terminal HA epitope tag.
HeLa cels were infected with VACV strain WR, vA56TAP (chapter 2, section
2.3), vA28iA56TAP (+ and ? IPTG), vA21iA56TAP (+ and ? IPTG), or mock infected.
VACV WR, with an untaged A56 was used as a negative control for nonspecific
interaction during afinity purification, while vA56TAP with a constitutively expresed
A28 was used as a positive control. The stocks of vA28iA56TAP and vA21iA56TAP
were prepared in the presence of IPTG so that the virus particles contained A28 and
were infectious, but synthesis of A28 or A21 during the next cycle depended on the
adition of IPTG. At 24 h post infection, the cels were lysed with Triton X-10
detergent and the post-nuclear supernatant was subjected to TAP on streptavidin and
calmodulin Sepharose columns. The proteins in the final eluate were concentrated,
separated by SDS-PAGE, and detected by Western blotting with specific antibodies.
Western bloting of the starting material of cels infected with vA28iA56TAP
and vA21iA56TAP, confirmed synthesis of A28 and A21, respectively, depended on
IPTG (Figure 3-1, PreTAP). The slightly slower electrophoretic migration of A28 in
the lysate of vA28iA56TAP was due an HA epitope tag on the C terminus of the
65
Figure 3-1:  A56/K2 physically associate with A16.
 HeLa cels were mock infected (M) or infected with VACV WR, vA56TAP,
vA28iA56TAP (+ and ? IPTG) and vA21iA56TAP (+ and ? IPTG). After 24 h the cels
were lysed with Triton X-10 and the A56 protein was afinity purified sequentialy
over streptavidin and calmodulin Sepharose. The starting material (PreTAP) and
purified samples (TAP) were separated by SDS-PAGE and analyzed by Western
bloting using antibodies to the entry proteins: A16, A28, A21 and L5 as wel as
antibodies to the A56 and K2 proteins. Secondary antibodies conjugated to
horseradish peroxidase were used for detection by chemiluminescence. The
recombinant viruses are listed at the top and proteins targeted by the antibodies are
listed in the center.
66
protein.  Repression of either A28 or A21 had no effect on the synthesis or stability of
the other proteins examined. A56 and K2 were resolved as multiple bands due to
alternative initiation codons for A56 as well as glycosylation.
Folowing afinity purification of A56 from vA56TAP, vA28iA56TAP (+IPTG)
and vA21iA56TAP (+ IPTG) the A56 protein was observed to interacted with K2 as
wel as the EFC, as represented by A16, A28, A21 and L5 (Figure 3-1, TAP).
However, when A28 was represed by omiting IPTG, K2 and A16 stil co-purified
with A56 but only trace amounts of A21 and L5 were detected (Figure 3-1, TAP).
Similarly, when A21 was represed, K2 and A16 were detected after afinity
purification of A56 but only trace amounts of A28 and L5. These results likely
indicate the fuly asembled EFC is not required for interaction with A56/K2 and
sugest A16 alone or a subcomplex of polypeptides that includes A16 binds to
A56/K2. However, the asociation of A56/K2 with the four other entry proteins (G3,
G9, H2 and J5) was not examined because apropriate antibodies for detection were
not available at the time of the experiment.
3.3.2 A16 and G9 selectively copurify with A56/K2.
To determine if G3, G9, H2, and J5 were important for the interaction
betwen the EFC and A56/K2 several aditional recombinant viruses were
constructed with epitope tags on G9 or J5. In adition, antibodies were acquired to
G3 and H2. The recombinant virus vA28iA56TAPJ5Flag encoded an inducible A28,
TAP-taged A56, and J5 with the Flag epitope fused to the N terminus. Cels were
infected separately with VACV WR, vA56TAP or vA28iA56TAPJ5Flag (+ and ? IPTG).
After 24 h, the cels were lysed and the starting material was analyzed along with the
67
afinity-purified samples. Western blots of the starting material confirmed A28 was
stringently represed in the absence of IPTG, furthermore both J5 and G3 were
detected using Flag tag and G3 peptide antibody, respectively (Figure 3-2A, PreTAP).
The true J5 band could be distinguished from the uper and lower background bands
by its absence from cels infected with VACV WR and vA56TAP. The core protein
A3 (p4b) was used as a negative specificity control for the affinity purification. Bands
coresponding to A56, K2, A16, A28, A21, L5 and G3 were detected after afinity
purification of A56 from cels infected with vA56TAP (Figure3-2A, TAP). The same
proteins, as wel as J5 Flag, were detected after afinity purification of cels infected
with vA28iA56TAPJ5Flag in the presence of IPTG (Figure 3-2A, TAP). However, in
the absence of IPTG, cels infected with vA28iA56TAPJ5Flag did not expres the A28
protein and only K2 and A16 co-purified with A56. Therefore, neither J5 nor G3
interacted with A56/K2 when the A28 protein is represed and the EFC does not
assemble.
The second virus constructed was vA28iA56TAPG93XFlag. As its name implies,
expresion of A28 was inducible, A56 was TAP-taged, and G9 had thre copies of
the Flag tag (at the C-terminus). Cels were infected with VACV WR or
vA28iA56TAPG93XFlag (+ and ? IPTG). At 24 h after infection, the cels were lysed
and analyzed directly or after streptavidin afinity purification, as the calmodulin step
was not found to be required. Western blots of the starting material from cels
infected with vA28iA56TAPG93XFlag (- IPTG) showed A28 was represed while both
the epitope taged G93XFlag and H2 were expresed (Figure 3-2B, PreTAP). The use
of 3 copies of the Flag epitope enhanced the detection of G9 over background bands.
68
Figure 3-2:  A16 and G9 selectively co-purify with A56/K2.
(A) HeLa cels were mock infected (M) or infected individualy with VACV WR,
vA56TAP, vA28iA56TAPJ5Flag with (+) or without (?) IPTG. After 24 h, the A56
protein was isolated from the infected cel lysates by binding sucesively to
streptavidin and calmodulin beads. Western bloting was performed on the starting
material (PreTAP) and afinity purified proteins (TAP). (B) BS-C-1 cels were mock
infected (M) or infected with VACV WR or vA28iA56TAPG93XFlag (+ or ? IPTG).
After 24 h, the A56 protein was isolated by binding to streptavidin Sepharose.
Western bloting was performed on the starting material (Start) and afinity purified
proteins (Streptavidin) as above.
69
Figure 3-3: A56/K2 binds to affinity purified A16 and G9.
 BS-C-1 cels were mock infected (M) or infected with VACV WR, vA28iA163XFlag
(+ or ? IPTG), vA28iG93XFlag (+ or ? IPTG). Infected cels were harvested after 24h
and lysed with Triton X-10. The lysate was cleared by centrifugation and the A16
and G9 polypeptides were isolated by binding to agarose beads conjugated with Flag
antibody. The eluate (Flag IP) and starting material (Start) were separated by SDS-
PAGE and analyzed by Western bloting with antibodies to the viral proteins A56,
K2, A28, A16 and A21 as indicated in the center. The viruses are indicated at the top
of the figure.
70
A56, K2, A16, G9, A28 and H2 were detected after afinity purification of proteins
from cels infected with vA28iA56TAPG93XFlag ( + IPTG), but only K2, A16 and G9
co-purified with A56 in the absence of IPTG (Figure 3-2B, TAP). The A3 core
protein (p4b) was not detected in the presence or absence of IPTG. Colectively, the
affinity purification experiments indicated that of the eight EFC proteins, A16 and G9
interacted most directly with A56/K2.
3.3.3A56/K2 copurify with A16/G9.
The reciprocal experiment was caried out to confirm the interaction of A16
and G9 with A56/K2. To implement this, two aditional epitope taged viruses were
constructed. Both recombinant VACVs inducibly expresed A28, while G9 and A16
had a 3XFlag epitope atached to the C terminus in vA28iG93XFlag and
vA28iA163XFlag, respectively. Cels were infected with VACV WR, vA28iG93XFlag
(+ and ? IPTG) or vA28iA163XFlag (+ and ? IPTG). After 24 h, the cells were collected
and the postnuclear supernatant was incubated overnight with anti-Flag antibody
covalently linked to agarose beads, which were then washed extensively prior to
elution of the bound material. The eluted proteins were resolved by SDS-PAGE and
detected by Western bloting. Analysis of the starting material indicated that A28 was
stringently regulated and that each of the constructs expressed A56, K2, A16 and A21
(Figure 3-3, Start). The slower migration of A16 from samples infected with
vA28iA163XFlag compared to wild type was due to the 3XFlag epitope. A21 was
analyzed as a representative EFC protein to confirm that the complex was not
asembled in the absence of A28. As anticipated, A16 or G9 interacted with A21 only
when A28 was synthesized (+ IPTG) (Figure 3-3, Flag IP). Nevertheless, A16 and G9
71
interacted with A56 and K2 even when A28 was represed (Figure 3-3, Flag IP).
Thus, the interaction of A56/K2 with A16 and G9 ocured regardles of the afinity
tag and whether it resided on A56, A16 or G9.
3.3.3 Both A16 and G9 are required for  their association with A56/K2.
In the above experiments, A16 and G9 always co-purified with A56/K2 and
vice versa. Aditional recombinant viruses were constructed to determine whether
expresion of both A16 and G9 were required for a stable interaction with A56/K2.
The recombinant vA16iA56TAPG93XFlag expresed an inducible form of A16, TAP-
taged A56 and G9 with a 3X Flag tag. Cels were infected with either VACV WR or
vA16iA56TAPG93XFlag (+ and ? IPTG) for 24 h and the postnuclear supernatent was
analyzed directly or after streptavidin afinity purification. Analysis of the starting
material demonstrated the stringent represion of A16 in the absence of IPTG (Figure
3-4A, Start). Importantly, the other EFC proteins examined, namely G9, A21 and L5,
were stable even in the absence of A16. Curiously, the faster migrating A56 band
predominated in the lysates of cels infected with vA16iA56TAPG93XFlag sugesting
initiation predominantly at the second start codon but this was independent of IPTG.
In the presence of IPTG, A56 interacted with K2, A16, G9, A21 and L5 as shown by
their copurification (Figure 3- 4A, Streptavidin). In contrast, only K2 interacted with
A56 when synthesis of A16 was represed. Therefore, G9 canot interact
independently with A56/K2.
 Recombinant vG9iA56TAP, which expresed an inducible form of G9 with an
HA epitope tag and TAP-taged A56, was used to test whether A16 alone was able to
interact with A56/K2.  Western blotting showed that K2, A16, A28, A21, and
72
Figure 3-4:  Both A16 and G9 are required for binding A56/K2.
(A) BS-C-1 cels were mock infected (M) or infected with VACV WR or
vA16iA56TAPG93xFlag (+ or ? IPTG). The cels were lysed with Triton X-10
after 24 h and the A56 protein was isolated by binding to streptavidin beads. The
starting material (Start) and the afinity purified proteins (Streptavidin) were resolved
by SDS-PAGE and analyzed by Western bloting using antibodies to the viral
proteins indicated on the side. (B) HeLa cels were infected with vG9iHA56TAP (+
and ? IPTG) and the A56 protein was tandem afinity purified. The starting material
(Pre-TAP) and the purified proteins (TAP) were analyzed as in panel A.
73
L5 co-purified with A56 when cels were infected in the presence of IPTG (Figure 3-
4B). However, only K2 co-purified with A56 when cels were infected in the absence
of IPTG. Because of the relatively weak signal produced by the antibody to A16, this
analysis was repeated in two independent experiments. With a more intense A16
signal in the +IPTG lane, a low amount of the protein was observed to co-purifying
with A56 when G9 was represed. Therefore, both A16 and G9 are neded for
efficient interaction of either with A56/K2.
3.3.4 Association of A56/K2 with G9 requires A16.
Next, the reciprocal experiment was caried out to determine if A56/K2 co-
purified with Flag-taged G9 in the absence of A16. Cels were mock infected or
infected with VACV WR or vA16iA56TAPG93XFlag (+ and ? IPTG). After 24 h, the G9
protein was purified from the postnuclear supernatent by incubating with the Flag
antibody conjugated to agarose beads. The beads were washed and the bound proteins
were eluted, separated by SDS-PAGE and analyzed by Western bloting. GAPDH
served as a control for loading and non-specific binding. Analysis of the lysate prior
to imunopurification confirmed the stringent control of A16 expresion (Figure 3-5,
Start). When A16 was expresed (+ IPTG), the EFC represented by A28 and G9 as
wel as A56 and K2 co-purified with G9 (Figure 3-5, Flag IP). When A16 synthesis
was represed, however, A28, A56 or K2 failed to co-purified with G9. Therefore
A56/K2 did not stably bind to G9 in the absence of A16.
3.3.5 A16 and G9 stably associate with each other in uninfected cells.
The inability of G9 or A16 to independently associate with A56/K2 suggested
74
Figure 3-5: A16 is required for G9 to bind A56/K2.
Cels were mock infected (M) or infected with VACV WR or vA16iA56TAPG93XFlag
(+ or ? IPTG). Infected BS-C-1 cels were harvested at 24 h and lysed with Triton X-
10. Flag antibody conjugated to agarose beads was used to purify the G9 protein.
The bound material (Flag IP) was eluted from the agarose beads and separated along
with the starting material (Start) by SDS-PAGE and then transfered to nitrocelulose.
Western bloting was preformed using antibodies to the proteins A56, K2, A16, A28,
the Flag epitope or GAPDH as indicated.
75
Figure 3-6: A16 and G9 interact in uninfected cells.
(A) 293T cels were transfected with empty vector or plasmid DNA expresing A16
with a C-terminal influenza HA epitope (A16-HA) or co-transfected with plasmids
expresing A16-HA and G9 with a C-terminal 3XFlag tag (G9-Flag). After 48 h the
cels were lysed with Triton X-10, the postnuclear supernatant was incubated with
the Flag antibody bound to beads, and the captured proteins were analyzed by
Western bloting with the anti-HA antibody. (B) Cels were transfected with empty
vector, G9-Flag or co-transfected with A16-HA and G9-3XFlag. The lysates were
incubated with anti-HA antibody bound to beads and the captured proteins were
analyzed by Western blotting with the anti-Flag antibody.
76
these two EFC polypeptides existed as a heterodimer or higher order multimer. To
test this hypothesis, A16 and G9 were codon optimized for expresion in human cels
and taged with a C-terminal influenza HA epitope or a 3XFlag epitope, respectively.
A16HA and G93XFlag were cloned separately into pcDNA3.1 under control of the
human cytomegalovirus major imediate-early promoter. Human 293T cels were
transfected with the individual plasmids or co-transfected with both. After 48 h, the
cels were lysed with Triton X-10 and synthesis of the recombinant proteins was
demonstrated by SDS-PAGE and Western bloting of portions of the postnuclear
supernatants (Figure 3-6A, Start; Figure 3-6B, Start). Aditional portions of the
postnuclear supernatants were incubated with agarose conjugated to Flag or HA
antibody. The beads were washed extensively and the eluted proteins analyzed by
SDS-PAGE and Western bloting. The A16HA protein was detected after the Flag
imunoprecipitation only when coexpresed with G93XFlag (Figure 3-6A, Flag IP).
Likewise, the G93XFlag was detected after the HA imunoprecipitation only when
coexpresed with A16HA (Figure 3-6B, HA IP). Thus, A16 and G9 can asociate with
each other in the absence of other viral proteins.
3.4Discussion
An unusual feature of VACV reproduction is that the infectious virus particles
are asembled in the cytoplasm, rather than at the plasma membrane, and
subsequently transported to the periphery and exocytosed. Large numbers of progeny
virus particles remain adherent to the cell surface and these are chiefly responsible for
virus spread to neighboring cels [198]. It is believed that syncytia form when large
numbers of virus particles ?fuse-back? i.e. deposit their fusion proteins into the
77
plasma membrane of the parent cel. The fact that only smal numbers of syncytia
form normaly implies a negative regulation of fuse-back. The EV membrane
surounding the MV may form one barier to fuse-back, although MVs with broken
EV membranes are detected on the surface of cels [181]. The A56 and K2
polypeptides, which form a heteromultimer on the cel surface and EV membrane
may provide another barier since a syncytial phenotype ocurs with nul mutants of
either [167-170, 172, 173]. It is posible A56/K2 could regulate fuse-back by
preventing the disruption of the EV membrane and exposure of the MV or the
subsequent interaction of the MV and plasma membranes. The ability of A56/K2 to
interact with the EFC sugests the later mechanism is important although the EFC is
porly characterized both structuraly and functionaly. In fact, it is not known
whether the EFC directly mediates fusion or is simply a positive regulator.
There are at least eight proteins within the EFC and numerous protein-protein
interactions are neded to form a stable complex [84]. To determine the binding
partners of A56/K2 individual proteins of the EFC were represed to destabilize the
entry complex. Both A16 and G9 bound A56/K2, however alone these proteins
interacted weakly with A56/K2 sugesting that a complex of A16 and G9 is ned for
the interaction with A56/K2. The interaction betwen A16 and G9 was confirmed by
coimunoprecipitation of the two polypeptides folowing transfection of expresion
vectors into uninfected cels. A model (Figure 3-7) depicts A56/K2 in the plasma
membrane of an infected cel interacting with A16 and G9 of the EFC in the viral
membrane.
78
A16 and G9 apear to have two roles. Each is required for membrane fusion
and virus entry as wel as for interaction with A56/K2. Since viruses with mutations
in A56 or K2 form syncytia, it semed posible that modifications of A16 or G9 that
perturb the interaction with A56/K2 could result in a similar phenotype.
In conclusion, the presence of A56/K2 in the plasma membrane provides a
way of diferentiating infected from uninfected cels, presumably ensuring that
VACVs preferentially fuse with the latter.
79
Figure 3-7:  Model by which A56/K2 inhibit infected cell fusion.
 G9 and A16 are anchored in MV membrane in asociation with EFC. A56/K2 is
anchored in plasma membrane through the transmembrane domain of A56. The
interaction of A56/K2 with A16 and G9 is postulated to prevent fusion of the MV
particle with the plasma membrane. Fusion may be prevented as a resulting o f the
interaction of A56/K2 with the EFC which could prevent activation of the viral
fusion proteins.
80
Chapter 4: Cels expresing A56 and K2 show reduced virus entry
and fusion with VACV infected cells
4.1 Introduction
In the absence of either A56 or K2 infected cels fuse spontaneously at neutral
pH [167-170, 172, 173].  A56 and K2 are regulators of cel-cel fusion, a proces
thought to be the consequence of superinfection by cel surface EV [19]. In chapter
2 both A56 and K2 were shown to asociate with proteins of the EFC. A56 was
unable to bind the EFC in the absence of K2 and vice versa. This sugested a
relationship betwen binding of A56/K2 to the EFC and inhibition of cel fusion.
Although the EFC is composed of at least eight proteins; only A16 and G9 were
important for interacting with A56/K2 (chapter 3). Both A16 and G9 were neded to
interact with A56/K2 as neither protein alone bound eficiently. A16 and G9
interacted in transfected cells independent of any additional viral proteins.  These data
support a model by which A56/K2 bind to the EFC to prevent infected cell fusion.
When cels are mixed after single infection with IHD-J (A56+) or IHD-W
(A56-) cel fusion ocurs only betwen cels infected with IHD-W (A56-) [167]. To
provide suport for a biological function for the interaction betwen A56/K2 and the
EFC cels expresing A56/K2, or each protein individualy were mixed with cels
infected with v"A56"K2. Cels expresing both A56 and K2 were resistant to fusion
compared to cels expresing A56 or K2 alone.  Expresion of A56/K2 also
corelated with reduced virus entry. These results strongly sugest A56/K2 inhibits
cell fusion and virus entry likely by interacting with the EFC.
81
4.2Material and Methods
4.2.1 Cells and virus.
BS-C-1 (ATC CL-26), HeLa S3 (ATC CL-2.2) suspension cels were
grown as described in chapter 2, section 2.2.1. Growth of human 293T cels was
described in chapter 3, section 3.2.1. Construction of v"A56"K2 was described in
chapter 2, section 2.3. vFire-WR has ben described previously in [94].  General
procedures for preparing and titrating stocks were done as previously described in
chapter 2, section 2.2.1.
4.2.2 Purification of VACV.
HeLa cels were infected at an MOI of 3 vFire-WR and incubated for 2 day at
37 
o
C. VACV MV was isolated by mechanical disruption of HeLa S3 cels and
subjected to sedimentation twice through a 36% sucrose cushion. Virus was
resuspended in 1mM Tris-HCl (pH 9.0) and titered as described in chapter 2, section
2.2.1.
4.2.3 Codon optimization of A56 and K2.
The DNA sequence for the VACV WR A56R and K2L genes was optimized
(Geneart, Regensburg, Germany) to improve RNA procesing and translation by
altering codon usage and G-C content. The codon optimized A56R and K2L genes
were PCR amplified.  For A56, oligonucleotides were designed to apend the DNA
sequence for a V5 epitope tag to the C terminus of the ORF. The PCR products of
A56V5 and K2 were cloned into the directional TOPO vector pcDNA 3.1 (Invitrogen)
and sequenced to confirm proper insertion and sequence.
82
4.2.4 Transfection.
The day prior to transfection 2x10
5
 293T cels were seded per wel of a 48
wel plate in 30 !L of 10% DMEM lacking hygromycin. A transfection mixture
was prepared as folows: 1.5 !L of Lipofectamine 200 (Invitrogen) was mixed with
50 !L of Opti-Mem (Invitrogen) and incubated 5 min at rom temperature. In a
separate tube, 50 !L of Opti-Mem was combined with the DNA. After 5 min the two
solutions were mixed and incubated a minimum of 25 min at rom temperature.  The
diferent samples for transfection were configured as folows: 90 ng of total DNA,
10 ng of which is the P1-FLUC plasmid that encodes the firefly luciferase gene
expresed from the late P1 viral promoter. Cels were cotransfected with by ading
40 ng of A56 plasmid and 40 ng for K2. Cels transfected with only a single
plasmid consisted of 40 ng of either A56, K2, or VSVG with the remaining 40 ng
of DNA suplemented with empty vector. Once transfected the cels were grown for
24 h at 37 
o
C with 5% CO
2
 at which point the medium was changed and the cels
were incubated for an additional 24h prior to analysis.
4.2.5 Antibody staining and flow cytometry.
The folowing antibodies were used: anti-A56 monoclonal antibody 1H831
(provided by Alan L Schmaljohn), anti-K2 monoclonal antibody 4A11-4A3 (provided
by Richard Moyer), Cy5-conjugated donkey anti-mouse. (Jackson ImmunoResearch).
Antibody staining was performed as folows: cels were resuspended and peleted in
bench top centrifuge for 20 sec at 5k x g and supernatent was removed. The primary
antibody (anti-A56 or anti-K2), diluted in 10% DMEM, was incubated with cels for
15 mins at which point the cels were washed twice with 50 !L DPBS without Ca
83
and Mg (Quality Biological). Secondary antibody (diluted 1:30 in 10% DMEM)
was incubated 15 min with the cels after which the cel were washed twice with 50
!L DPBS. Samples were acquired on a FACSCalibur flow cytometer (BD
Imunocytometry Systems) and analyzed using FlowJo software (TreStar, San
Carlos, CA).
4.2.6 Quantification of cell-cell fusion.
2x10
5
 BS-C-1 were seded per wel of 24 wel plate. The folowing day, cels
were infected at an MOI of 5 in 2.5% EMEM with v"A56"K2. Separately, 293T
cels were cotransfected with A56/K2 or transfected individualy with A56, K2,
empty vector or the VSVG glycoprotein fused to GFP. Al transfections also
contained 10 ng of p1-Fluc plasmid. After 48 h the transfected cels were
resuspended imediately prior to ading to the infected cel monolayer at a
concentration of 5x10
5
/mL in 10%DMEM with 40 !g cytosine arabinose (AraC).
The medium was removed from the infected cel monolayers at 18 h postinfection
and replaced with 250 !L of medium coresponding to 1.25x10
5
 293T cels. The
cels were incubated with the infected monolayer for 4 h at 37 
o
C and then lysed by
ading 10 !l of 3.5X cel culture lysis bufer (Promega). Cels were incubated with
lysis bufer for 10 min at rom temperature with constant rotation. A 20 !l portion of
the lysate was mixed with 10 !l of luciferase asay substrate (Promega), and activity
was quantified on a Berthold Sirius luminometer.
4.2.7 Measuring virus entry
vFire-WR peleted over 2X sucrose cushions was diluted in 10% DMEM to
84
6x10
6
 Pfu/ml. 50 ?l of virus inoculum was aded per wel of a 48 wel plate. At 2 h
post-infection, medium was removed and cels were lysed by ading 10 ?L of cel
culture lysis bufer and incubating 10 min at rom temperature with constant rotation.
A 20 ?l portion of lysate was removed and incubated with 10 ?l of luciferase asay
substrate, Luc activity was quantified on a Berthold Sirius luminometer.
4.3Results
4.3.1 Expression of A56 and K2 in transfected cells.
The A56 and K2 proteins localize to the plasma membrane of infected cels
and are also found in the EV membrane. A56 is required for membrane retention of
K2 in both infected as wel as transfected cels [173, 174]. A56 and K2 inhibit cel-
cel fusion posibly through an interaction with proteins of the EFC. Cel surface
localization of A56 and K2 is important for inhibiting cel fusion which is suported
by studies which reveal: i) deletion of the membrane anchor of A56 [173], i) deletion
of the signal sequence from K2 [174] or ii) adition of antibodies to A56 [172] and
K2 [173] lead to cell-cell fusion.
Cel surface localization of A56 and K2 was analyzed by flow cytometry.
Prior to expresion in mamalian cels the A56 and K2 genes were codon optimized.
VACV genes are normaly expresed in the cytoplasm and may contain sequences
inhibitory to nuclear expresion. The optimized A56 and K2 genes were individualy
cloned into pcDNA3.1 under control of the human cytomegalovirus major
imediate-early promoter. Cels were cotransfected with plasmids expresing A56
and K2, or with a single plasmid encoding A56, K2 or empty vector.  At 48 h post-
85
Figure 4-1: Cell surface expression of A56 and K2.
293T cels were cotransfected with plasmids expresing A56 and K2 (A, B), or
transfected with a single plasmid for empty vector, A56 (C), or K2 (D). At 48hr
postransfection cels were stained with A56 antibody (A, C) or K2 antibody (B, D),
washed and incubated with secondary antibody conjugated to Cy5 prior to analysis by
flow cytometry. Staining of cels transfected with empty vector is represented within
the gray shaded area.  Mean fluorescent intensity (MFI) is listed below the abscissa.
86
transfection the cels were stained with antibodies to A56 or K2, washed and then
stained with secondary antibodies conjugated with a fluorophore. Cells were not fixed
so as to retain the integrity of the plasma membrane and folowing staining the
samples were analyzed by flow cytometry. Both the antibodies used to detect A56
and K2 were of mouse origin preventing simultaneously analysis of A56 and K2.
Instead, cels transfected with A56 and K2 were divided, with a portion stained with
A56 antibody and a portion with the K2 antibody. Cels cotransfected with both A56
and K2 plasmid displayed abundant surface staining of both proteins (Figure 4-
1A+B). Cels transfected with an A56 plasmid also exhibited extensive surface
staining for A56. The gray area represents antibody staining of cels transfected with
empty vector. The mean fluorescence intensity of cels expresing K2 alone was
reduced 7 fold when compared to cels expresing K2 with A56. These results agre
wel with previous reports that have demonstrated A56 is important for anchoring K2
to the plasma membrane [173].
4.3.2 A56/K2 expresion corelates with reduced fusion with virus
induced syncytia.
Cel fusion ocurs spontaneously at neutral pH among cels infected with
viruses that lack either the A56R or K2L gene. If cels are mixed after single
infection with IHD-J (A56+) or IHD-W (A56-) cel fusion ocurs only betwen cels
infected with IHD-W (A56-) [167]. This indicated acquisition of A56 was asociated
with inhibiting cel fusion, but did not demonstrate A56 (along with K2) was
sufficient to inhibit cell fusion.
87
To determine if A56 and K2 were suficient to inhibit fusion, 293T cels
were cotransfected with plasmids expresing A56 and K2, or transfected with a single
plasmid encoding A56, K2, VSVG glycoprotein fused to EGFP, or empty vector. A
plasmid containing the firefly Luc gene under control of a late viral promoter was
included in al transfections to monitor cel fusion. At 48 h post-transfection the cels
were resuspended in medium containing the viral DNA replication inhibitor AraC to
prevent virus late gene expresion which may ocur after the cels are mixed with the
infected monolayer. The transfected 293T cels were aded to a monolayer of
BS-C-1 cels that had ben infected 18 h previously with v"A56"K2. Extensive cel
fusion had developed in the infected BS-C-1 monolayer by 18 h postinfection. The
cels were incubated with the monolayer for 4 h, lysed and Luc activity was
measured.
Data is expresed as a ratio of the Luc activity relative to A56/K2. Cels
expresing both A56 and K2 displayed the lowest Luc activity, which would be
expected if binding to the EFC inhibits fusion. Comparing the Luc value of cels
expresing A56/K2 to the values obtained for cels expresing only A56 or K2
indicated a 3.72 and 5.53 fold increase in Luc activity, respectively, indicating cels
expresing only A56 or K2 were unable to inhibit fusion (Figure 4-2). These data are
consistent A56 or K2 individualy being unable to eficiently bind the EFC. The ratio
of Luc value for cels expresing just A56 (3.72) was lower than the value obtained
from cels expresing K2 alone (5.53) , VSVG (5.61) or empty vector (6.63),
however the value was wel above what was observed for expresion of A56/K2
(1.00). The VSVG glycoprotein and empty vector were used as negative controls
88
Figure 4-2: Efect of A56 and K2 expresion on fusion betwen transfected cels
and VACV induced syncytia.
293T cels were cotransfected with plasmids for A56 and K2 or transfected with a
single plasmid expresing A56, K2, VSVG or empty vector. Al transfections
included firefly luciferase plasmid as a reporter for cel-cel fusion. At 48 h post-
transfection the cels were resuspended and aded to a monolayer of BSC1 cels that
had ben infected for 18 h with v"A56"K2. After 4 hrs the cels were lysed and
asayed for firefly luciferase. Data is represented as a ratio of relative light units
(RLU) relative to A56/K2 sample. Experiments were preformed in quadruplicate and
data points represent the mean (listed below sample names) ? standard erors of the
means.
89
 as cels transfected with either plasmid were not expected to inhibit cel fusion. A56
was observed to weakly interact with proteins of the EFC (Figure 2-5), which may
acount for the slight reduction in Luc activity. Never the les, optimal inhibition of
cel fusion required both A56 and K2. These results demonstrate that uninfected cels
are able to fuse with infected cels and cels expresing both A56 and K2 reduce the
extent of fusion.
4.3.3 A56 and K2 expression reduce virus infection.
 The proceses of virus-cel fusion and cel-cel fusion require the conserved
multiprotein EFC, indicated A56/K2 may be able to regulate virus entry in adition to
cel-cel fusion. There curently is no asay available to directly quantify virus
fusion, however early gene expresion has ben used to study entry of VACV. The
VACV vFire-WR expreses the firefly Luc gene from an early-late promoter and Luc
synthesis begins almost imediate after virus entry. Although this is a post-fusion
asay it has ben used previously to characterize the efect of low pH on virus entry
[94, 95]. 293T cels were cotransfected with plasmids for A56 and K2 or
transfected with a single plasmid for A56, K2, VSVG glycoprotein fused to GFP, or
empty vector. At 48 h post-transfection cels were infected at an MOI of 1 with
vFire-WR, 2 h later the cells were lysed and the Luc activity was measured.
The data is reported as a ratio of Luc value compared to infection of cels
transfected with vector alone. Cels transfected with both A56 and K2 displayed only
27% of the Luc activity of cels transfected with vector alone (Figure 4-3). Cels
expressing only A56 or K2 failed to effectively inhibit virus entry to the extent of
90
Figure 4-3:  Effect of A56 and K2 expression on VACV entry.
293T cels were cotransfected with plasmids expresing A56 and K2 or transfected
with a single plasmid for A56, K2, VSVG or empty vector. At 48hr post-transfection
cels were infected at an MOI of 1 with vFire-WR. After 2hrs, luciferase activity was
measured and expresed as relative light units (RLU). The data is the ratio of RLU
compared to vector.  Experiments were done in quadruplicate with the mean (listed
below the sample names) ? standard errors of the means.
91
cels expresing both proteins. In fact, cels expresing K2 showed an increase in
Luc activity (1.57 fold), while a slight reduction in Luc expresion was noted with
cells expressing A56 alone (68%) similar to what was noted for inhibition of cell-cell
fusion.  Cells expressing the VSVG glycoprotein displayed similar Luc activity (1.00)
compared to cels transfected with empty vector. These results indicate that A56/K2
in addition to reducing cell-cell fusion also appear to reduce virus entry as measure by
early gene expression.
4.4Discussion
Cel fusion is trigered by low pH treatment of infected cels [165, 16] or
ocurs spontaneous at neutral pH with viruses that are deleted for A56R or K2L gene
[167-170, 172, 173]. Cel fusion depends on cel surface EV and is hypothesized to
involve ?fuse back? of EV, in which the viral EFC is depositing into the plasma
membrane [19]. Viruses unable to form EV do not triger cel fusion [152] and
fusion fails to ocur when components of the viral EFC are represed [80]. A56 and
K2 have ben shown to bind the viral EFC. This interaction was shown to depend on
expresion of both A56 and K2 (chapter 2) and the fusion regulatory proteins
(A56/K2) were identified to interact specificaly with A16 and G9 entry proteins
(chapter 3). Through this interaction a model was developed by which A56/K2 binds
the EFC to inhibit cell-cell fusion (Figure 3-7).
Previous experiments involving mixing of cels individualy infected with IHD-J
(A56+) or IHD-W (A56-) demonstrated cell fusion occurs only between cells infected
with IHD-W (A56-) [167]. In these experiments cels expresing A56 (and
presumably K2) were resistant to cel fusion. To extend this observation and
92
demonstrate A56/K2 are suficient to prevent fusion, cels were transfected and
shown to expres both A56 and K2 on their cel surface. These cels were aded to a
monolayer of cels that had formed syncytia and cel fusion was monitored by
activation of Luc. Cels expresing both A56 and K2 fused porly exhibiting only
20% of Luc activity relative to cels transfected with empty vector. In comparison,
cels expresing only A56 or K2 were unable to inhibit cel fusion with Luc value 3.7
fold and 5.5 fold higher, respectively, compared to cels expresing A56/K2. These
results were consistent with the inability of A56 or K2 alone to bind the EFC and
confirmed i) uninfected cels are able to fuse with syncytia form by infected cels and
i) expresion of A56 and K2 corelated with reduction in cel fusion with virus
trigered syncytia. This provides the first evidence that A56 and K2 are suficient to
inhibit cel fusion.  Cel-cel fusion and virus-cel fusion can be view as very similar
proceses. Entry of VACV was examined in cels expresing A56 and K2 by early
gene expresion.  A56 and K2 reduced early gene expresion by 70% compared to
cels transfected with empty vector. Expresion of A56 showed only a 30%
reduction, while K2 actualy increased early gene expresion. The increase in Luc
expresion observed for cels expresing K2 was unexpected. Aditional experiments
are required to explain the increase in Luc activity. Perhaps the increase is related to
the SERPIN motif within K2. A minor portion of A56 was observed to bind to the
EFC in the absence of K2, which may partialy acounted for a slight reducing in
virus entry for cels expresing A56 (Figure 2-5). These data ofer the first evidence
for a biological significance to the interaction between A56/K2 and the EFC.
93
Chapter 5:  Conclusions and Future Direction
5.1 Conclusions
In this disertation, I have sought to understand the mechanism by which A56
and K2 proteins inhibit infected cel-cel fusion. Deletion of A56 or K2 is asociated
with spontaneous neutral pH syncytia formation at late times during infection.
Spontaneous cel fusion requires EV and is likely mediated by superinfection of cel
surface EV. Entry and fusion of VACV requires a conserved multiprotein complex
located within the MV membrane.
My disertation project focused on identification of the protein interactions of
A56 and K2 related to the ability of these proteins to regulate infected cel fusion.
Tandem afinity purification revealed A56 interacted with proteins of the VACV
EFC. A56 asociates with K2 so it was conceivable that K2 may also bind the EFC.
An interaction betwen K2 and the EFC was confirmed by co-purification of the EFC
with the K2. Further experiments revealed A56 failed to bind the EFC in the absence
of K2. Interestingly, K2 displayed a similar dependence on A56 to bind the EFC. To
validate an interaction betwen A56/K2 and the EFC the A28 protein was isolated by
tandem afinity purification and Western blot confirmed A56 and K2 asociated with
the afinity purified EFC. However, in the absence of K2, the A56 protein failed to
co-purify with the EFC. The results reported in chapter 2 provided the first evidence
for an interaction betwen A56/K2 and the viral EFC. More importantly, it sugested
binding of A56/K2 to the EFC might regulate infected cel fusion. The inability of
A56 or K2 to bind the EFC independently agred wel with the requirement for both
proteins to inhibit cell fusion suggesting the two processes may be related.
94
A56 is important for anchoring K2 to the plasma and EV membrane. The two
proteins likely interact during transport through the secretory pathway, although the
domains involved in their interaction have not ben wel defined. A region betwen
amino acids 145-373 of K2 is important for binding A56, while mutations that abolish
the protease inhibitory activity of K2 have no efect on interactions with A56 [173].
K2 with truncations of the N and C termini failed to inhibit cel fusion, sugesting
truncations may prevent the protein from asuming a particular conformation
important for regulating cel fusion. In our hands, similar modifications of K2
generally eliminated the anti-fusion activity of the protein.
Neither the stoichiometry of the interaction betwen A56 and K2 nor whether
the two proteins are constantly asociated is known. Afinity purification of A56
identified a number of aditional proteins that may compete with K2 for binding to
A56. In particular the interaction betwen A56 and C3 could have potential benefit
for the virus in avoiding complement-mediated lysis of infected cels or virus
particles.
A direct interaction with the EFC could not be atributed to either A56 or K2
individualy. Perhaps the conformation of A56 and K2 is altered upon asociating
with one another. For example, binding of K2 with A56 may alter the conformation
of A56 and alow it to asociate with the EFC, or the oposite may be true.
Alternatively, a conformation formed by the complex of A56/K2 may asociate with
the EFC. Stil another posibility is that A56 and K2 both bind weakly to the EFC so
that a complex stable enough for isolation is only detected when the two are
asociated. A minor form of A56 was observed to asociate with the EFC even in the
95
absence of K2 (Figure 2-5), sugesting A56 may directly bind the EFC. Western blot
analysis of A56 indicates the protein migrates as multiple bands, likely related to
various states of glycosylation or alterative translation initiation.  Whether a particular
glycosylated form preferentialy asociates with K2 and the EFC was not adresed.
A soluble complex of A56 and K2 secreted from VACV infected cels was isolated
and shown to bind the EFC. The higher molecular weight form of A56 predominated
in the soluble complex consistent with fuly glycosylated protein being secreted from
the cel. This high molecular weight species of A56 was sensitive to digestion with
proteinase K, indicating this form is likely present on the cel surface [172]. A56 and
K2 may also form higher oligomeric structures on the cel surface that could
potentially bind more efficiently to the viral EFC due to increased avidity.
Once it was identified that A56 and K2 interacted with the EFC the next step
was to determine which proteins within the EFC mediated the interaction. Two
scenarios were considered: i) A56/K2 associates with the entire EFC, perhaps through
the interface formed by protein interactions within the complex, or alternatively i)
individual proteins of the EFC interact directly with A56/K2. The viral EFC is
composed of at least eight proteins. To determine which of these the protein were
important for binding A56/K2, a series of conditional lethal viruses were constructed.
The viral EFC has been shown not to form when synthesis of A21 or A28 is repressed
[84]. Afinity purification of A56 in the absence of either A21 or A28 revealed
interactions with the EFC were limited to A16 and G9. This sugested that only a
subset of the EFC was required to interact with A56/K2.
96
Since both A16 and G9 co-purified with A56/K2 it was unknown which of the
two proteins directly mediated the interaction. To ases this, synthesis of A16 or G9
was represed and afinity purification of A56/K2 was performed. In the absence of
A16, the G9 protein did not asociate with A56/K2, while in the absence of G9, the
A16 protein did not co-purified with A56/K2. The co-purification of G9 and A16
with A56/K2 sugested a direct asociation betwen A16 and G9. This was further
suported by the inability of either A16, or G9 to bind individualy to A56/K2. To
investigate the potential interaction betwen A16 and G9 the two proteins were
expresed by transfection in uninfected cels. A16 imunoprecipitated G9, and A16
was shown to co-purified with G9. This result was the first to demonstrate an
interaction betwen two proteins of the EFC. An interaction betwen H2 and A28
has ben infered, due in part to the ability of the two proteins to asociate in the
absence of A16, although the proteins had not yet ben shown to interact in the
absence of a virus infection [84]. A curent focus of the lab is to identify aditional
protein interaction within the viral EFC.
The identification and characterization of an interaction betwen the EFC and
A56/K2 was important for understanding the mechanism for regulation of cel fusion,
however biological significance for this interaction stil remained to be established.
The working hypothesis is cels expresing A56 and K2 may be refractory to cel-cel
fusion or infection. A56/K2 are predicted to interact with the EFC at the cel surface.
Therefore, cels transfected with A56 and K2 were monitored for surface expresion
of the two proteins by flow cytometry. Both A56 and K2 localized to the surface of
transfected cels. In the absence of A56, a significant decrease was observed in the
97
amount of cel surface K2, consistent with the requirement of A56 for retention of K2
on the plasma membrane. Therefore the A56 and K2 proteins behave normaly in
these transfected cells.
Cels infected with v"A56"K2 normaly exhibit extensive cel fusion. Our
thought was thatcels transfected with A56/K2 would exhibit resistance to fusing with
the syncytia in much the same way that cels infected with wild-type virus expresing
A56/K2 do not fuse with A56 negative infected cels. Inded, cels transfected with
A56/K2 displayed greatly reduced fusion with the syncytia monolayer (as monitored
by Luc activity) compared to cels transfected with an empty vector. In comparison,
cels expresing either A56 alone or K2 alone had a 3 to 5 fold increase, respectively,
in Luc activity compared to cels expresing both A56/K2. This was consistent with
the requirement of A56/K2 to bind the EFC. Expresion of A56 and K2 did not
eliminate the ability of the cel to fuse with the syncytia, but significantly reduced the
amount fusion.
The proces of cel-cel fusion and virus-cel fusion both depend on the viral
EFC. Since expresion of A56/K2 reduced fusion of cels with the syncytia, the
infectivity of cels expresing A56/K2 may also be reduced. To test this, cels were
infected with a VACV expresing the Luc gene to measure virus entry. Cels
expresing both A56/K2 displayed a dramatic reduction in Luc activity compared to
cels transfected with vector alone. This indicates cels expresing A56/K2 display a
reduction in fusion with syncytia and entry of VACV.
There is no obvious benefit gained by superinfection of cels indicating the
ability to regulate infected cel fusion may ofer several advantages to the VACV.
98
Expresion of A56/K2 may aid the virus in distinguishing betwen infected and
uninfected cels. Alternatively, the development of large multinucleated syncytia
may lead to premature apoptosis. Other viruses, notably HIV and influenza virus,
have mechanisms to prevent superinfection by either downregulating or removal of
cel proteins important for initiating virus entry and infection. Certain strains of
herpesvirus are known to form syncytia in cel culture. Syncytia formation is
asociated with mutations the viral glycoproteins gK [20] and gB [201] as wel as
mutations in UL20 [202].  The mechanisms by which the mutations inhibit cel-cel
fusion are unknown, perhaps these mutations alter traficking or enhance surface
expresion of the viral fusion proteins. It has ben proposed that syncytia formation
in herpesviruses may be detrimental to the virus in vivo [203].
In conclusion, the A56 and K2 proteins are required for inhibition of infected
cel fusion. Afinity purification of A56 and K2 identified an interaction with the
viral EFC, sugesting this asociation may be important for inhibition of infected cel
fusion. Cels expresing A56 and K2 were resistant to fusion with virus induced
syncytia and displayed a similar resistance to virus entry. Aditional studies should
reveal the mechanism by which binding of A56/K2 to A16/G9 inhibit virus fusion.
5.2Future Directions
Identification and characterization of the interaction betwen A56/K2 and the
EFC provides the first evidence for a molecular mechanism for the regulation of
infected cel fusion. Much remains to be determined with respect to poxvirus entry
and fusion, in particular how the interaction of A56/K2 with the EFC regulates cel-
cel fusion. Although the EFC is required for virus-cel and cel-cel fusion it is
99
uncertain whether the EFC directly mediates fusion or has an acesory role, perhaps
serving as a scafold for asembly of the fusion protein(s).  Never the les, VACV
apears to have evolved a mechanism of regulating cel fusion through the interaction
of A56/K2 with EFC.
Antibodies to A56 and K2 triger cel fusion similar to deletion of the
respective protein. The antibodies may triger cel fusion through several
mechanism: i) the antibodies disrupt interactions betwen A56/K2 and the EFC or i)
the antibodies triger endocytosis of A56 or K2, down regulating surface expresion.
The former is the more interesting outcome as the antibody epitopes could be maped
to identify domains within A56 and K2 important for the interaction with the EFC.
 A cel line expresing A56 and K2 would be prefered over transfection,
which is asociated with variation in the number of cels transfected as wel as the
level of protein expresion. A cel line expresing A56 and K2 would be anticipated
to display a similar inhibition of virus entry as transfection and could be used to
scren for viral mutants. In particular, viral mutants that triger cel fusion in the
presence of A56/K2 or are unable to bind the fusion regulatory proteins may be
identified. Alternatively, a soluble complex of A56 and K2, at a high enough
concentration, may inhibit virus entry and provide a means of identifying viral
mutants resistant to the anti-fusion activity of A56/K2.
VACV has evolved a mechanism for regulation of cel fusion facilitated by
interaction with A56/K2. This indicates A16 and G9 may represent a novel target for
development of antipoxvirus agents. A beter understanding of the interactions of
A16/G9 with other proteins within the EFC may shed light on a mechanism by which
100
A56/K2 inhibit cel-cel fusion leading to development of smal molecule inhibitors
of virus entry.
Neutral pH cell fusion caused by deletion of A56 and K2 is likely mediated by
superinfection of cel surface EV, although direct evidence is lacking. Electron
microscopy has ben utilized to examine entry of VACV and images have ben
captured documenting fusion of both MV [91-93] and EV [97] particles with the cel
membrane.  It may be difficult to capture the presumably rare asynchronous fuse back
of EV, however during the course of superinfection the MV membrane merges with
the plasma membrane. Therefore, imunoelectron microscopy could be used to
examine the plasma membrane for an increase in MV proteins when cels are infected
with an A56 or K2 deletion virus.
Many aditional questions with regard to A56/K2 regulation remain to be
answered, for example. What is the stoichiometry of the interaction between A56/K2?
Is glycosylation of A56 important for binding A16/G9? What components of the
EFC do A16 and G9 asociate with? How does binding of A56/K2 to A16/G9 inhibit
fusion (steric hindrance)? How is the EFC involved in virus fusion? Deletion of A56
and K2 is asociated with cel-cel fusion in cel culture, but does cel-cel fusion
ocur in vivo? Are there aditional mechanisms for inhibiting superinfection? A56
and K2 are primarily expresed during late gene expresion, sugesting there may be
an aditional mechanism for preventing superinfection during early and intermediate
gene expresion.  If this is the case, why are these mechanisms unable to prevent EV
superinfection?
101
Finaly, the work that I have presented in this disertation has provided a
foundation to beter understand the mechanism by which VACV regulates infected
cel fusion. Hopefuly, the questions I have adresed over the course of my research
have provided the poxvirus field with information neded to further examine the
mechanism of virus entry and fusion.
102
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