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MAPPING AND PHENOTYPIC CHARACTERIZATION OF TEMPERATURE
SENSITIVE VACCINIA VIRUS MUTANTS Cts6 AND Cts9
BRADLEY PAUL DULLING
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
Bradley Paul Dilling
I thank my wonderful wife, Sarah, for all her help and encouragement throughout
our time together. I have wonderful parents, to who I owe a great deal for their support
and guidance. I thank them for emphasizing the importance of education and persistence.
I would like to thank all my friends and colleagues in the Condit Lab, Mr. Steve
Cresawn, Dr. Susan D'Costa, Dr. Sayuri Kato, Dr. Nissin Moussatche, Audra Strahl, Dr.
Hendrik Nollens, Dr. Cindy Prins, and Ms. Tommie Albright for all their help. I
especially would like thank my graduate advisor, Dr. Rich Condit, for all his advice and
counsel during my proj ect. Rich is an incredible person to know and work for. I would
also like to thank the rest of my committee (Dr. Sue Moyer and Dr. Donna Duckworth)
for their input into my proj ect.
TABLE OF CONTENTS
ACKNOWLEDGMENT S ................. ................. iii........ ....
LIST OF FIGURES .............. ....................vi
KEY TO SYMBOLS ........................... .......vii
AB S TRAC T ......_ ................. ..........._..._ viii..
1 INTRODUCTION ................. ...............1.......... ......
General Vaccinia Background ................. ......... ...............1.......
Virus Life Cycle ................... ...............1..
Virion Structure and Entry .............. ...............1.....
Early Gene Expression .............. ...............4.....
DNA Replication ................. .......... ...............4.......
Postreplicative Gene Expression ................. ...............5............ ....
M orphogenesis .............. ...............6.....
The Problem............... ...............9.
2 MATERIALS AND METHODS ................. ...............11................
Cell s and Viruses ................. ................. 11......... ...
M arker Rescue ................. ...............11.......... ......
DNA Sequence Analysis ................. ...............11................
Electron Microscopy............... ...............1
Recombinant DNA Clones .............. ...............13....
A ntibodies............... ........... .........1
Purification of Virus Particles ......._. ......... __. ......__ ...._. ...........14
W western Blots ............_... ...... ......... ... ...............14...
Protein Pulse Label and Pulse Chase Assays .............. ...............15....
Transcription by Permeabilized Virions ................. ...............15................
3 RE SULT S ................. ...............16.......... .....
Mapping of the Mutations ................. ...............16.......... ....
Sequencing Data ................. ...............17.................
Protein Synthesis .............. ...............22....
Protein Processing .............. ...............22....
Electron Microscopy............... ..............2
Immunodetection of A28 Protein ............. ...... ....__ .........._......28
Virion Transcription .............. ...............30....
Particle to Infectivity Ratio............... ...............30.
4 DI SCUS SSION ....__. ................. .......__. .........3
LIST OF REFERENCES ......__................. .......__. .........3
BIOGRAPHICAL SKETCH .............. ...............42....
LIST OF FIGURES
1. Vaccinia virus life cycle............... ...............3..
2. Vaccinia virus morphogenesi s ................. ...............7.......... .....
3. Marker rescue of Cts6.................. ...............18...............
4. Nucleotide alignment of A28L. ......_._............. ......_._ ...._. .........._.20
5. Amino acid sequence alignment of A28 ......... ......... ................ ...............21
6. Viral protein synthesis. ............. ...............23.....
7. Viral protein processing. .............. ...............25....
8. Electron micrograph pictures. .............. ...............26....
9. Electron micrograph pictures. .............. ...............27....
10. Immunodetection of A28 protein ................. ...............29...............
11. In vitro transcription activity. ............. ...............3..... 1
KEY TO SYMBOLS
aa amino acid
oC degrees centigrade
DNA deoxyribonucleic acid
mRNA messenger ribonucleic acid
m.o.i. multiplicity of infection
OD optical density
ORF open reading frame
PCR polymerase chain reaction
RNA ribonucleic acid
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
MAPPING AND PHENOTYPIC CHARACTERIZATION OF TEMPERATURE
SENSITIVE VACCINIA VIRUS MUTANTS Cts6 AND Cts9
Bradley Paul Dilling
Chair: Richard C. Condit
Major Department: Molecular Genetics and Microbiology
To further our understanding of morphogenesis in vaccinia virus, our laboratory is
investigating several temperature sensitive mutants that display a "normal" phenotype.
Normal mutants show no defects in viral DNA replication or protein synthesis, and thus
are likely to be defective in virus assembly. In this report we describe two temperature
sensitive mutants (Cts6 and Cts9) comprising a single complementation group.
Cts6 and Cts9 were mapped to the A28L gene by marker rescue. DNA sequence
analysis of the mutant A28L gene reveals a C-terminal frameshift mutation in both
mutants, predicting that both encode C-terminal truncated proteins. Western blot analysis
with A28L specific polyclonal antibody shows that the A28L protein is contained in
virions. Metabolic labeling of proteins in infected cells, and pulse chase analysis of
labeled proteins, shows that viral protein synthesis and proteolytic processing of virion
precursor proteins in Cts6 and Cts9 infected cells is indistinguishable from a wild type
virus infection at the nonpermissive temperature. Electron microscopic analysis of virus
infected cells shows that wild type and mutant virus infections were indistinguishable at
both the permissive and nonpermissive temperatures. In an in vitro transcription assay
purified mutant particles produced at the nonpermissive temperature were able to
transcribe at the same level as wild type. However the purified particles at the
nonpermissive temperature were not infectious at the permissive temperature, suggesting
a defect in cell entry. We hope that continued study of Cts6 and Cts9 will lead to a further
understanding of vaccinia virus cell entry.
General Vaccinia Background
Vaccinia virus is a member of the Poxviridae family. It contains a large, double-
stranded DNA genome, and replicates solely in the cytoplasm of vertebrate cells.
Vaccinia virus is known mostly for its use in the eradication of smallpox, caused by
another member of the Poxviridae family, variola virus. The vaccinia genome (192 kB)
encodes over 200 polypeptides, half of which are incorporated into the virion. Because
poxviruses carry out replication and transcription of a DNA genome in the cytoplasm, it
is an ideal model system for studying mRNA synthesis and processing. Vaccinia virions
have a smooth, rounded rectangular appearance, and measure about 350 x 270 nm under
cryoelectron microscopy. The intracellular mature virion (IMV) is the most abundant
infectious particle, composed of a dumbbell-shaped core, surrounded by a lipoprotein
bilayer. The core contains the viral genome, enzymes required for transcription and
replication in the cytoplasm, and additional proteins thought to have structural roles
(Moss, 2001). This unique structure of vaccinia virus is assembled during an intricate
morphogenesis process that is the focus of this study.
Virus Life Cycle
Virion Structure and Entry
Although IMV is the most abundant form of the virus, three others are produced
during the life cycle. Intracellular mature virions are well suited to mediate transport
among hosts, but are not well suited for spreading the virus within the host because of its
vulnerability to complement and antibody. Other forms that exist are intracellular
enveloped virus (IEV), cell-associated enveloped virus (CEV), and extracellular
enveloped virus (EEV) (Smith et al., 2002). These different infectious forms of the virus,
combined with the fact that cellular receptors and viral attachment proteins have not been
identified, complicate studies of virus entry into the cell. Although IMVs are commonly
used in the laboratory to infect cells, they are only released after cell lysis. The IMVs are
thought to enter the cell by fusion with the plasma membrane or vesicles. The EEVs and
CEVs are more important in cell-to-cell spread. The EEV is thought to enter by a
mechanism that involves endocytosis followed by low-pH disruption of the EEV outer
membrane and then fusion of the released IMV with endosomal membrane. Only four
IMV proteins have been implicated in cell entry and penetration: L1R, A27L, D8R, and
H3L. Difficulties with studying EEV entry led to only identifying two EEV proteins:
A34R, B5R (Moss, 2001). The mechanisms of vaccinia cell entry and penetration are
still under investigation.
Figure 1 is an overview of the vaccinia virus life cycle. It is generally accepted that
the final result of virus entry is the delivery of the core into the cytoplasm of the cell. It
has been shown that the uncoating of viral cores is blocked by prevention of transcription
or translation, suggesting the requirement for a virus-induced or virus-encoded protein. A
putative 23-kD protein with trypsin-like activity was partially purified from infected cells
and is thought to have a role in uncoating of the cores. Before the complete uncoating of
the virus core takes place, early genes are transcribed, including DNA polymerase, RNA
polymerase, growth factors, immune defense molecules, and intermediate transcription
Figure 1. Vaccinia virus life cycle. One of the infectious forms of virus attaches to the
cell, penetrates the membrane, and releases the core into the cytoplasm (1).
The cores synthesize early mRNAs that are translated into a variety of
proteins including intermediate transcription factors and DNA replication
factors (2). The virion uncoats (3), the DNA genome is replicated (4), and
intermediate genes are expressed (5). Intermediate genes encode late
transcription factors, allowing for late gene transcription (6). Late genes
encode early transcription factors, enzymes, and virion structural proteins.
Newly assembled virions (7) undergo maturation (8) to form intracellular
mature virions. These virions are wrapped by modified Golgi membranes
(9) and transported to the periphery of the cell. Fusion of the outer
membrane with the plasma membrane results in the release of extracellular
enveloped virions (10).
factors (Moss, 2001). Because enzymes that carry out early gene transcription and
modification of early RNAs are packaged in virions, it is possible to perform an in vitro
transcription reaction with purified virus particles. The purified virus particles can be
permeabilized with neutral detergent, incubated with nucleoside triphosphates, and they
will produce authentic fully modified early mRNA (Condit et al., 2002).
Early Gene Expression
During a vaccinia infection, gene expression is regulated at the level of
transcription. There are three classes of gene expression: early, intermediate, and late.
Each gene class has its own distinct promoters and cognate trans-acting factors, mostly
virus-coded. The regulation of vaccinia genes can be thought of as a "cascade" because
the factors required for initiation of each gene class are encoded primarily by the genes of
the preceding class. A virus-coded, multi-subunit, RNA polymerase carries out the RNA
synthesis. This RNA polymerase is packaged into the virion late during the infection,
and is synthesized throughout the infection. The RNA polymerase exists in two forms:
one is specific for early genes, and the other is specific for intermediate and late genes.
These two forms of RNA polymerase have eight subunits in common, ranging from 7 kD
to 147 kD. The early gene-specific form has an additional 94 kD subunit, which is
required for recognition of early promoters (Condit et al., 2002).
As previously discussed, virus uncoating takes place after early genes are
transcribed. Many early gene products are factors required for replication of the viral
genome. One of the most unique aspects of poxviruses is that DNA replication occurs
entirely in the cytoplasm of the infected cell, a characteristic that is only shared with
African swine fever virus. A discrete cytoplasmic area of replication free of cellular
organelles (termed factory areas, or virus factories) has been located using light and
electron microscopy. Some of the important virus-encoded DNA replication proteins are
the DNA polymerase, nucleoside triphosphatase, DNA glycosylase, B IR protein kinase,
and DNA ligase. DNA replication begins 1 to 2 hours after infection, and results in about
10,000 copies of the genome, about half of which are packaged into virions. Vaccinia
virus contains a unique genome structure: a double stranded genome with covalently
closed inverted terminal repetitions. Currently there are still large gaps in the
understanding of its DNA replication. The accepted model for replication proposes that
vaccinia replicates through concatemer intermediates, with the hairpin termini having an
essential role. Replication of the genome then allows for the transcription of intermediate
and late genes (Moss, 2001).
Postreplicative Gene Expression
Inhibitors of DNA replication prevent the transcription of intermediate and late
genes; thus these genes are referred to as postreplicative. As discussed earlier, the RNA
polymerase that is responsible for intermediate and late genes contains eight of the nine
subunits that are present in the early RNA polymerase. Transcription of intermediate
genes requires five early gene products termed Vaccinia Intermediate Transcription
Factors. Intermediate genes encode proteins that are involved in immune defense, virus
morphogenesis, and late transcription. Late genes require four viral intermediate gene
products called Vaccinia Late Transcription Factors. Late genes also require one or more
host proteins. The products of late genes include RNA polymerase, immune defense
proteins, early gene transcription factors, poly(A) polymerase, and virion morphogenesis
proteins. Intermediate and late gene mRNAs are thought to be capped and polyadenylated
by the same enzymes responsible for modifying early mRNAs (Condit et al., 2002). After
the late genes are expressed and the DNA concatemers have been resolved, assembly of
new virus particles begins (discussed in detail in the next section).
Investigations of morphogenesis using electron microscopy have reported that the
formation of new virions takes place in the virus factories that were discussed earlier.
Figure 2 is an overview of vaccinia morphogenesis. The first visible structures to appear
are called crescents, which are composed of lipid and virus coded protein (Smith et al.,
2002). There is ongoing debate about the origins of the crescents. Some believe that
crescents are synthesized de novo and that they have no apparent continuity with cellular
organelles (Dales et al., 1968;Grimley et al., 1970). Others believe that the crescents are a
pair of tightly apposed membranes that are derived from cisternae originating from the
intermediate compartment between the endoplasmic reticulum and the Golgi complex
(Sodeik et al., 2002). The de novo model for membrane synthesis contradicts the
principle that membranes grow from existing membranes. In contrast, the double
membrane theory fits what we do know about membrane biosynthesis (Smith et al.,
2002). Crescents continue to mature in spherical structures called immature virions (IV)
and are filled with a matrix that exists in the virus factory. The crescents contain late viral
membrane proteins, while the electron dense matrix enclosed in them contains viral core
proteins. The electron dense matrix appears to condense, viral DNA is packed into the IV
and DNA nucleoids appear. Particles with nucleoids are termed immature virions with
nucleoid (IVN). The packing of DNA into the IV does require several virus-encoded
proteins. The next step is the maturation of the IVN into IMV, which is a process that
coincides with the proteolytical cleavage of core proteins such as 4a, 4b, and p25.
Figure 2. Vaccinia virus morphogenesis begins with the appearance of the viroplasm
or virus factory followed by the emergence of crescents (1). The crescents
mature and into IVs (2) and are packed with DNA (3). The DNA and viral
matrix inside the IV condense to form IVN (4). The IVN then undergo a
morphogenesis into IMV (5). The IMV are then rapped with two additional
membranes to form IEV (6). The IEV move to the periphery of the cell and
the outer membranes fuses with the cell membrane exposing the CEV on the
cell surface (7). The CEV may be released from the cell as EEV (8).
The cleavage of these proteins triggers the transition from the spherical IVN to the brick-
shaped Intracellular mature virus (IMV), and the generation of the distinct core structure
of the IMV (Sodeik et al., 2002).
After IMV formation the particles move to sites where they are wrapped in a
double layer of membrane to form an intracellular enveloped virus (IEV). The movement
of the IMV from the virus factory to where they are wrapped in additional membranes
this is thought to be dependent upon A27 protein and microtubules. Some of the IMV are
not wrapped in additional membranes and stay in the cell until cell lysis. At later times
during infection a larger portion of the IMV remain unwrapped, possibly due to the
depletion of wrapping membranes. The IEV form of the virus is an intermediate between
the IMV and the cell-associated enveloped virus (CEV). The IEV form functions to
transfer the virus to the cell periphery with the help of the Fl2 protein, and to release the
virus particle from the cell with a membrane that shields the sensitive IMV particle from
antibody and complement (Smith et al., 2002).
Once the IEV reaches the cell surface the outer envelope fuses with the cell
membrane allowing the CEV to be exposed on the cell surface. The A36 protein has been
shown to be present in the IEV and not in the CEV. A36 protein was shown to be
concentrated on the cytosolic face of the plasma membrane beneath CEV and is thought
to aid in the emersion of CEV on the cell surface by the formation of actin tails (van Eijl
et al., 2000). Along with the A36 protein the F l2, A33, A34, and B5R proteins are all
involved in actin tail formation. Actin tails are able to grow considerable distances from
the cell surface and aid in virus penetration into surrounding cells. As actin tails grow
longer they may detach from the cell with the CEV still at its tip, alternatively the CEV
may be released to give Extracellular enveloped virus (EEV). Both the EEV and the
CEV form of the virus contain 3 membranes surrounding the core. A33, A34, A36, and
B5R proteins are also involved in the release of the EEV. Although EEV represents only
a small fraction of virus infectivity, it is biologically very important for long-range virus
spread. Not only does the EEV form have a higher infectivity than other forms, but also it
is resistant to complement and neutralization by antibody.
Temperature sensitive (ts) vaccinia virus mutants have been an integral part to
understanding viral morphogenesis. Many ts mutants that have defects in different stages
of morphogenesis have been isolated and described. A ts mutation in the H5 gene
produces a virus that has "curdled virosomes" and does not produce any of the structures
of morphogenesis (Demasi et al., 2000). The F10 protein has been implicated in the
formation of viral crescents through the use of a ts mutant (Wang et al., 1995). A ts
mutant that contains a J1 defect is able to produce crescents, however, the mutant forms
aberrant, empty IVs (Chiu et al., 2002). A ts mutant with a mutation in the IS gene
produces non-infectious IMVs (Fathi et al., 1991).
A collection of 65 temperature sensitive vaccinia virus mutants have been isolated
and subsequently described (Condit et al., 1981;Condit et al., 1983). The goal of creating
this collection was to have a thorough, systematic genetic analysis of vaccinia virus
genes. Complementation analysis was performed on all 65 mutants, showing that these
mutants comprise 32 complementation groups. These complementation groups were then
analyzed to see if they were able to synthesize DNA and proteins at the nonpermissive
temperature. The results of these tests produced four phenotypes: (1) normal, which
contains mutants that were able to synthesize DNA and early and late proteins at the
same level as wild type virus. (2) DNA-negative are mutants that cannot synthesize DNA
and are only able to synthesize early proteins. (3) Defective late mutants display normal
DNA, and early protein synthesis, but late protein synthesis is either weak, delayed, or
both. (4) Abortive late mutants are also able to synthesize DNA and early proteins,
however, their late protein synthesis begins and later aborts. Within this collection of
mutants is a complementation group that contains Cts6 and Cts9, which display wild type
DNA and protein synthesis, and thus were placed in the normal category (Condit et al.,
1983). Mutants that are classified as "normal" usually are defective in morphogenesis.
Cts6 was mapped by marker rescue, using a cosmid clone library, to the right half of the
A fragment of the HindIII restriction map (Thompson et al., 1986). The purpose of this
research proj ect is to continue the preliminary characterization of this complementation
group to obtain a better understanding of viral morphogenesis. This includes the genetic
mapping of the mutants to a single open reading frame and sequencing this gene to
determine the nature of the mutations. A phenotypic characterization will also be
described which includes protein synthesis and processing, virion transcription and
infectivity, and protein detection in infected cells and purified virions. Electron
microscopy analysis of the mutants is also examined in order to get a detailed look at
MATERIALS AND METHODS
Cells and Viruses
The BSC40 cells and wild type vaccinia virus strain WR, Cts6 and Cts9, the
methods for cell and virus culture, virus infection, and plaque titration have been
previously described (Condit et al., 1981;Condit et al., 1983). The non-permissive
temperature for mutant infections was maintained between 39.5oC and 40oC, which will
be labeled in figures as 40oC for convenience.
Marker rescue was performed as previously described (Meis et al., 1991;
Thompson et al., 1986). The cosmid clones used for the initial mapping and later as a
positive control have been previously characterized (Thompson et al., 1986). B. Luttge
and R. Moyer generously supplied PCR primer sets also used for the initial mapping and
as positive and negative controls. Alternatively, PCR products used in the rescue were
generated using primers that specifically amplified A27L, A28L, and A29L open reading
frames (ORFs). PCR products were also generated using primers that specifically
amplified A25L-A26.4L, A25L-A27L, A27L-A28L, A27L-A29L, and A28L-A29L
ORFs. Integrated DNA Technologies, Inc. (IDT Coralville, IA) synthesized all
oligonucleotides used as primers.
DNA Sequence Analysis
DNA sequence of the A27L and A28L genes from wt, Cts6, and Cts9 viruses was
obtained by direct sequencing of PCR products amplified from total infected cell DNA.
Total infected cell DNA was isolated using the Qiagen Dneasy miniprep spin columns
(Qiagen) according to the manufacturer' s instructions for isolation from cells in culture as
previously described (Latner et al., 2000). Plasmids pT-Adv-A28L and pET-16b-A28L
construction is described below. Their isolation from Escherichia coli culture was done
using the Sigma GenElute High Performance Plasmid Maxiprep Kit (Sigma) following
the manufacturer' s instructions for vacuum format. All DNA mentioned above was
sequenced by the University of Florida ICBR DNA Sequencing Core Laboratory.
Confluent monolayers ofBSC40 cells on 60 mm dishes were infected with wt or
mutant viruses at an m.o.i. of 10. The cells were incubated at either 31 oC or 40 oC for 24,
36, or 48 h. At the appropriate time postinfection the cells were washed with 4 mL of 0. 1
M sodium cacodylate buffer, pH 7.4, then 2 mL of 2% glutaraldehyde in 0. 1 M sodium
cacodylate buffer was added to each dish, the dishes were then incubated at room
temperature for 1 hour with occasional rocking. The cells were then scraped from the
dishes, pelleted at 700 g for 5 min, resuspended in 0.1 M sodium cacodylate buffer, and
stored at 4 oC until further processing. The cells were collected by centrifugation, post-
fixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol solutions and
100% acetone, and embedded in Embed-812 resin mix. En bloc staining was done using
uranyl acetate solution in 75% ethanol. After thin sectioning, the samples were post
stained in uranyl acetate and Reynolds lead acetate. After mounting the samples were
viewed under a Hitachi H7000 transmission electron microscope. Fixating, embedding,
thin sectioning, staining, and microscopy were done with the assistance of the University
of Florida ICBR Electron Microscopy Core Laboratory.
Recombinant DNA Clones
The PET-16b-A28 contains the A28L open reading frame and was created as
follows. The A28L gene was PCR amplified from vaccinia WR DNA using primers that
add a Ndel site at the 5' end and a BamHI site at the 3' end. This PCR product was then
ligated into the 3' T overhangs of the pT-Adv vector (Clontech) making pT-Adv-A28L.
pT-Adv-A28L was then digested with Ndel and BamHI, the fragment containing A28L
was isolated and cloned into Ndel-BamHI cut pET-16b (Novagen) making pET-16b-
The pET-16b-A28L was used for the bacterial overexpression of polyhistidine-
tagged A28 according to the pET system manual (Novagen). Briefly, Rosetta(DE3)pLysS
containing pET-16b-A28L were grown in 50 mL LB containing 34 Cpg/mL ampicillin and
34 Cpg/mL chloramphenicol at 37oC overnight. We used 25 mL of the preculture to
inoculate a 500 mL culture that was grown at 37oC. After the OD590 Of the culture
reached 0.6, the culture was induced by the addition of IPTG (0. 1M stock) to a final
concentration of 1mM and incubated for 4 hours at 37oC. Bacterial cells were
sedimented, and the pellets were resuspended in 20 mL BugBuster Protein Extraction
Reagent (Novagen) and incubated at room temperature for 10 min. After freezing and
thawing the sample was treated with 125 units of RQ 1 RNase-Free DNase (Promega).
The inclusion bodies were then isolated and washed according to Antibodies (Harlow et
al., 1988). The inclusion body preparation was then sent to Strategic Biosolutions for
polyclonal antibody production using the standard two rabbit 70-day protocol. This
antibody will be referred to as the anti-protein antibody for convenience. An anti-peptide
antibody was also created by Sigma Genosys. This antibody was made from the peptide
DRRVQDVNDTISDVKQKWRC, which spans amino acids 56-75 of A28. This antibody
will be referred to as the anti-peptide antibody for convenience.
Purification of Virus Particles
Confluent monolayers of BCS40 cells on 150 mm dishes were infected with wt,
Cts6, or Cts9 viruses at an m.o.i. of 10 at 40oC. 24 h after infection cells were harvested
and purification was performed by sedimentation through preformed discontinuous
sucrose density gradients as previously described (Joklik, 1962b). Purified virions were
quantified by optical density at 260 nm (1 OD260 = 64 Cpg virus). Infectivity of the
purified virus was assayed by plaque titration on BSC40 cells at 31 oC or 40 oC.
After separation of proteins on 12% SDS-PAGE or 10-20% linear gradient precast
(Bio-Rad) gels, the proteins were transferred to nitrocellulose membrane (Bio-Rad) at
100V for 1hr. The blots were then blocked overnight in 5% nonfat dry milk dissolved in
0.05 M Tris, pH 7.5; 0.15 M NaCl; 0.1% Tween-20 (TBST). The blots were washed
twice in TBST for 5 min and then re-blocked in a solution of 3% BSA in TBS (TBST
without Tween-20). Blots were then washed twice in 0.02 M Tris, pH 7.5; 0.5 M NaCl;
0.5% Tween-20, 0.2% TritonX-100 (TBST-T) and washed once for 5 min in TBS. The
blots were then incubated with the anti-peptide antibody at a dilution of 1:1000 in 3%
BSA, TBS or with the anti-protein antibody at a dilution of 1:500 in 3% BSA, TBS for 1
hr. The blot was then washed one time quickly, one time for 10 min, and twice for 5 min
each in TB ST-T. The blots were then incubated with a donkey anti-rabbit IgG HRP -
linked Whole Ab (Amersham Biosciences) at a dilution of 1:2000 in 5% milk, TBST for
1 hr. The blots were then washed in TBST-T one time quickly, one time for 10 min, and
twice for 5 min. Two subsequent washes with TBST for 5 min were performed before
detection with ECL Western Blotting Detection Reagents (Amersham Biosciences) and
exposure to X-ray film.
Protein Pulse Label and Pulse Chase Assays
Protein pulse labeling of infected cells with trans-35S labeled methionine (ICN
Biochemical), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
and audoradiography were done as previously described (Condit et al., 1981). In protein
pulse chase experiments the pulse-labeled cells were washed and incubated in serum
containing medium for various times. At times 0, 3, 6, 9, 12 hrs the cells were harvested
and analyzed as described above.
Transcription by Permeabilized Virions
Permeabilized virion transcription experiments were done as previously described
by Gershowitz and Moss (Gershowitz et al., 1979) and Gross and Shuman (Gross et al.,
1996). To measure total incorporation into RNA reactions containing 60 mM Tris-HCI
pH 8.0, 0.05% nonidet P-40 (NP40), 10 mM dithiothreitol (DTT), 10 mM MgCl2, 5 mM
ATP, 1 mM UTP, 1 mM GTP, 0.2 mM oC 32P-CTP (3000 Ci/mmol Perkin Elmer) and 0.3
OD260 Of Virus were incubated at 30oC, and 50 Cl1 aliquots were precipitated directly with
5% TCA. The virion associated versus released RNA was measured using identical
reactions except the 50 ul aliquots were diluted into 200 ul stop solution (50 mM Tris-
HCI pH 8.0, 10 mM DTT, 10 mM EDTA, 0.05% NP40) and centrifuged for 3 min in a
microfuge. The supernatants (released RNA) were then removed and precipitated in 5%
TCA. The pellets (core associated RNA) were resuspended in 0.2mL of a buffer
containing 50 mM Tris-HC1, pH 8.0, 0.1% SDS, and precipitated with 5% TCA. TCA
perceptible radioactivity was determined by liquid scintillation counting.
Mapping of the Mutations
The temperature sensitive vaccinia virus mutants Cts6 and Cts9 comprise a
complementation group that has a "normal" phenotype, that is, they display wild type
protein and DNA synthesis (Condit et al., 1983). Not only were these mutants shown to
be in the same gene by complementation analysis, but they were also shown to have an
undetectable recombination frequency, <1.6 X 10-4, which suggests that the mutations lie
close together (Condit et al., 1981). Using a library of overlapping cosmid clones,
Thompson and Condit showed that Cts6 mapped to the right half of the HindIII A
fragment of the vaccinia genome (Thompson et al., 1986). Further preliminary mapping
(R. Condit, personal communication) showed that the mutation could be narrowed down
to a 5 kB region of the genome. This approach used PCR products generated off vaccinia
wild type DNA using primers designed and generously donated by B. Luttge and R
Moyer. The genes included in this 5 kB region are the A25L A26.4L (A-type inclusion
proteins), A27L (IMV membrane protein), A28L (unknown function), and A29L (35 kD
subunit of RNA polymerase) (Fig. 3). Unlike cowpox that encodes a functional A-type
inclusion (ATI) gene, vaccinia virus has a homolog that is disrupted into several ORFs
and is not functional. Because the A-type inclusion is likely to be non-essential and
because of the preliminary phenotype of the mutants, A27L and A28L were the most
likely candidate genes for the location of the mutation.
The first strategy that was used to rescue the mutants to a single ORF was
to generate PCR products that specifically amplified each ORF, treating the ATI reading
frames as a single ORF. These PCR products were then used in a one-step marker rescue
experiment as described in the materials and methods. Attempts using this approach did
not result in a significant rescue with any of the PCR products (data not shown). The next
strategy taken was to generate overlapping PCR products that incorporated two or more
genes. PCR products were made that specifically amplified A25L-A26.4L, A25L-A27L,
A27L-A28L, A27L-A29L, and A28L-A29L ORFs. These PCR products along with the
single ORF products were used in a one-step marker rescue experiment and several of the
products resulted in significant rescue. Fig. 3 shows a subset of the results showing that
rescue did not occur with products containing A25L-A27L, A28L-A29L, or with just
A27L or A28L, however there is rescue with products that contains A27L and A28L. The
experiment was performed with both Cts6 and Cts9 with similar results, however, only
Cts6 is shown in Fig. 3. Because of the orientation of A27L and A28L this result suggests
that the mutations must be at the 5' end of A27L or at the 3' end of A28L.
To determine the precise mutation that causes the temperature sensitivity in Cts6
and Cts9, the A27L and A28L genes from both viruses were sequenced. Figure 4 shows
the nucleotide sequence alignment of A28L from Cts6, Cts9, and wt. The mutant viruses
have wild type A27L genes and they both have a two nucleotide deletion in the A28L
gene. Both mutants are missing the 394th and 395th HUClCOtides of the 43 8-nucleotide
A28L gene, which in the wild type are both cytosine residues. This data is consistent with
the marker rescue results, which suggested that the mutations could be located at the 3'
end of A28L. The protein translation of Cts6 and Cts9 is shown in Fig. 5, which shows
Figure 3. Marker rescue of Cts6. At the top is a 7020 bp region of the vaccinia genome where Cts6 has been previously mapped,
which includes genes A25L to A29L. The gene name and known protein function are indicated above each gene. The
middle of the figure shows a schematic representation of a subset of PCR products used in the rescue of Cts6. The
gene(s) included in each PCR product are indicated to the right of the product and each product is numbered to the left.
The bottom of the figure shows a subset of dishes used in the rescue with the corresponding PCR product labeled below
h Q\ 00
trI CO N 00
--N M rr 4 t
Cts6-A28L (1) ATGAACTCTCTATCAATTTTTTTTATTGTGGTAGCGACGGCGGTG
Cts9-A28L (1) ATGAACTCTCTATCAATTTTTTTTATTGTGGTAGCGACGGCGGTG
wt-A28L (1) ATGAACTCTCTATCAATTTTTTTTATTGTGGTAGCGACGGCGGTG
Cts6-A28L (51) TTTACTTTTTATCCAGGGTTACTCAATATATGAAAATATGGCATAT
Cts9-A28L (51) TTTACTTTTTATCCAGGGTTACTCAATATATGAAAATATGGCATAT
wt-A28L (51) TTTACTTTTTATCCAGGGTTACTCAATATATGAAAATATGGCATAT
Cts6-A28L (101) AGGAATTTAATGCTACTCATGCAGCATTCGAATATTCAAAACTGT
Cts9-A28L (101) AGGAATTTAATGCTACTCATGCAGCATTCGAATATTCAAAACTGT
wt-A28L (101) AGGAATTTAATGCTACTCATGCAGCATTCGAATATTCAAAACTGT
Cts6-A28L (151) GGAACACCGGCATTAGATAGGAGAGTTCAAGATGTCAACGAAATC
Cts9-A28L (151) GGAACACCGGCATTAGATAGGAGAGTTCAAGATGTCAACGAAATC
wt-A28L (151) GGAACACCGGCATTAGATAGGAGAGTTCAAGATGTCAACGAAATC
Cts6-A28L (201) TGATGTAAAGCAAAAGTGGAGATGTGTGGTTTATCCAGGAACGTG
Cts9-A28L (201) TGATGTAAAGCAAAAGTGGAGATGTGTGGTTTATCCAGGAACGTG
wt-A28L (201) TGATGTAAAGCAAAAGTGGAGATGTGTGGTTTATCCAGGAACGTG
Cts 6-A2 8L (251) TAT CCGCTT CCATATTT GGATTT CAGGCAGAAGTT GGACCCAATAATACT
Ct s 9-A2 8L (251) TAT CCGCTT CCATATTT GGATTT CAGGCAGAAGTT GGACCCAATAATACT
wt -A2 8L (251) TAT CCGCTT CCATATTT GGATTT CAGGCAGAAGTT GGACCCAATAATACT
Cts6-A28L (301) AGATCCATTAGAAAATTTAACACGATGCAACAATGTATAGATACT
Cts9-A28L (301) AGATCCATTAGAAAATTTAACACGATGCAACAATGTATAGATACT
wt-A28L (301) AGATCCATTAGAAAATTTAACACGATGCAACAATGTATAGATACT
Cts6-A28L (351) TTCTGATGTTATTAACATCAATATTTATAATCCATGTGTTGA-AT
Cts9-A28L (351) TTCTGATGTTATTAACATCAATATTTATAATCCATGTGTTGA-AT
wt-A28L (351) TTCTGATGTTATTAACATCAATATTTATAATCCATGTGTTGACAT
401 438 ^
Cts6-A28L (399) TAAATAACGCAGAGTGTCAGTTTCTAAAATCTGTACTT
Cts9-A28L (399) TAAATAACGCAGAGTGTCAGTTTCTAAAATCTGTACTT
wt-A28L (401) TAAATAACGCAGAGTGTCAGTTTCTAAAATCTGTACTT
Figure 4. Nucleotide alignment of A28L from wild type, Cts6, and Cts9 viruses. In
Cts6 and Cts9 cytosine residues located a positions 394 and 395 are deleted,
which are indicated by a ^ below the sequence.
Figure 5. Amino acid sequence alignment of A28 protein from wild type, Cts6, and
Cts9 (A). The first 131 amino acids encoded are identical in mutant and wild
type viruses. The mutation, which is indicated by a ^ under the sequence,
causes a frameshift which results in three aa substitutions followed by a stop
codon, which causes a C-terminal truncation. (B) Wild type and mutant A28
proteins. A transmembrane domain, indicated by a red box and labeled TM,
is located between the 4th and 26th amino acids. The green box at the C-
terminal end of the mutant protein represents the three substitutions encoded
by the mutant viruses, which begins at aa 132. The Cs inside the proteins
represent conserved cysteines (see discussion).
Wild Type (146 aa)
Cts6 & Cts9 (134 aa)
that the mutants produce C-terminal truncated proteins. The wild type protein is 146
amino acids (aa) in length, while the mutant form of the protein is only 134 aa in length.
The two deletions cause a frameshift and after the first 131 aa are translated correctly 3 aa
are substituted before a stop codon is reached.
Cts6 and Cts9 have been previously shown to have a "normal" phenotype due to
their wild type levels of DNA and protein synthesis (Condit et al., 1983). In order to
confirm the protein synthesis results both mutants were assayed for their pattern of
protein expression in infected cells. Cells were infected with Cts6, Cts9, or wt and
incubated at 31oC or 40oC, and proteins were pulse-labeled at various times post-
infection with 35S-methionine. The proteins were then separated by SDS-PAGE and
detected by autoradiography (Fig. 6). The wt virus represents normal protein synthesis at
31oC or 40oC. Both Cts6 and Cts9 show protein synthesis that looks like wild type. Cell
host proteins are seen at 0 h post-infection and are subsequently shut off by 3 h post-
infection with the appearance of early proteins. Late viral protein synthesis is seen by 6 h
post-infection and persists throughout the experiment. The mutants displayed a normal
pattern of protein synthesis, however, some of the films appear lighter due to a technical
problem during exposure.
The processing of several viral proteins is associated with morphogenesis from
IVN to IMV (Klemperer et al., 1997). Virus morphogenesis is arrested at the IVN stage
with a mutation in a protease that is implicated in cleavage of virion proteins (Byrd et al.,
2002;Kane et al., 1993). Therefore, virion protein processing is a good marker for
detecting defects in virus assembly. In order to test whether the maj or core polypeptides
9 12 03 6 912
Figure 6. Viral protein synthesis in wild type, Cts6, and Cts9 infected cells. Cells were
infected at 31oC and 40oC, and pulse-labeled for 15 min with trans-35S
methionine. Cells were harvested, and the proteins were separated by a 10%
SDS-PAGE and autoradiographed. The autoradiograms are shown above.
The infecting virus is indicated along the bottom, and temperature of
incubation is indicated at the left. Approximate molecular weights, in
kilodaltons, are shown to the right of the gels. The time of the pulse labeling
is indicated, in hours, above each lane.
0 3 6912
0 3 6912
03 6 912
are processed correctly Cts6 and Cts9 were assayed in a pulse chase experiment. Cells
were infected with wt, Cts6, or Cts9 and incubated at 31oC or 40oC for 8 h and pulse
labeled with 35S-methionine. At various times after labeling the cells were chased in the
presence of unlabeled amino acids, harvested, and analyzed by SDS-PAGE and
autoradiography (Fig. 7). Normal proteolytic processing can be observed by the decrease
in intensity of the p4a and p4b proteins, 102 and 72 kD respectively. The products of
these cleavages result in the appearance of two more bands 4a and 4b, 62 and 60 kD
respectively. The results show that Cts6 and Cts9 are able to process p4a and p4b into
their respective products 4a and 4b. The disappearance of the 102 kD band is not as
prominent as usual. This could be a result of an insignificant removal of the label, which
was gently done only once to ensure the integrity of the infected cell monolayer.
However the appearance of the 60 and 62 kD bands are strong evidence that the proteins
are being processed normally.
Transmission electron microscopy was used to further investigate Cts6 and Cts9 to
see if they are able to assemble normal looking particles in vivo. Briefly, B SC40 cells
were infected with wt, Cts6, or Cts9 at 31oC or 40oC for 24, 36, or 48 h and then fixed
and processed for electron microscopy. Figures 8 & 9 show that Cts6 and Cts9 are
indistinguishable from wt at both 3 1oC and 40oC. All stages of vaccinia virus
morphogenesis were observed including viral crescents, immature virions, immature
virions with nucleoids, and intracellular mature virions.
0 3 6912 0 3 6912 03 6 912
0 36d912 0 3 6912 0 36d912
wt Cts6 Cts9
Figure 7. Viral protein processing in infected cells. 8 hours after infection cells were
labeled with trans-35S methionine, then chased with unlabeled amino acids
for up to 12 hours. Cells were harvested and separated on a 12% SDS-
PAGE and autoradiographed. The autoradiograms are shown. The infecting
virus is indicated along the bottom, and temperature of incubation is
indicated at the left. Approximate molecular weights, in kilodaltons, are
shown to the right of the gels. The length of the chase is indicated, in hours,
above each lane.
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Figure 8. Electron micrograph pictures of cells infected with CtsG (A) or wt (B) at 40
"C. Cells were infected with an m.o.i. of 10 and processed for electron
microscopy 24 h postinfection. Arrows indicate examples of the different
stages of morphogenesis: crescent (C), immature virions (IV), immature
virions with nucleoid (IVN), and intracellular mature virion (IMV).
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Immunodetection of A28 Protein
To further characterize the mutants, they were examined by western blot using
A28 specific antibodies. Two antibodies were used in these western blots, an anti-peptide
antibody and an anti-protein antibody, prepared as described in materials and methods.
Infected cell lysates were first examined with anti-protein antibody. Briefly, cells were
infected with wt, Cts6, or Cts9 and incubated at 40oC for various times, then harvested
and the proteins separated by SDS-PAGE. After transferring the proteins to a
nitrocellulose membrane the blots were incubated with a 1:500 dilution of anti-protein
antibody, then incubated with an anti-rabbit antibody and detected with ECL detection
reagent and autoradiography (Fig. 10 A).
A band the correct size for A28, about 15 kD, was detected in all three viruses late
in the infection. This band was not seen in the mock, 3 h post infection, or 6 h post
infection lanes, however it was seen in the 9 h post infection and the 12 h post infection
lanes. The viruses were grown at 40oC and purified, as described in materials and
methods and were examined with the anti-peptide antibody. Briefly, 0.2 OD units of
purified virus was run on a 10-20% gradient gel, and transferred to a nitrocellulose
membrane. The membrane was incubated with a 1:1000 dilution of the anti-peptide
antibody, then incubated with a secondary anti-rabbit antibody and Einally detected with
ECL detection reagent and autoradiography (Fig. 10 B). A band the correct size was
detected in purified virions in all three viruses. The two mutant viruses seemed to contain
a slightly smaller protein, which would be consistent with the sequencing data. The
amount of A28 protein in the mutant viruses also seemed to be much less than wild type.
/ _A ~Ce
15I -- -e
Figure 10. Immunodection of A28 protein. (A) Western blot of infected cell time
course. Proteins were separated on 12% SDS-PAGE and detected with anti-
protein antibody. The infecting virus and time harvested after infection, in
hours, is indicated above gel. Approximate molecular weight marker, in
kilodaltons, is shown on the left. (B) Western blot of purified virus. Proteins
were separated on a 10-20% gradient gel and detected with anti-peptide
antibody. The purified virus is indicated above the gel and approximate
molecular weight, in kilodaltons, is shown on the left.
3 h p.I.
6 h pl.I.
9 h pJ.I
1 2h pJ..
Purified vaccinia particles can be treated with NP40 and DTT to permeabilize them
by removing the outer membrane, but leaving the enzyme-containing core structurally
and functionally intact. These permeablilized virions are capable of carrying out
transcription and modification of early viral mRNA (Hassett et al., 1997). Purified virions
that were grown at 40oC, as described in materials and methods were permeabilized with
a NP40 and DTT solution and incubated with NTPs, and a 32P-CTP at 30oC for various
times. At each time point the cores were spun down and the supernatant, which contained
released transcripts, was TCA precipitated, filtered, and counted in a liquid scintillation
counter. The cores were then disrupted in a SDS solution, TCA precipitated, filtered, and
counted in a liquid scintillation counter. The results (Fig. 11) show that the mutants are
able to synthesis and release early mRNA. The mutants do show a reduced amount of
mRNA compared to the wild type, however it is only about a 2-fold difference.
Particle to Infectivity Ratio
In order to further study Cts6 and Cts9, purified particles grown at 40oC were
assayed at 3 1oC to see if they were infectious at the permissive temperature. Briefly,
BSC40 cells were infected with a serial dilution of purified virus, and grown at 40oC for
seven days in order to determine plaque forming units per mL (pfu/mL). Using the
standard 1 OD260 = 1.2 x 1010 particles/mL (Joklik, 1962a), the concentration of virus
particles was determined. The particle to infectivity ratio was then calculated for wt,
Cts6, andCts9. Wt virus has a particle to infectivity ratio of 39.2 particles/plaque forming
unit. Cts6 has a ratio of 11,200 particles/plaque forming unit. Cts9 has a ratio of 10,000
particles/plaque forming unit. This suggests that mutant viruses are almost 250 fold less
infectious as the wild type virus.
Figure 11. In vitro transcription activity of wild type (A), Cts6 (B), and Cts9 (C)
purified virions. Purified vaccinia virus particles were incubated for various
times, indicated on the x axis, with detergent and ribonucleside
triphospahtes The incorporation of oC 32P-CTP into RNA was determined,
which is indicated on the y axis.
0 20 40 E
0 20 40
E 120 -
0 80 -
. 60 -
o 20 -
0 20 40
Temperature sensitive (ts) vaccinia mutants have played a crucial role in the
understanding of vaccinia virus morphogenesis. Ts mutants have implicated many genes
involved in viral morphogenesis including H5 (Demasi et al., 2000), F10 (Wang et al.,
1995), J1 (Chiu et al., 2002), and 16 (Grubisha et al., 2003). The purpose of this study
was to use the ts vaccinia virus mutants Cts6 and Cts9 in order to further our
understanding of morphogenesis. Cts6 and Cts9 are a result of hydroxylamine
mutagenesis and random plaque screening. They have been previously described to have
a "normal" phenotype, based on their ability to synthesize DNA and proteins at wild type
levels (Condit et al., 1983), which makes them candidates for defects in viral
The genetic mapping of the mutants to a single open reading frame was the first
goal of this proj ect. Cts6 and Cts9 have been previously mapped by marker rescue to the
right side of the HindIII A fragment (Thompson et al., 1986). R. Condit further mapped
Cts6 and Cts9, using PCR products generated with primers donated by R. Moyer and B.
Luttge, to a 5 kB region of the vaccinia genome shown at the top of Fig. 3. This region of
the genome includes genes A25L through A29L. Some poxviruses, such as cowpox and
fowlpox, encode a protein that causes IMVs to become occluded in a dense protein
matrix called A-type inclusions (ATI). This ATI protein is not essential and vaccinia has
a frameshift mutation that disrupts the ATI gene into five smaller ORFs (Cooper et al.,
1981). These ORFs are named A25L, A26.1L, A26.2L, A26.3L, and A26.4L and were
treated as a single ORF for mapping purposes. These genes were not prime candidates for
containing the mutation, because ATI is not an essential protein. The A29L gene encodes
the 3 5 kD subunit of the RNA polymerase. A29L was also thought not to contain the
mutation because Cts6 and Cts9 were shown to display normal protein synthesis (Condit
et al., 1983), while an RNA polymerase mutant is defective in the synthesis of late viral
mRNA and proteins (Hooda-Dhingra et al., 1989). The two candidate genes left in the
region are A27L and A28L, which are the two genes most likely to contain the mutations.
A27L protein encodes a IMV protein involved in morphogenesis (Sanderson et al., 2000).
Nothing was known about the A28L gene at the time of the mapping. PCR products that
specifically amplified each open reading frame in this 5 kB region were used in a marker
rescue experiment but was unsuccessful. The next approach used was to create PCR
products that contained more than one ORF. PCR products were made that included
A25L-A27L, A27L-A28L, A27L-A29L, and A28L-A29L, and used in another maker
rescue along with the single ORF products. The mutants could only be rescued with
products that span both A27L and A28L, but not with either gene individually. Both
A27L and A28L are oriented to the left, suggesting that the mutations lie either at the
beginning of A27L or at the end of A28L.
The region spanning A27L and A28L from wt, Cts6, and Cts9 was sequenced and
aligned. TheA27L gene was identical in all three viruses. Differences were found
between the wt and mutants sequences in the A28L gene. Surprisingly the mutant viruses
have the exact same sequence for the A28L gene. Several explanations exist for both
mutants having identical A28 sequences; the most likely of them is that these viruses are
siblings generated by spontaneous mutations and were present in the wild type virus
stock used in the mutagenesis. The mutations comprise deletion of the 394th and 395th
nucleotides of the A28L gene. A28L is present in all poxviruses and with its high
sequence conservation it is predicted to play an essential role in the virus life cycle
(Senkevich et al., 2004a). This result fits our marker rescue results well because the first
deletion is only 45 nucleotides away from the stop codon. This data is the best example
from our lab of the amount of overhang required for a successful recombination in
vaccinia virus. The translation of the mutant A28 protein results in a C-terminally
truncated protein. The mutations cause a frameshift after the first 131 aa are translated
there are three aa substitutions before a stop codon is reached.
As discussed earlier, Cts6 and Cts9 have a preliminary phenotype of "normal,"
which means they are able to synthesize DNA and proteins at wild type levels. Before
further characterization of the mutants could take place confirmation of these preliminary
results was completed. In order to look at the protein synthesis of the mutants a pulse
label assay, as described in materials and methods, was performed on the mutants. Cts6
and Cts9 display a normal pattern of protein synthesis, confirming the preliminary protein
synthesis results. Because late protein synthesis requires DNA replication (Moss, 2001)
these results are sufficient to confirm the normal DNA synthesis phenotype.
Viral protein processing of core proteins such as 4a, 4b, and p25 coincides with
the maturation of the IVN into IMV (Sodeik et al., 2002). The next step in the
characterization of the mutants was to check the mutants for their ability to process these
core proteins. Although we were anticipating the mutants to be defective, they displayed
wild type levels of protein processing. These results suggest that any morphogenesis
defect present in Cst6 and Cts9 exists after the formation of IMVs.
Because the mutants are able to process viral core proteins we wanted to examine
the viral particles under an electron microscope to determine whether normal looking
IMVs were made. We also wanted to see if we could discover any other defects in the
morphogenesis pathway the mutants might contain. The mutants appeared normal
throughout all the stages of viral morphogenesis at the nonpermissive temperature.
Mutant IMVs made at the nonpermissive temperature were then purified for further
Purified particles and infected cells were checked for the presence of the A28
protein. Cts6 and Cts9 have the same protein expression pattern of A28 as wild type virus
in infected cells. The A28 protein is expressed at late times during a vaccinia infection.
Sequence analysis of A28L reveals that the A of the start codon is part of a TAAA, which
is a typical promoter of poxvirus late genes (Davison et al., 1989). A28 contains an early
transcription termination sequence, TTTTTAT, prohibiting A28 expression at early times
(Ink et al., 1989). Analysis of 0. 1 OD units of purified virions revealed that the mutants
are able to package the A28 protein into the virion, however, less protein is present than
in wild type virions. Because the mutants cannot package A28 at wild type levels they
may have a morphogenesis defect during assembly. The mutant protein that is packaged
into the virion is slightly smaller than the wild type protein, which was predicted by the
sequencing data. This suggests that the mutant protein is stable, however, it is not
functional at the nonpermissive temperature.
All the enzymes necessary for transcription and modification of early mRNAs are
packaged into the virion. Thus, it is possible to permeabilize purified particles with a
neutral detergent and incubate them with nucleoside triphosphates and they will produce
authentic fully modified early mRNAs in vitro (Condit et al., 2002). Cts6 and Cts9
purified particles are both able to transcribe mRNAs in vitro, however, only about half as
efficient as wild type. This is further evidence that although the mutant particles appear
normal they may not be assembled correctly resulting in slightly diminished transcription.
The existence of multiple infectious forms of virus has complicated the study of
virus entry. IMVs are the form of virus usually used by investigators to infect cells in the
laboratory and it is possible to purify these particles from infected cells to study them
(Moss, 2001). The purified IMV particles made at the nonpermissive temperature were
assayed to see if they are able to infect cells at the permissive temperature. We found that
the mutant particles were about 1,000 times less infectious than wild type particles. Cts6
and Cts9 are capable of all viral functions once inside of the cell. However, mutant
virions made at the nonpermissive temperature are not infectious at the permissive
temperature. This suggested to us that the mutants are not able to enter the cell, which
means that A28 has a role in virus entry into the cell.
Very recently Senkevich et al. (2004) published two papers about A28L and its
role in vaccinia biology. The papers used an A28 hemagglutinin tagged inducible
knockout virus to demonstrate many of the same results we have shown. A28 is
expressed as a late protein, is essential for virion infectivity, and replication. A28 is not
required for RNA and protein synthesis, protein processing, or the assembly of
intracellular and extracellular virions. Further investigation revealed A28L to be a
substrate of the viral disulfide bond formation enzyme and other aspects that our study
did not address. A28 was shown to be anchored in the IMV membrane by a N-terminal
transmembrane domain, while the C-terminus is exposed on the surface and is predicted
to contain two intramolecular disulfide bonds at four conserved cysteines. An extensive
investigation of A28's role in virus entry revealed that it is not required for binding to the
cell, however, A28 is required for cell rounding and cytopathic effects. A28 defieient
virions have a defect in penetration and membrane uncoating. The A28 defieient virions
also were not able to cause vaccinia virus-induced cell fusion (Senkevich et al.,
2004b; Senkevich et al., 2004a).
The predicted intramolecular bonds of A28 are relevant to our studies using Cts6
and Cts9. The conserved cyteines thought to be involved in the bonds are shown in Fig.
5. The terminal cysteine is not present in the mutant protein, which means that one of the
disulfide bonds cannot be formed and may have a functional effect on the protein. Further
investigations with Cts6 and Cts9 need to be conducted to see if any differences are
present between the ts mutants and the A28 knockout virus. Vaccinia virus-induced cells
fusion is thought to mimic events during virus entry, and A28 is required for virus-
induced cell fusion (Senkevich et al., 2004b). This suggests that A28's role in virus entry
is through fusion with the plasma membrane. A27 is another IMV protein that has been
implicated in cell fusion (Vazquez et al., 1998). A27 and A28 may work together in
fusion and entry, however, A27-defieient IMV retain nearly complete (Hsiao et al., 1999)
or partial (Vazquez et al., 1999) infectivity. A27 has also been shown to function in
binding to cell surface glycoaminoglycans (Vazquez et al., 1999). Although the
mechanism of action is still undetermined, A28 is an essential vaccinia protein that
functions in cell fusion and entry.
LIST OF REFERENCES
Byrd, C. M., Bolken, T. C., and Hruby, D. E. (2002). The vaccinia virus I7L gene product
is the core protein proteinase. J. Virol. 76, 8973-8976.
Chiu, W. L. and Chang, W. (2002). Vaccinia virus J1R protein: a viral membrane protein
that is essential for virion morphogenesis. J. Yirol. 76, 9575-9587.
Condit, R. C. and Motyczka, A. (1981). Isolation and preliminary characterization of
temperature-sensitive mutants of vaccinia virus. Virology 113, 224-241.
Condit, R. C., Motyczka, A., and Spizz, G. (1983). Isolation, characterization, and
physical mapping of temperature-sensitive mutants of vaccinia virus. Yirology 128, 429-
Condit, R. C. and Niles, E. G. (2002). Regulation of viral transcription elongation and
termination during vaccinia virus infection. Biochim.Biophys.Acta 1577, 325-336.
Cooper, J. A., Wittek, R., and Moss, B. (1981). Extension of the transcriptional and
translational map of the left end of the vaccinia virus genome to 21 kilobase pairs.
J. Yirol. 39, 733-745.
Dales, S. and Mosbach, E. H. (1968). Vaccinia as a model for membrane biogenesis.
Virology 35, 564-583.
Davison, A. J. and Moss, B. (1989). Structure of vaccinia virus late promoters.
J.M~ol.Biol. 210, 771-784.
Demasi, J. and Traktman, P. (2000). Clustered charge-to-alanine mutagenesis of the
vaccinia virus H15 gene: isolation of a dominant, temperature-sensitive mutant with a
profound defect in morphogenesis. J. Virol. 74, 2393-2405.
Fathi, Z. and Condit, R. C. (1991). Phenotypic characterization of a vaccinia virus
temperature-sensitive complementation group affecting a virion component. Virology
Gershowitz, A. and Moss, B. (1979). Abortive transcription products of vaccinia virus are
guanylylated, methylated, and polyadenylylated. J. Virol. 31, 849-853.
Grimly, P. M., Rosenblum, E. N., Mims, S. J., and Moss, B. (1970). Interruption by
Rifampin of an early stage in vaccinia virus morphogenesis: accumulation of membranes
which are precursors of virus envelopes. J. Virol. 6, 519-533.
Gross, C. H. and Shuman, S. (1996). Vaccinia virions lacking the RNA helicase
nucleoside triphosphate phosphohydrolase II are defective in early transcription. J. Yirol.
Grubisha, O. and Traktman, P. (2003). Genetic analysis of the vaccinia virus I6 telomere-
binding protein uncovers a key role in genome encapsidation. J. Yirol. 77, 10929-10942.
Harlow, E. and Lane, D. (1988). "Antibodies: a laboratory manual." Cold Spring Harbor
Hassett, D. E., Lewis, J. I., Xing, X., DeLange, L., and Condit, R. C. (1997). Analysis of
a temperature-sensitive vaccinia virus mutant in the viral mRNA capping enzyme
isolated by clustered charge-to-alanine mutagenesis and transient dominant selection.
Virology 238, 391-409.
Hooda-Dhingra, U., Thompson, C. L., and Condit, R. C. (1989). Detailed phenotypic
characterization of five temperature-sensitive mutants in the 22- and 147-kilodalton
subunits of vaccinia virus DNA-dependent RNA polymerase. J. Yirol. 63, 714-729.
Hsiao, J. C., Chung, C. S., and Chang, W. (1999). Vaccinia virus envelope D8L protein
binds to cell surface chondroitin sulfate and mediates the adsorption of intracellular
mature virions to cells. J. Yirol. 73, 8750-8761.
Ink, B. S. and Pickup, D. J. (1989). Transcription of a poxvirus early gene is regulated
both by a short promoter element and by a transcriptional termination signal controlling
transcriptional interference. J. Virol. 63, 4632-4644.
Joklik, W. K. (1962a). The preparation and characteristics of highly purified
radioactively labelled poxvirus. Biochim.Biophys.Acta 61, 290-301.
Joklik, W. K. (1962b). The purification fo four strains of poxvirus. Virology 18, 9-18.
Kane, E. M. and Shuman, S. (1993). Vaccinia virus morphogenesis is blocked by a
temperature-sensitive mutation in the I7 gene that encodes a virion component. J. Yirol.
Klemperer, N., Ward, J., Evans, E., and Traktman, P. (1997). The vaccinia virus 11
protein is essential for the assembly of mature virions. J. Yirol. 71, 9285-9294.
Latner, D. R., Xiang, Y., Lewis, J. I., Condit, J., and Condit, R. C. (2000). The vaccinia
virus bifunctional gene J3 (nucleoside-2'-O-)- methyltransferase and poly(A) polymerase
stimulatory factor is implicated as a positive transcription elongation factor by two
genetic approaches. Virology 269, 345-355.
Meis, R. J. and Condit, R. C. (1991). Genetic and molecular biological characterization of
a vaccini a virus gene which renders the virus dependent on i satin-b eta-thi osemi carb azone
(IBT). Virology 182, 442-454.
Moss, B. (2001). Poxviridae: The Viruses and Their Replication. In "Fields Virology" (D.
M. Knipe and P. M. Howley, Eds.), pp. 2849-2884. Lippincott Williams & Wilkins,
Sanderson, C. M., Hollinshead, M., and Smith, G. L. (2000). The vaccinia virus A27L
protein is needed for the microtubule-dependent transport of intracellular mature virus
particles. J.Gen. Virol. 81 Pt 1, 47-58.
Senkevich, T. G., Ward, B. M., and Moss, B. (2004a). Vaccinia virus A28L gene encodes
an essential protein component of the virion membrane with intramolecular disulfide
bonds formed by the viral cytoplasmic redox pathway. J. Virol. 78, 2348-23 56.
Senkevich, T. G., Ward, B. M., and Moss, B. (2004b). Vaccinia virus entry into cells is
dependent on a virion surface protein encoded by the A28L gene. J. Yirol. 78, 2357-2366.
Smith, G. L., Vanderplasschen, A., and Law, M. (2002). The formation and function of
extracellular enveloped vaccinia virus. J.Gen. Yirol. 83, 2915-2931.
Sodeik, B. and Krijnse-Locker, J. (2002). Assembly of vaccinia virus revisited: de novo
membrane synthesis or acquisition from the host? Trends M~icrobiol. 10, 15-24.
Thompson, C. L. and Condit, R. C. (1986). Marker rescue mapping of vaccinia virus
temperature-sensitive mutants using overlapping cosmid clones representing the entire
virus genome. yirology 150, 10-20.
van Eijl, H., Hollinshead, M., and Smith, G. L. (2000). The vaccinia virus A36R protein
is a type Ib membrane protein present on intracellular but not extracellular enveloped
virus particles. Virology 271, 26-36.
Vazquez, M. I. and Esteban, M. (1999). Identification of functional domains in the 14-
kilodalton envelope protein (A27L) of vaccinia virus. J. Yirol. 73, 9098-9109.
Vazquez, M. I., Rivas, G., Cregut, D., Serrano, L., and Esteban, M. (1998). The vaccinia
virus 14-kilodalton (A27L) fusion protein forms a triple coiled-coil structure and interacts
with the 21-kilodalton (Al7L) virus membrane protein through a C-terminal alpha-helix.
J. Yirol. 72, 10126-10137.
Wang, S. and Shuman, S. (1995). Vaccinia virus morphogenesis is blocked by
temperature-sensitive mutations in the F10 gene, which encodes protein kinase 2. J. Yirol.
Bradley Dilling was born in State College, Pennsylvania in 1979, and moved to
Cape Coral Florida in 1986, where he graduated from Mariner High School in 1997. He
graduated from the University of South Florida (Tampa) in 2001, with a bachelor' s
degree in biology. He received his Master of Science degree in 2004. He plans on
furthering his academic career in dental school at the University of Florida and eventually
plans on practicing dentistry in South Florida.