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Mapping and Phenotypic Characterization of Temperature Sensitive Vaccinia Virus Mutants Cts6 and Cts9


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MAPPING AND PHENOTYPIC CHARAC TERIZATION OF TEMPERATURE SENSITIVE VACCINIA VIRUS MUTANTS Cts6 AND Cts9 By BRADLEY PAUL DILLING A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Bradley Paul Dilling

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iii ACKNOWLEDGMENTS 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 fo r 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 Kat o, Dr. Nissin Moussatch Audra Strahl, Dr. Hendrik Nollens, Dr. Cindy Prins, and Ms. Tommie Albright for all their help. I especially would like thank my graduate advi sor, Dr. Rich Condit, for all his advice and counsel during my project. Rich is an incred ible person to know and work for. I would also like to thank the rest of my committ ee (Dr. Sue Moyer and Dr. Donna Duckworth) for their input into my project.

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iv TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF FIGURES...........................................................................................................vi KEY TO SYMBOLS........................................................................................................vii ABSTRACT.....................................................................................................................vi ii CHAPTER 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 Morphogenesis......................................................................................................6 The Problem..................................................................................................................9 2 MATERIALS AND METHODS...............................................................................11 Cells and Viruses........................................................................................................11 Marker Rescue............................................................................................................11 DNA Sequence Analysis............................................................................................11 Electron Microscopy...................................................................................................12 Recombinant DNA Clones.........................................................................................13 Antibodies...................................................................................................................13 Purification of Virus Particles....................................................................................14 Western Blots..............................................................................................................14 Protein Pulse Label and Pulse Chase Assays.............................................................15 Transcription by Permeabilized Virions.....................................................................15 3 RESULTS...................................................................................................................16 Mapping of the Mutations..........................................................................................16 Sequencing Data.........................................................................................................17

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v Protein Synthesis........................................................................................................22 Protein Processing......................................................................................................22 Electron Microscopy...................................................................................................24 Immunodetection of A28 Protein...............................................................................28 Virion Transcription...................................................................................................30 Particle to Infectivity Ratio.........................................................................................30 4 DISCUSSION.............................................................................................................33 LIST OF REFERENCES...................................................................................................39 BIOGRAPHICAL SKETCH.............................................................................................42

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vi LIST OF FIGURES Figure page 1. Vaccinia virus life cycle................................................................................................... 3 2. Vaccinia virus morphogenesis.........................................................................................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.......................................................................................31

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vii APPENDIX KEY TO SYMBOLS aa amino acid Ccarboxy oC degrees centigrade DNA deoxyribonucleic acid h hour kB kilobase kD kilodalton M molar l microliter min minute mL milliliter mm millimeter mRNA messenger ribonucleic acid m.o.i. multiplicity of infection Namino nm nanometer OD optical density ORF open reading frame PCR polymerase chain reaction RNA ribonucleic acid

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viii Abstract of Dissertation Pres ented 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 CHARAC TERIZATION OF TEMPERATURE SENSITIVE VACCINIA VIRUS MUTANTS Cts6 AND Cts9 By Bradley Paul Dilling May 2004 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 mu tants 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 repor t we describe two temperature sensitive mutants (Cts6 and Cts9) co mprising a single complementation group. Cts6 and Cts9 were mapped to the A28L gene by marker rescue. DNA sequence analysis of the mutant A28L gene reveal s a C-terminal frameshift mutation in both mutants, predicting that both en code C-terminal truncated prot eins. Western blot analysis with A28L specific polyclonal antibody shows that the A28L protei n is contained in virions. Metabolic labeling of proteins in infected cells, and pulse chase analysis of labeled proteins, shows that viral protein sy nthesis and proteolytic processing of virion precursor proteins in Cts6 and Cts9 infected cells is indist inguishable from a wild type virus infection at the nonpermissive temperatur e. Electron microscopic analysis of virus

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ix infected cells shows that wild type and mutant virus infectio ns were indistinguishable at both the permissive and nonpermissive temperatur es. In an in vitr o transcription assay purified mutant particles produced at the nonpermissive temperature were able to transcribe at the same level as wild t ype. 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 st udy of Cts6 and Cts9 will lead to a further understanding of vaccinia virus cell entry.

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1 CHAPTER 1 INTRODUCTION General Vaccinia Background Vaccinia virus is a member of the Poxviridae family. It contains a large, doublestranded 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 fo r studying mRNA synthesis and pr ocessing. Vaccinia virions have a smooth, rounded recta ngular appearance, and meas ure about 350 x 270 nm under cryoelectron microscopy. The intracellular mature virion (IMV) is the most abundant infectious particle, composed of a dum bbell-shaped core, surr ounded by a lipoprotein bilayer. The core contains the viral genom e, enzymes required for transcription and replication in the cytoplasm, and additional proteins thought to have structural roles (Moss, 2001). This unique structure of vacci nia 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 vi rions are well suited to mediate transport among hosts, but are not well suited for spreadin g the virus within the host because of its

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2 vulnerability to complement and antibody. Ot her forms that exist are intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV), and extracellular enveloped virus (EEV) (Smith et al., 2002). Thes e 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 ar e only released after cell lysis. The IMVs are thought to enter the cell by fusion with the pl asma 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 follo wed 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 identifyi ng 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 core s is blocked by preven tion of transcription or translation, suggestin g the requirement for a virus-induced or virus-encoded protein. A putative 23-kD protein with tr ypsin-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 ar e transcribed, including DNA polymerase, RNA polymerase, growth factors, immune defense molecules, and intermediate transcription

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3 Figure 1. Vaccinia virus life cycle. One of the inf ectious forms of virus attaches to the cell, penetrates the membrane, and releas es the core into the cytoplasm (1). The cores synthesize early mRNAs that are translated into a variety of proteins including intermediate tran scription factors and DNA replication factors (2). The virion uncoats (3), the DNA genome is replicated (4), and intermediate genes are e xpressed (5). Intermediate genes encode late transcription factors, allowing for late gene transcription (6). Late genes encode early transcription factors, en zymes, and virion st ructural proteins. Newly assembled virions (7) undergo ma turation (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 re sults in the release of extracellular enveloped virions (10).

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4 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 par ticles. The purified vi rus particles can be permeabilized with neutral detergent, incuba ted with nucleoside triphosphates, and they will produce authentic fully modified early mRNA (Condit et al., 2002). Early Gene Expression During a vaccinia infection, gene expre ssion is regulated at the level of transcription. There are thr ee classes of gene expression: early, intermediate, and late. Each gene class has its own di stinct promoters and cognate trans -acting factors, mostly virus-coded. The regulation of vaccinia gene s can be thought of as a “cascade” because the factors required for initiati on of each gene class are enco ded primarily by the genes of the preceding class. A virus-coded, multi-s ubunit, 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 ei ght 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 pr omoters (Condit et al., 2002). DNA Replication As previously discussed, virus uncoati ng takes place after early genes are transcribed. Many early gene pr oducts are factors required fo r replication of the viral genome. One of the most unique aspects of poxviruses is that DNA replication occurs entirely in the cytoplasm of th e infected cell, a characteristic that is only shared with African swine fever virus. A discrete cytopl asmic area of replication free of cellular

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5 organelles (termed factory ar eas, or virus factories) has been located using light and electron microscopy. Some of the important virus-encoded DNA rep lication proteins are the DNA polymerase, nucleoside triphosphatase DNA glycosylase, B1 R protein kinase, and DNA ligase. DNA replication begins 1 to 2 hours after infection, an d results in about 10,000 copies of the genome, about half of wh ich are packaged into virions. Vaccinia virus contains a unique genome structure: a double stranded genom e with covalently closed inverted terminal re petitions. Currently there ar e still large gaps in the understanding of its DNA replic ation. The accepted model for replication proposes that vaccinia replicates through concatemer intermediates, with the hairpin termini having an essential role. Replication of th e 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 refe rred 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 pol ymerase. 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 Fact ors. Late genes also require one or more host proteins. The products of late gene s include RNA polymerase, immune defense proteins, early gene transcri ption factors, poly(A) polymer ase, and virion morphogenesis proteins. Intermediate and late gene mRNA s are thought to be capped and polyadenylated by the same enzymes responsible for modifying early mRNAs (Condit et al., 2002). After

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6 the late genes are expressed and the DNA concatemers have been resolved, assembly of new virus particles begins (discusse d in detail in the next section). Morphogenesis Investigations of morphoge nesis using electron microsc opy have reported that the formation of new virions takes place in the vi rus factories that we re discussed earlier. Figure 2 is an overview of vacci nia morphogenesis. The first vi sible structures to appear are called crescents, which are composed of lip id and virus coded protein (Smith et al., 2002). There is ongoing debate about the orig ins 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 e ndoplasmic reticulum and the Golgi complex (Sodeik et al., 2002). The de novo model for membrane synthesis contradicts the principle that membranes grow from ex isting membranes. In contrast, the double membrane theory fits what we do know a bout membrane biosynthesis (Smith et al., 2002). Crescents continue to mature in spheri cal 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 matr ix 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. Partic les with nucleoids are termed immature virions with nucleoid (IVN). The packing of DNA into th e 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.

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7 Figure 2. Vaccinia virus morphogenesis begins w ith the appearance of the viroplasm or virus factory followed by the emerge nce of crescents (1). The crescents mature and into IVs (2) and are pack ed 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 m ove 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 rel eased from the cell as EEV (8).

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8 The cleavage of these proteins triggers the transition from the spherical IVN to the brickshaped Intracellular mature virus (IMV), and the generation of the di stinct 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 intracel lular enveloped virus (IEV). The movement of the IMV from the virus factory to wher e they are wrapped in additional membranes this is thought to be dependent upon A27 pr otein 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 th e help of the F12 prot ein, 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 th e outer envelope fu ses 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 formati on of actin tails (van Eijl et al., 2000). Along with the A36 protein th e F12, A33, A34, and B5R proteins are all involved in actin tail formati on. 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 th e CEV still at its tip, alternatively the CEV

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9 may be released to give Extracellular enve loped virus (EEV). Both the EEV and the CEV form of the virus contain 3 membra nes surrounding the core. A33, A34, A36, and B5R proteins are also involve d in the release of the EEV. Although EEV represents only a small fraction of virus infec tivity, it is biologically very important for long-range virus spread. Not only does the EEV form have a highe r 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 mutant s 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 de fect is able to produce crescents, however, the mutant forms aberrant, empty IVs (Chiu et al., 2002). A ts mutant with a mutation in the I8 gene produces non-infectious IMVs (Fathi et al., 1991). The Problem A collection of 65 temperature sensitive vacc inia 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 perfor med 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 s ynthesize DNA and proteins at the nonpermissive temperature. The results of these tests produced four phenotypes: (1) normal, which contains mutants that were able to synthe size DNA and early and late proteins at the

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10 same level as wild type virus. (2) DNA-ne gative are mutants that cannot synthesize DNA and are only able to synthesi ze early proteins. (3) Defective late mutants display normal DNA, and early protein synthesis, but late pr otein synthesis is either weak, delayed, or both. (4) Abortive late mutant s are also able to synthe size DNA and early proteins, however, their late protein synt hesis begins and later aborts Within this collection of mutants is a complementation group that contai ns Cts6 and Cts9, whic h display wild type DNA and protein synthesis, and thus were plac ed in the normal ca tegory (Condit et al., 1983). Mutants that are classified as “norma l” usually are defective in morphogenesis. Cts6 was mapped by marker rescue, using a cosm id clone library, to the right half of the A fragment of the HindIII restriction map (T hompson et al., 1986). The purpose of this research project is to continue the prelimin ary characterization of this complementation group to obtain a better understa nding of viral morphogenesis. This includes the genetic mapping of the mutants to a single open r eading frame and sequencing this gene to determine the nature of the mutations. A phenotypic characterization will also be described which includes protein synthesi s and processing, virion transcription and infectivity, and protein detection in inf ected cells and purified virions. Electron microscopy analysis of the mutants is also examined in order to get a detailed look at their morphogenesis.

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11 CHAPTER 2 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 cu lture, virus infection, and plaque titration have been previously described (Condit et al., 1 981;Condit et al., 1983). The non-permissive temperature for mutant infecti ons was maintained between 39.5oC and 40oC, which will be labeled in figures as 40oC for convenience. Marker Rescue Marker rescue was performed as prev iously described (Meis et al., 1991; Thompson et al., 1986). The cosmid clones us ed 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 contro ls. 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, A 27L-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.

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12 Total infected cell DNA was isolated using the Qiagen Dneasy miniprep spin columns (Qiagen) according to the manuf acturer’s instructions for isola tion 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 Performan ce Plasmid Maxiprep Kit (Sigma) following the manufacturer’s instructions for vac uum format. All DNA mentioned above was sequenced by the University of Flor ida ICBR DNA Sequencing Core Laboratory. Electron Microscopy Confluent monolayers of BSC40 cells on 60 mm dishes were inf ected with wt or mutant viruses at an m.o.i. of 10. Th e cells were incubated at either 31 oC or 40 oC for 24, 36, or 48 h. At the appropriate time postinfect ion 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 rock ing. 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, postfixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol solutions and 100% acetone, and embedded in Embed-812 resi n mix. En bloc staining was done using uranyl acetate solution in 75% ethanol. After thin sectioning, the samples were post stained in uranyl acetate a nd Reynolds lead acetate. After mounting the samples were viewed under a Hitachi H7000 transmission electron micros cope. Fixating, embedding, thin sectioning, staining, and microscopy were done with the assistance of the University of Florida ICBR Electron Mi croscopy Core Laboratory.

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13 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 NdeI site at the 5’ end and a BamHI s ite 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 NdeI and BamHI, the fragment containing A28L was isolated and cloned into NdeI-Bam HI cut pET-16b (Novagen) making pET-16bA28L. Antibodies The pET-16b-A28L was used for the bact erial overexpression of polyhistidinetagged A28 according to the pET system ma nual (Novagen). Briefly, Rosetta(DE3)pLysS containing pET-16b-A28L were grown in 50 mL LB containing 34 g/mL ampicillin and 34 g/mL chloramphenicol at 37C overnight We used 25 mL of the preculture to inoculate a 500 mL culture that was grown at 37C. After the OD590 of the culture reached 0.6, the culture was induced by the a ddition of IPTG (0.1M stock) to a final concentration of 1mM and incubated for 4 hours at 37C. Bact erial cells were sedimented, and the pellets were resuspende d in 20 mL BugBuster Protein Extraction Reagent (Novagen) and incubated at room temperature for 10 mi n. After freezing and thawing the sample was treated with 125 un its of RQ1 RNase-Free DNase (Promega). The inclusion bodies were then isolated and washed according to Antibodies (Harlow et al., 1988). The inclusion body preparation was th en sent to Strategic Biosolutions for polyclonal antibody production us ing the standard two rabbi t 70-day protocol. This antibody will be referred to as the anti-pro tein antibody for convenience. An anti-peptide antibody was also created by Sigma Genosys. This antibody was made from the peptide

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14 DRRVQDVNDTISDVKQKWRC, which spans ami no 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 sedime ntation through preformed discontinuous sucrose density gradients as previously desc ribed (Joklik, 1962b). Pu rified virions were quantified by optical density at 260 nm (1 OD260 = 64 g virus). Infectivity of the purified virus was assayed by pla que titration on BSC40 cells at 31 oC or 40 oC. Western Blots After separation of proteins on 12% SDS-PAGE or 10-20% linear gradient precast (Bio-Rad) gels, the proteins were transferre d to nitrocellulose membrane (Bio-Rad) at 100V for 1hr. The blots were then blocked ove rnight 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-block ed 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) a nd washed once for 5 min in TBS. The blots were then incubated with the anti-pe ptide 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 qui ckly, one time for 10 min, and twice for 5 min each in TBST-T. The blots were then inc ubated 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 TB ST-T one time quickly, one time for 10 min, and twice for 5 min. Two subsequent washes w ith TBST for 5 min were performed before

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15 detection with ECL Western Blotting Detect ion Reagents (Amersham Biosciences) and exposure to X-ray film. Protein Pulse Label and Pulse Chase Assays Protein pulse labeling of in fected cells with trans-35S labeled methionine (ICN Biochemical), sodium dodecyl sulfate-polya crylamide gel electrophoresis (SDS-PAGE), and audoradiography were done as previously described (Condit et al., 1981). In protein pulse chase experiments the pulse-labeled ce lls were washed and incubated in serum containing medium for various times. At tim es 0, 3, 6, 9,12 hrs the cells were harvested and analyzed as described above. Transcription by Permeabilized Virions Permeabilized virion transcription experime nts 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-HCl 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 32P-CTP (3000 Ci/mmol Perkin Elmer) and 0.3 OD260 of virus were incubated at 30oC, and 50 l aliquots were precipitated directly with 5% TCA. The virion associated versus re leased RNA was measured using identical reactions except the 50 ul aliquots were diluted into 200 ul stop solution (50 mM TrisHCl pH 8.0, 10 mM DTT, 10 mM EDTA, 0.05 % NP40) and centrifuged for 3 min in a microfuge. The supernatants (released RNA) we re then removed and precipitated in 5% TCA. The pellets (core associated RNA) were resuspended in 0.2mL of a buffer containing 50 mM Tris-HCl, pH 8.0, 0.1% SDS, and precipitated with 5% TCA. TCA perceptible radioactivity was determ ined by liquid scintillation counting.

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CHAPTER 3 RESULTS Mapping of the Mutations The temperature sensitive vaccinia virus mutants Cts6 and Cts9 comprise a complementation group that has a “normal” phe notype, that is, they display wild type protein and DNA synthesis (Condi t et al., 1983). Not only were these mutants shown to be in the same gene by complementation analys is, 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 (Thomps on et al., 1986). Further preliminary mapping (R. Condit, personal communica tion) showed that the mutation could be narrowed down to a 5 kB region of the genome. This appro ach 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 A 25L – 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 di srupted into several ORFs and is not functional. Because the A-type inclusion is lik ely 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.

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17 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 we re 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 resc ue with any of the PCR products (data not shown). The next strategy taken was to generate overlapping P CR products that incor porated two or more genes. PCR products were made that speci fically amplified A 25L-A26.4L, A25L-A27L, A27L-A28L, A27L-A29L, and A28L-A29L OR Fs. These PCR products along with the single ORF products were used in a one-step ma rker 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 contai ning 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 orientat ion of A27L and A28L this result suggests that the mutations must be at the 5’ e nd of A27L or at th e 3’ end of A28L. Sequencing Data 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 nucleotides of the 438-nucleotide A28L gene, which in the wild type are both cyto sine 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

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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 i ndicated above each gene. The middle of the figure shows a schematic re presentation of a subset of PCR products used in the rescue of Cts6. The gene(s) included in each PCR product are i ndicated 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 corres ponding PCR product labeled below the dish.

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19

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20 1 50 Cts6-A28L (1) ATGAACTCTCTATCAATTTTTTTTATTGTGGTAGCGACGGCTGCGGTGTG Cts9-A28L (1) ATGAACTCTCTATCAATTTTTTTTATTGTGGTAGCGACGGCTGCGGTGTG wt-A28L (1) ATGAACTCTCTATCAATTTTTTTTATTGTGGTAGCGACGGCTGCGGTGTG 51 100 Cts6-A28L (51) TTTACTTTTTATCCAGGGTTACTCAATATATGAAAATTATGGCAATATTA Cts9-A28L (51) TTTACTTTTTATCCAGGGTTACTCAATATATGAAAATTATGGCAATATTA wt-A28L (51) TTTACTTTTTATCCAGGGTTACTCAATATATGAAAATTATGGCAATATTA 101 150 Cts6-A28L (101) AGGAATTTAATGCTACTCATGCAGCATTCGAATATTCAAAATCTATAGGT Cts9-A28L (101) AGGAATTTAATGCTACTCATGCAGCATTCGAATATTCAAAATCTATAGGT wt-A28L (101) AGGAATTTAATGCTACTCATGCAGCATTCGAATATTCAAAATCTATAGGT 151 200 Cts6-A28L (151) GGAACACCGGCATTAGATAGGAGAGTTCAAGATGTCAACGACACAATTTC Cts9-A28L (151) GGAACACCGGCATTAGATAGGAGAGTTCAAGATGTCAACGACACAATTTC wt-A28L (151) GGAACACCGGCATTAGATAGGAGAGTTCAAGATGTCAACGACACAATTTC 201 250 Cts6-A28L (201) TGATGTAAAGCAAAAGTGGAGATGTGTGGTTTATCCAGGAAACGGTTTTG Cts9-A28L (201) TGATGTAAAGCAAAAGTGGAGATGTGTGGTTTATCCAGGAAACGGTTTTG wt-A28L (201) TGATGTAAAGCAAAAGTGGAGATGTGTGGTTTATCCAGGAAACGGTTTTG 251 300 Cts6-A28L (251) TATCCGCTTCCATATTTGGATTTCAGGCAGAAGTTGGACCCAATAATACT Cts9-A28L (251) TATCCGCTTCCATATTTGGATTTCAGGCAGAAGTTGGACCCAATAATACT wt-A28L (251) TATCCGCTTCCATATTTGGATTTCAGGCAGAAGTTGGACCCAATAATACT 301 350 Cts6-A28L (301) AGATCCATTAGAAAATTTAACACGATGCAACAATGTATAGACTTTACATT Cts9-A28L (301) AGATCCATTAGAAAATTTAACACGATGCAACAATGTATAGACTTTACATT wt-A28L (301) AGATCCATTAGAAAATTTAACACGATGCAACAATGTATAGACTTTACATT 351 400 Cts6-A28L (351) TTCTGATGTTATTAACATCAATATTTATAATCCATGTGTTGTA--AAATA Cts9-A28L (351) TTCTGATGTTATTAACATCAATATTTATAATCCATGTGTTGTA--AAATA wt-A28L (351) TTCTGATGTTATTAACATCAATATTTATAATCCATGTGTTGTACCAAATA 401 438 ^^ Cts6-A28L (399) TAAATAACGCAGAGTGTCAGTTTCTAAAATCTGTACTT Cts9-A28L (399) TAAATAACGCAGAGTGTCAGTTTCTAAAATCTGTACTT wt-A28L (401) TAAATAACGCAGAGTGTCAGTTTCTAAAATCTGTACTT Figure 4. Nucleotide alignment of A28L from wild type, Ct s6, 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.

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21 A 1 50 Cts6-A28 (1) MNSLSIFFIVVATAAVCLLFIQGYSIYENYGNIKEFNATHAAFEYSKSIG Cts9-A28 (1) MNSLSIFFIVVATAAVCLLFIQGYSIYENYGNIKEFNATHAAFEYSKSIG wt-A28 (1) MNSLSIFFIVVATAAVCLLFIQGYSIYENYGNIKEFNATHAAFEYSKSIG 51 100 Cts6-A28 (51) GTPALDRRVQDVNDTISDVKQKWRCVVYPGNGFVSASIFGFQAEVGPNNT Cts9-A28 (51) GTPALDRRVQDVNDTISDVKQKWRCVVYPGNGFVSASIFGFQAEVGPNNT wt-A28 (51) GTPALDRRVQDVNDTISDVKQKWRCVVYPGNGFVSASIFGFQAEVGPNNT 101 146 Cts6-A28 (101) RSIRKFNTMQQCIDFTFSDVININIYNPCVVKYK-----------Cts9-A28 (101) RSIRKFNTMQQCIDFTFSDVININIYNPCVVKYK-----------wt-A28 (101) RSIRKFNTMQQCIDFTFSDVININIYNPCVVPNINNAECQFLKSVL ^ B Figure 5. Amino acid sequence alignment of A28 protein from wild type, Cts6, and Cts9 (A). The first 131 amino acids enc oded 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 trunca tion. (B) Wild type and mutant A28 proteins. A transmembrane domain, i ndicated by a red box and labeled TM, is located between the 4th and 26th amino acids. The green box at the Cterminal end of the mutant protein repr esents the three substitutions encoded by the mutant viruses, which begins at aa 132. The Cs inside the proteins represent conserved cysteines (see discussion).

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22 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 frames hift and after the first 131 aa ar e translated correctly 3 aa are substituted before a stop codon is reached. Protein Synthesis Cts6 and Cts9 have been previously s hown 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 mu tants 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-l abeled at various times postinfection with 35S-methionine. The proteins were then separated by SDS-PAGE and detected by autoradiography (Fig. 6). The wt vi rus represents normal protein synthesis at 31oC or 40oC. Both Cts6 and Cts9 show protein s ynthesis that looks like wild type. Cell host proteins are seen at 0 h post-infecti on and are subsequently shut off by 3 h postinfection with the appearance of early proteins Late viral protein synthesis is seen by 6 h post-infection and persists throughout the e xperiment. The mutants displayed a normal pattern of protein synthesis, however, some of the films appe ar lighter due to a technical problem during exposure. Protein Processing The processing of several viral proteins is associated with morphogenesis from IVN to IMV (Klemperer et al., 1997). Virus morphogenesis is arrest ed 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). Ther efore, virion protein pro cessing is a good marker for detecting defects in virus asse mbly. In order to test whethe r the major core polypeptides

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23 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 al ong 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.

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24 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, ha rvested, 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 a nd 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 th e infected cell monolayer. However the appearance of the 60 and 62 kD ba nds are strong evidence that the proteins are being processed normally. Electron Microscopy 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, BSC40 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 31oC and 40oC. All stages of vaccinia virus morphogenesis were observed including vira l crescents, immature virions, immature virions with nucleoids, and intracellular mature virions.

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25 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% SDSPAGE and autoradiographed. The auto radiograms are shown. The infecting virus is indicated along the bottom, and temperature of incubation is indicated at the left. Approximate mo lecular weights, in kilodaltons, are shown to the right of the gels. The lengt h of the chase is indicated, in hours, above each lane.

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26 A B Figure 8. Electron micrograph pictures of cells inf ected with Cts6 (A) or wt (B) at 40 oC. Cells were infected with an m .o.i. of 10 and processed for electron microscopy 24 h postinfection. Arrows i ndicate examples of the different stages of morphogenesis: cresents (C ), immature virions (IV), immature virions with nucleoid (IVN), and in tracellular mature virion (IMV).

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27 A B Figure 9. Electron micrograph pictures of cells inf ected with Cts9 (A) or wild type (B) at 40 oC. Cells were infected with an m .o.i. of 10 and processed for electron microscopy at 24 h postinfection. The a rrow points to an example of an intracellular mature virion (IMV).

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28 Immunodetection of A28 Protein To further characterize the mutants, they were examined by western blot using A28 specific antibodies. Two antibodies were us ed in these western blots, an anti-peptide antibody and an anti-protein an tibody, 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 dete cted 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 w ith a 1:1000 dilution of the anti-peptide antibody, then incubated with a secondary anti-rabbit antibody and finally detected with ECL detection reagent and autoradiography (Fig. 10 B). A band the correct size was detected in purified virions in all three viru ses. 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 al so seemed to be much less than wild type.

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29 Figure 10. Immunodection of A28 protein. (A) We stern blot of infected cell time course. Proteins were separated on 12 % SDS-PAGE and detected with antiprotein antibody. The infecting virus a nd time harvested after infection, in hours, is indicated above gel. Approx imate molecular weight marker, in kilodaltons, is shown on the left. (B) West ern blot of purified virus. Proteins were separated on a 10-20% gradient gel and detected with anti-peptide antibody. The purified virus is indica ted above the gel and approximate molecular weight, in kiloda ltons, is shown on the left.

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30 Virion Transcription Purified vaccinia particles can be treate d with NP40 and DTT to permeabilize them by removing the outer membrane, but leaving the enzyme-containing core structurally and functionally intact. These permeablili zed 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 32P-CTP at 30oC for various times. At each time point the cores were spun down and the supernatant, which contained released transcripts, was TC A precipitated, filtered, and count ed 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, howeve r 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 31oC 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 inf ectivity ratio of 39.2 particles/plaque forming unit. Cts6 has a ratio of 11,200 particles/pla que forming unit. Cts9 has a ratio of 10,000 particles/plaque forming unit. This suggests that mutant vi ruses are almost 250 fold less infectious as the wild type virus.

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Figure 11. In vitro transcription activity of wild type (A), Ct s6 (B), and Cts9 (C) purified virions. Purified vaccinia virus particles were incubated for various times, indicated on the x axis, w ith detergent and ribonucleside triphospahtes The incorporation of 32P-CTP into RNA was determined, which is indicated on the y axis.

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32 A 0 20 40 60 80 100 120 140 0204060 Time (min)Incorporation CMP (pmole) Core Release B 0 20 40 60 80 100 120 140 0204060 Time (min)Incorporation CMP (pmole) Core Release C 0 20 40 60 80 100 120 140 0204060 Time (min)Incorporation CMP (pmole) Core Release

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33 CHAPTER 4 DISCUSSION Temperature sensitive (ts) vaccinia muta nts have played a crucial role in the understanding of vaccinia virus morphogenesis. Ts mutants have implicated many genes involved in viral morphogene sis including H5 (Demasi et al., 2000), F10 (Wang et al., 1995), J1 (Chiu et al., 2002), and I6 (Grubi sha 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 prot eins at wild type levels (Condit et al., 1983), which makes them candidates for defects in viral morphogenesis. The genetic mapping of the mutants to a single open reading frame was the first goal of this project. Cts6 a nd Cts9 have been previously mapped by marker rescue to the right side of the Hi ndIII A fragment (Thompson et al ., 1986). R. Condit further mapped Cts6 and Cts9, using PCR products generate d with primers donated by R. Moyer and B. Luttge, to a 5 kB region of th e 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 disr upts 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

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34 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 35 kD subunit of the RNA polymerase. A 29L was also thought not to contain the mutation because Cts6 and Cts9 were shown to display normal pr otein synthesis (Condit et al., 1983), while an RNA polymerase mutant is defective in the synthesis of late viral mRNA and proteins (Hooda-Dhi ngra et al., 1989). The two ca ndidate 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 morphogene sis (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 uns uccessful. 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, sugge sting 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 a ll 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

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35 stock used in the mutagenesis. The mutations comprise deletion of the 394th and 395th nucleotides of the A28L gene. A28L is pr esent 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 th e 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 muta nt A28 protein results in a C-terminally truncated protein. The mutations cause a fram eshift after the firs t 131 aa are translated there are three aa substitutions before a stop codon is reached. As discussed earlier, Cts6 and Cts9 ha ve a preliminary phenotype of “normal,” which means they are able to synthesize DNA a nd 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 me thods, was performed on the mutants. Cts6 and Cts9 display a normal patter n of protein synthesis, conf irming the preliminary protein synthesis results. Because late protein synt hesis requires DNA replication (Moss, 2001) these results are sufficient to conf irm the normal DNA synthesis phenotype. Viral protein processing of core protei ns 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 th e mutants for their ability to process these core proteins. Although we were anticipating the mutants to be defective, they displayed wild type levels of protei n processing. These results s uggest that any morphogenesis defect present in Cst6 and Cts9 ex ists after the formation of IMVs.

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36 Because the mutants are able to process vi ral core proteins we wanted to examine the viral particles under an electron microsco pe 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 stag es of viral morphogenesis at the nonpermissive temperature. Mutant IMVs made at the nonpermissive te mperature were then purified for further study. Purified particles and infected cells we re checked for the presence of the A28 protein. Cts6 and Cts9 have th e same protein expression pattern of A28 as wild type virus in infected cells. The A28 prot ein 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 poxvi rus late genes (Davison et al., 1989). A28 contains an early transcription termination seque nce, 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 th e A28 protein into the virion, however, less protein is present than in wild type virions. Because the mutants ca nnot package A28 at wild type levels they may have a morphogenesis defect during assemb ly. 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 transcript ion 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 nuc leoside triphosphates and they will produce

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37 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 evid ence that although the muta nt particles appear normal they may not be assembled correctly re sulting in slightly dimi nished 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 investig ators 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 ma de at the nonpermissive temperature were assayed to see if they are able to infect cells at the permissi ve 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 temperat ure 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) pub lished 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 extracel lular virions. Further investiga tion revealed A28L to be a substrate of the viral disulfide bond formati on enzyme and other aspects that our study did not address. A28 was shown to be anc hored in the IMV membrane by a N-terminal transmembrane domain, while the C-terminus is exposed on the surface and is predicted

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38 to contain two intramolecular disulfide bonds at four conserved cyst eines. 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 r ounding and cytopathic effects. A28 deficient virions have a defect in pe netration and membrane uncoati ng. The A28 deficient 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 ar e 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 th e 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 vi rus entry, and A28 is required for virusinduced 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 pr otein that has been implicated in cell fusion (Vazquez et al ., 1998). A27 and A28 may work together in fusion and entry, however, A27-deficient IMV reta in nearly complete (Hsiao et al., 1999) or partial (Vazquez et al., 1999) infectivit y. 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, A 28 is an essential vaccinia protein that functions in cell fusion and entry.

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39 LIST OF REFERENCES Byrd, C. M., Bolken, T. C., and Hruby, D. E. (2002). The vaccinia vi rus I7L gene product is the core protein proteinase. J.Virol. 76 8973-8976. Chiu, W. L. and Chang, W. (2002). Vaccinia vi rus J1R protein: a viral membrane protein that is essential for virion morphogenesis. J.Virol. 76 9575-9587. Condit, R. C. and Motyczka, A. (1981). Isol ation 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. Virology 128 429443. Condit, R. C. and Niles, E. G. (2002). Regul ation 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.Virol. 39 733-745. Dales, S. and Mosbach, E. H. (1968). Vacci nia as a model for membrane biogenesis. Virology 35 564-583. Davison, A. J. and Moss, B. (1989). Struct ure of vaccinia virus late promoters. J.Mol.Biol. 210 771-784. Demasi, J. and Traktman, P. (2000). Cluste red charge-to-alanine mutagenesis of the vaccinia virus H5 gene: isolation of a domin ant, temperature-sensitive mutant with a profound defect in morphogenesis. J.Virol. 74 2393-2405. Fathi, Z. and Condit, R. C. (1991). Phenot ypic characterization of a vaccinia virus temperature-sensitive complementati on group affecting a virion component. Virology 181 273-276. Gershowitz, A. and Moss, B. (1979). Abortive tr anscription products of vaccinia virus are guanylylated, methylate d, and polyadenylylated. J.Virol. 31 849-853. Grimley, P. M., Rosenblum, E. N., Mims, S. J., and Moss, B. (1970). Interruption by Rifampin of an early stage in vaccinia virus morphogenesi s: accumulation of membranes which are precursors of virus envelopes. J.Virol. 6 519-533.

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40 Gross, C. H. and Shuman, S. (1996). Vaccinia virions lacking the RNA helicase nucleoside triphosphate phosphohydrolase II ar e defective in early transcription. J.Virol. 70 8549-8557. Grubisha, O. and Traktman, P. (2003). Genetic analysis of the vaccini a virus I6 telomerebinding protein uncovers a key role in genome encapsidation. J.Virol. 77 10929-10942. Harlow, E. and Lane, D. (1988). "Antibodies : a laboratory manual." Cold Spring Harbor Laboratory. Hassett, D. E., Lewis, J. I., Xing, X., DeLange L., and Condit, R. C. (1997). Analysis of a temperature-sensitive vaccinia virus mu tant in the viral mRNA capping enzyme isolated by clustered charge-to-alanine mu tagenesis and transient dominant selection. Virology 238 391-409. Hooda-Dhingra, U., Thompson, C. L., and C ondit, R. C. (1989). Detailed phenotypic characterization of five temperature-sens itive mutants in the 22and 147-kilodalton subunits of vaccinia virus DNAdependent RNA polymerase. J.Virol. 63 714-729. Hsiao, J. C., Chung, C. S., and Chang, W. ( 1999). Vaccinia virus envelope D8L protein binds to cell surface chondroitin sulfate a nd mediates the adsorp tion of intracellular mature virions to cells. J.Virol. 73 8750-8761. Ink, B. S. and Pickup, D. J. (1989). Transcri ption of a poxvirus early gene is regulated both by a short promoter element and by a tran scriptional 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 purificati on fo four strains of poxvirus. Virology 18 9-18. Kane, E. M. and Shuman, S. (1993). Vacci nia virus morphogenesis is blocked by a temperature-sensitive mutation in the I7 gene that encodes a virion component. J.Virol. 67 2689-2698. Klemperer, N., Ward, J., Evans, E., and Tr aktman, P. (1997). The vaccinia virus I1 protein is essential for the assembly of mature virions. J.Virol. 71 9285-9294. Latner, D. R., Xiang, Y., Lewis, J. I., Cond it, 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 pos itive 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 vaccinia virus gene which renders the virus dependent on isatin-beta-thiosemicarbazone (IBT). Virology 182 442-454.

PAGE 50

41 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, Philadelphia. Sanderson, C. M., Hollinshead, M., and Smit h, G. L. (2000). The vaccinia virus A27L protein is needed for the mi crotubule-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 w ith intramolecular disulfide bonds formed by the viral cytoplasmic redox pathway. J.Virol. 78 2348-2356. 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.Virol. 78 2357-2366. Smith, G. L., Vanderplasschen, A., and Law, M. (2002). The formation and function of extracellular enveloped vaccinia virus. J.Gen.Virol. 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 Microbiol. 10 15-24. Thompson, C. L. and Condit, R. C. (1986). Marker rescue mapping of vaccinia virus temperature-sensitive mutants using overlapp ing cosmid clones representing the entire virus genome. Virology 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 ex tracellular enveloped virus particles. Virology 271 26-36. Vazquez, M. I. and Esteban, M. (1999). Iden tification of functional domains in the 14kilodalton envelope protein (A27L) of vaccinia virus. J.Virol. 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 (A17L) virus membrane protein through a C-terminal alpha-helix. J.Virol. 72 10126-10137. Wang, S. and Shuman, S. (1995). Vaccini a virus morphogenesis is blocked by temperature-sensitive mutations in the F 10 gene, which encodes protein kinase 2. J.Virol. 69 6376-6388.

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42 BIOGRAPHICAL SKETCH Bradley Dilling was born in State College, Pennsylvania in 1979, and moved to Cape Coral Florida in 1986, where he gra duated 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 receive d 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.


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MAPPING AND PHENOTYPIC CHARACTERIZATION OF TEMPERATURE
SENSITIVE VACCINIA VIRUS MUTANTS Cts6 AND Cts9














By

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


2004































Copyright 2004

by

Bradley Paul Dilling
















ACKNOWLEDGMENTS

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


page


ACKNOWLEDGMENT S ................. ................. iii........ ....


LIST OF FIGURES .............. ....................vi


KEY TO SYMBOLS ........................... .......vii


AB S TRAC T ......_ ................. ..........._..._ viii..


CHAPTER


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


Figure pg

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















APPENDIX
KEY TO SYMBOLS

aa amino acid

C- carboxy

oC degrees centigrade

DNA deoxyribonucleic acid

h hour

kB kilobase

kD kilodalton

M molar

Cl1 microliter

mmn minute

mL milliliter

mm millimeter

mRNA messenger ribonucleic acid

m.o.i. multiplicity of infection

N- amino

nm nanometer

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

By

Bradley Paul Dilling

May 2004

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.















CHAPTER 1
INTTRODUCTION

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).

DNA Replication

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).

Morphogenesis

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.


























J


IMV


IVN


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).


Viroplas m


SCrescents









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).

The Problem

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

their morphogenesis.














CHAPTER 2
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

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.

Electron Microscopy

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-

A28L.

Antibodies

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.

Western Blots

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.















CHAPTER 3
RESULTS

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.

Sequencing Data

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
the dish.










h Q\ 00
NNN


trI CO N 00
NNMNNN





1Q 1


0..e









r...
Soo


--N M rr 4 t


















1 50
Cts6-A28L (1) ATGAACTCTCTATCAATTTTTTTTATTGTGGTAGCGACGGCGGTG
Cts9-A28L (1) ATGAACTCTCTATCAATTTTTTTTATTGTGGTAGCGACGGCGGTG
wt-A28L (1) ATGAACTCTCTATCAATTTTTTTTATTGTGGTAGCGACGGCGGTG
51 100
Cts6-A28L (51) TTTACTTTTTATCCAGGGTTACTCAATATATGAAAATATGGCATAT
Cts9-A28L (51) TTTACTTTTTATCCAGGGTTACTCAATATATGAAAATATGGCATAT
wt-A28L (51) TTTACTTTTTATCCAGGGTTACTCAATATATGAAAATATGGCATAT
101 150
Cts6-A28L (101) AGGAATTTAATGCTACTCATGCAGCATTCGAATATTCAAAACTGT
Cts9-A28L (101) AGGAATTTAATGCTACTCATGCAGCATTCGAATATTCAAAACTGT
wt-A28L (101) AGGAATTTAATGCTACTCATGCAGCATTCGAATATTCAAAACTGT
151 200
Cts6-A28L (151) GGAACACCGGCATTAGATAGGAGAGTTCAAGATGTCAACGAAATC
Cts9-A28L (151) GGAACACCGGCATTAGATAGGAGAGTTCAAGATGTCAACGAAATC
wt-A28L (151) GGAACACCGGCATTAGATAGGAGAGTTCAAGATGTCAACGAAATC
201 250
Cts6-A28L (201) TGATGTAAAGCAAAAGTGGAGATGTGTGGTTTATCCAGGAACGTG
Cts9-A28L (201) TGATGTAAAGCAAAAGTGGAGATGTGTGGTTTATCCAGGAACGTG
wt-A28L (201) TGATGTAAAGCAAAAGTGGAGATGTGTGGTTTATCCAGGAACGTG
251 300
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
301 350
Cts6-A28L (301) AGATCCATTAGAAAATTTAACACGATGCAACAATGTATAGATACT
Cts9-A28L (301) AGATCCATTAGAAAATTTAACACGATGCAACAATGTATAGATACT
wt-A28L (301) AGATCCATTAGAAAATTTAACACGATGCAACAATGTATAGATACT
351 400
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.



































~ n


1 50
(1) MNSLSIFFIVVATAAVCLLFIQGYSIYENYGNIKEFNATHAFYKI
(1) MNSLSIFFIVVATAAVCLLFIQGYSIYENYGNIKEFNATHAFYKI
(1) MNSLSIFFIVVATAAVCLLFIQGYSIYENYGNIKEFNATHAFYKI
51 100
(51) GTPALDRRVQDVNDTISDVKQKWRCVVYPGNGFVSASIFGFAVPN
(51) GTPALDRRVQDVNDTISDVKQKWRCVVYPGNGFVSASIFGFAVPN
(51) GTPALDRRVQDVNDTISDVKQKWRCVVYPGNGFVSASIFGFAVPN
101 146
(101) RSIRKFNTMQQCIDFTFSDVININIYNPCVVKYK---------
(101) RSIRKFNTMQQCIDFTFSDVININIYNPCVVKYK---------
(101) RSIRKFNTMQQCIDFTFSDVIININYNPCVVPNINNAECQFKV


Cts6-A28
Cts9-A28
wt-A28

Cts6-A28
Cts9-A28
wt-A28

Cts6-A28
Cts9-A28
wt-A28


1 4


26


132


1 4


26


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.

Protein Synthesis

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.

Protein Processing

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


















-102
- 72


- 35

- 25




.102
- 72



- 25


9 12 03 6 912


oso~


iLr* ~IS~~-.'


Cts6


wt


Cts9


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


31oC









40oC


0 36d912


I Y,
Ir ~r

iri~ci~i~E









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.

Electron Microscopy

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


1102
72
62


0 36d912 0 3 6912 0 36d912




IllI



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.


310C









A

q :.,t.
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."" r uf-~ii~j~~ i~~~-:;;~;;~JI"-~fii:
rr
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li C'
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:I
;-~ ~ ~I .r..~~F~;i%~i ;"
rr,
~ : i f.
~
I- r
P ~~ ''
: e~ ~I rr. i
'~* ;'
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).







A






rc4~ ~ 500f nmllr)j.r~
BL (~r.~


;~M J~MV




Fiue .Elcrn irorppcurso cells infcte w itCs9()owidyp(B
at4 C el wr netdwiha O.of1 n rcssdfreeto
micrscop at 24hpsifcin h rowpit oa xml fa
inraelua maur viin(M )









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.

























EB


/ _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..


Y


1511


~r~8r~br~8r~8









Virion Transcription

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

Time (min)


^ 140

E 120
100

0 80
o~ 60

S40

0 20


-E Core
-H Release


0 20 40

Time (min)


^ 140

E 120 -
100
-
0 80 -
. 60 -

40
o 20 -

-01


-* Core
-m Release


140

o 120

- 100

S80
,o60

40

0 20

0


-+ Core
-a Release


0 20 40

Time (min)















CHAPTER 4
DISCUSSION

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

morphogenesis.

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

study .

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.
















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BIOGRAPHICAL SKETCH

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.