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Role of the Viral mRNA Capping Enzyme during Vaccinia Virus Infection

Permanent Link: http://ufdc.ufl.edu/UFE0022901/00001

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Title: Role of the Viral mRNA Capping Enzyme during Vaccinia Virus Infection
Physical Description: 1 online resource (110 p.)
Language: english
Creator: Shatzer, Amber
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: capping, infection, initiation, methyltransferase, mrna, termination, transcription, vaccinia, virus
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Eukaryotic cells and viruses cap mRNA transcripts to increase the stability and translatability of the transcripts. Vaccinia virus, the prototypic member for the poxviridae family, encodes a multifunctional, heterodimeric mRNA capping enzyme. The large subunit is encoded by D1R (97kDa) and the small subunit is encoded by D12L (33kDa). In addition to its role in mRNA capping, the viral capping enzyme has also been implicated by previous biochemical analysis to be an early transcription termination factor and an intermediate transcription initiation factor. A temperature sensitive mutant, Dts36, which has a mutation in the D1 protein, has been analyzed in vivo and in vitro. In the phenotypic analysis of Dts36, some early mRNA transcripts and proteins are present in reduced amounts. This is consistent with a defect in one of the enzymatic activities in the formation of the cap structure, most likely the methyltransferase activity. Some early viral transcripts were also longer than expected which is consistent with a defect in the termination function of the capping enzyme. A defect in intermediate gene transcription was also observed during a temperature shift-up experiment. These results represent the first time that the capping enzyme has been shown to be involved in early termination and intermediate initiation in vivo. The results of the phenotypic characterization were confirmed by biochemical analysis. Biochemical assays designed to examine the enzymatic activities of the mRNA capping enzyme were performed and the mutant D1 protein was found to be defective in the methyltransferase activity as predicted by the phenotypic analysis. The results from both analyses when taken together lead to the hypothesis that the mutant enzyme subunits may not be able to properly associate which could explain the capping defect as well as the defects observed in early transcription termination and intermediate transcription.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Amber Shatzer.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Condit, Richard C.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022901:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022901/00001

Material Information

Title: Role of the Viral mRNA Capping Enzyme during Vaccinia Virus Infection
Physical Description: 1 online resource (110 p.)
Language: english
Creator: Shatzer, Amber
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: capping, infection, initiation, methyltransferase, mrna, termination, transcription, vaccinia, virus
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Eukaryotic cells and viruses cap mRNA transcripts to increase the stability and translatability of the transcripts. Vaccinia virus, the prototypic member for the poxviridae family, encodes a multifunctional, heterodimeric mRNA capping enzyme. The large subunit is encoded by D1R (97kDa) and the small subunit is encoded by D12L (33kDa). In addition to its role in mRNA capping, the viral capping enzyme has also been implicated by previous biochemical analysis to be an early transcription termination factor and an intermediate transcription initiation factor. A temperature sensitive mutant, Dts36, which has a mutation in the D1 protein, has been analyzed in vivo and in vitro. In the phenotypic analysis of Dts36, some early mRNA transcripts and proteins are present in reduced amounts. This is consistent with a defect in one of the enzymatic activities in the formation of the cap structure, most likely the methyltransferase activity. Some early viral transcripts were also longer than expected which is consistent with a defect in the termination function of the capping enzyme. A defect in intermediate gene transcription was also observed during a temperature shift-up experiment. These results represent the first time that the capping enzyme has been shown to be involved in early termination and intermediate initiation in vivo. The results of the phenotypic characterization were confirmed by biochemical analysis. Biochemical assays designed to examine the enzymatic activities of the mRNA capping enzyme were performed and the mutant D1 protein was found to be defective in the methyltransferase activity as predicted by the phenotypic analysis. The results from both analyses when taken together lead to the hypothesis that the mutant enzyme subunits may not be able to properly associate which could explain the capping defect as well as the defects observed in early transcription termination and intermediate transcription.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Amber Shatzer.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Condit, Richard C.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022901:00001


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1 ROLE OF THE VIRAL mRNA CAPPING ENZYME DURING VACCINIA VIRUS INFECTION By AMBER NICOLE SHATZER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Amber Nicole Shatzer

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3 To my mother who was not able to see me complete my journey, but who I know is with me in spirit.

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4 ACKNOWLEDGMENTS There are many people that I feel that I need to acknowledge starting wi th m y family. My parents, Kim and Ed Shatzer, have loved me unconditionally and have supported all of the decisions I have made in my life. Without thei r love and support, I w ould not be the person who I am today. My younger brother, Ja red Shatzer, has always been a source of insp iration for me since he was born and I hope that he knows how mu ch I love him. My numerous aunts, uncles, and cousins, as well as close family friends also deserve much thanks. It is because of their love and support that I have been able to get through the hardships of the past year. I have had many wonderful times in graduate school with friends that I did not know until moving to Florida by myself five years ago. Wit hout the friendships forged in those first few weeks of graduate school, I am not sure if I co uld have survived being so far away from my family. I am especially thankful for my boyfriend, Tolga Barker, my best friend, Amanda DuBose, and my roommate, Russ During. We have had many good times together and I am extremely grateful for those times and the times that are yet to come. A big reason for my successes in graduate sc hool has been my mentor, Rich Condit, who took me in when I did not have a lab. He allowe d me to join his lab, and for the past five years he has taught me much about science and even more about life. He shaped me into th e scientist and, more importantly, the person I am today. For al l he has done, I will be eternally grateful. The Condit lab would not have been the Cond it lab without the in dividuals who I have had the privilege to work with for the past se veral years. Dr. Sayuri Kato, Dr. Steve Cresawn, Dr. Susan DCosta, and Dr. Nissin Moussatche have all taught me the techniques that I used in this study. Additionally, Travis Bainbridge, Ol ga Boyd, Alyson Brinker, Brad Dilling, Nicole Kay, Dr. Hendrik Nollens, Aparna Manoharan, Dr Cindy Prins, and Carson Rodeffer should be thanked for their contributions to the atmosphere of the lab and to this wo rk. I would also like to

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5 thank Dr. Ed Niles and Dr. Paul Golnick, who in my eyes are honorary lab members, for the advice and wisdom they shared with me about this project. I would also like to thank th e members of my supervisory committee (Dr. Dave Bloom, Dr. Art Edison, and Dr. Bert Flanegan) for thei r support and feedback on my project. Members of the Moyer and McFadden labs also deserve th anks for the feedback and insight they provided me with during joint lab meetings. Finally I w ould like to thank Joyce Conners and the entire fiscal staff of Molecular Gene tics and Microbiology for all the administrative support they have provided me.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF FIGURES.........................................................................................................................9ABSTRACT...................................................................................................................................11 CHAP TER 1 INTRODUCTION..................................................................................................................131.1Poxviruses.....................................................................................................................131.2Viral Life Cycle............................................................................................................. 141.2.1Viral Entry......................................................................................................... 141.2.2Viral Transcription............................................................................................ 161.2.2.1Viral RNA polymerase....................................................................... 171.2.2.2Early transcription.............................................................................. 181.2.2.3Intermediate and late transcription..................................................... 191.2.3Viral DNA Replication.....................................................................................211.2.4Virion Morphogenesis and Trafficking............................................................. 221.3Capping of mRNA........................................................................................................231.3.1Structure and Function of the mRNA Cap........................................................ 231.3.2Formation of the mRNA Cap............................................................................ 241.4Vaccinia Virus mRNA Capping Enzyme..................................................................... 251.4.1Enzymatic Activities of mRNA Capping.......................................................... 261.4.2Early Viral Transcription Termination.............................................................. 271.4.3Intermediate Viral Transcription Initiation....................................................... 271.5Previous Mutants in Capping Enzyme Subunits........................................................... 281.6Study Objectives...........................................................................................................292 MATERIALS AND METHODS........................................................................................... 332.1Methods for Phenotypi c Characterization..................................................................... 332.1.1Cells and Viruses..............................................................................................332.1.2Viral DNA Isolation and DNA Sequencing......................................................332.1.3Marker Rescue..................................................................................................342.1.4Virion Thermostability...................................................................................... 342.1.5 Metabolic Labeling of Protei ns and Gel Electrophoresis................................. 342.1.6Isolation of Viral RNA...................................................................................... 352.1.7 Synthesis of Riboprobes.................................................................................... 352.1.8 Northern Blot Analysis..................................................................................... 362.1.9 Western Blot Analysis...................................................................................... 362.1.10 Isolation of Viral DNA Replication Samples.................................................... 372.1.11Slot Blot Hybridization for Viral DNA Replication.........................................372.1.12AraC Experiment..............................................................................................382.1.13Shift-Up Experiment.........................................................................................38

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7 2.2Methods for Biochemical Characterization.................................................................. 382.2.1Virus Purification.............................................................................................. 382.2.2Preparation of Virion Extracts.......................................................................... 392.2.3In vitro Transcription by Permeabilized Virions.............................................. 392.2.4Enzyme-GMP Complex Formation in Permeabilized Virions and Virion Extracts..............................................................................................................402.2.5Coupled Transcription and Methyltr ansferase Assay in Permeabilized Virions...............................................................................................................402.2.6Uncoupled Methyltransferase Assay in Permeabilized Virion and Virion Extracts..............................................................................................................412.2.7Non-Specific RNA Polymerase Activity.......................................................... 413 PHENOTYPIC ANALYSIS OF A TEMPERA TURE SENSITIVE MUTANT IN THE LARGE SUBUNIT OF THE VACCINIA VIRUS MRNA CAPPING ENZYME ............... 433.1Introduction................................................................................................................... 433.2Results........................................................................................................................ ...443.2.1Marker Rescue and DNA Sequencing..............................................................443.2.2Growth Properties and Virion Thermostability of Dts36.................................. 453.2.3Basic Phenotypic Characterization of Dts36.....................................................463.2.3.1Viral protein synthesis........................................................................463.2.3.2Viral DNA synthesis........................................................................... 473.2.3.3Viral RNA synthesis........................................................................... 473.2.3.4Viral protein and RNA synthesi s in the presence of araC..................483.2.4Early gene transcription termination is defective in Dts36-infected cells........ 493.2.4.1Analysis of F11L transcript in Dts36 non-permissive infections.......493.2.4.2Analysis of the E9L, A20R, a nd D5R transcripts in Dts36 nonpermissive infections..........................................................................513.2.4.3Analysis of the F11, E9, A20, and D5 protein synthesis in Dts36 non-permissive infections................................................................... 513.2.5Intermediate Gene Transcription is Co mpromised in Dts36-infected Cells..... 523.3Summary.......................................................................................................................534 BIOCHEMICAL ANALYSIS OF A TEMPERATURE SENSITIVE MUTANT IN THE LARGE SUBUNI T OF THE VACCINIA VIRUS MRNA CAPPING ENZYME....... 694.1Introduction................................................................................................................... 694.2Results........................................................................................................................ ...694.2.1Analysis of D1 Protein in Virions and Virion Extracts.................................... 694.2.2Analysis of mRNA Capping Enzyme Activities in Permeabilized Virions...... 704.2.2.1In vitro transcription in permeabilized virions................................... 704.2.2.2Enzyme-GMP complex formation...................................................... 714.2.2.3Analysis of (guanine-N7 )-methyltransferase activity......................... 724.2.3Analysis of mRNA Capping Enzyme Activities in Virion Extracts................. 73

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8 4.2.3.1Non-specific RNA polymerase activity.............................................. 734.2.3.2Enzyme-GMP complex formation...................................................... 744.2.3.3Analysis of methyltransferase activity................................................ 744.3Summary.......................................................................................................................745 DISCUSSION.........................................................................................................................865.1Introduction................................................................................................................... 865.2Temperature Sensitivity................................................................................................ 865.3Early Viral Gene Transcription Termination................................................................ 875.4The Reactivation of Early Viral Messages Late during Viral Infection....................... 885.5DNA Negativity of Dts36............................................................................................. 905.6Intermediate Viral Gene Transcription in Dts36........................................................... 915.7mRNA Capping.............................................................................................................925.8mRNA Capping Enzyme Subunit Association............................................................. 945.9Future Studies...............................................................................................................95LIST OF REFERENCES...............................................................................................................99BIOGRAPHICAL SKETCH.......................................................................................................110

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9 LIST OF FIGURES Figure page 1-1 Vaccinia virus life cycle. ............................................................................................... ...301-2 Overview of vaccinia virus morphogenesis. .................................................................... 311-3 The mRNA cap 1 structure. ............................................................................................. 323-1 Marker rescue of Dts36. .................................................................................................. .543-2 The D1 protein. .......................................................................................................... ......553-3 One-step growth of IHDW and Dts36 viruses. ................................................................ 563-4 Virion thermostability of Dts36 and IHDW at 45C. ...................................................... 573-5 Basic phenotypic characteri zation of Dts36: protein synt hesis, D1 synthesis, and DNA replication. ..............................................................................................................583-6 Analysis of early, intermed iate and late RNA synthesis in cells infected with Dts36 or IHDW........................................................................................................................ ....603-7 Viral protein and mRNA synthesis in cells infected Dts36 or IHDW in the presence of araC. .............................................................................................................................613-8 Analysis of the early viral transcript, F11L in cells infected with Dts36 or IHDW. ...... 623-9 Primer walk experiment with various ri boprobes for the analysis of the longer than expected F11L transcripts..................................................................................................633-10 Analysis of the early viral transcript, E9 L, in cells infected with Dts36 or IHDW...........643-11 Synthesis of the F11, E9, A20, and D5 viral proteins during Dts36 and IHDW infections..................................................................................................................... .......653-12 Analysis of protein synthesis and DNA re plication in Dts36 a nd IHDW-infected cells during a shift-up experiment..............................................................................................663-13 Analysis of viral RNA s ynthesis in Dts36 and IHDW-infected cells during a shift-up experiment..................................................................................................................... .....674-1 SDS-PAGE and western blot analysis of purified IHDW and Dts36 virions....................764-2 Western blot analysis of IH DW and Dts36 virion extracts................................................ 774-3 In vitro transcription in permeabilized IHDW and Dts36 virions..................................... 78

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10 4-4 Enzyme guanylylate complex for mation in purified virions.............................................794-5 Coupled transcription and methyltransf erase assay in permeabilized IHDW and Dts36 virions......................................................................................................................804-6 Uncoupled methyltransferase assay schematic.................................................................. 814-7 Uncoupled methyltransferase assay in IHDW and Dts36 permeabilized virions. ........... 824-8 Non-specific RNA polymerase activity in IHDW and Dts36 virion extracts.................... 834-9 Enzyme-guanylylate complex formation in IHDW and Dts36 virion extracts. ...............844-10 Uncoupled methyltransferase assay in IHDW and Dts36 virion extracts.......................... 855-1 Structure of the carboxyl-terminus (aa 545844) of the large subun it of the vaccinia virus mRNA capping enzyme............................................................................................ 975-2 Structure of the interaction of the car boxyl-terminus of the la rge subunit with the entire small subunit of the vaccinia virus mRNA capping enzyme................................... 98

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ROLE OF THE VIRAL mRNA CAPPI NG ENZYME DURING VACCINIA VIRUS INFECTION By Amber Nicole Shatzer December 2008 Chair: Richard C. Condit Major: Medical SciencesImmunology and Microbiology Eukaryotic cells and viruses cap mRNA tr anscripts to increase the stability and translatability of the transcripts. Vaccinia virus, the prototypic member for the poxviridae family, encodes a multifunctional, heterodimeric mRNA capping enzyme. The large subunit is encoded by D1R (97kDa) and the small subunit is encoded by D12L (33kDa). In addition to its role in mRNA capping, the viral capping enzyme has also been implicated by previous biochemical analysis to be an early transcri ption termination factor and an intermediate transcription initiation factor. A temperature sensitive mutant, Dts36, which has a mutation in the D1 protein, has been analyzed in vivo and in vitro. In the phenotypic analysis of Dts36, some ear ly mRNA transcripts and proteins are present in reduced amounts. This is consistent with a defect in one of the enzymatic activities in the formation of the cap structure, most likely th e methyltransferase activ ity. Some early viral transcripts were also longer than expected which is consistent w ith a defect in the termination function of the capping enzyme. A defect in inte rmediate gene transcri ption was also observed during a temperature shift-up experi ment. These results represen t the first time that the capping enzyme has been shown to be involved in ea rly termination and intermediate initiation in vivo The results of the phenotypic characterization were confirmed by biochemical analysis.

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12 Biochemical assays designed to examine the enzymatic activities of the mRNA capping enzyme were performed and the mutant D1 protein was found to be defective in the methyltransferase activity as predicted by the phenotypic analysis. The results from both analyses when taken together lead to the hypothesis that the mutant enzyme subunits may not be able to properly associate which could explain th e capping defect as well as th e defects observed in early transcription termination and intermediate transcription.

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13 CHAPTER 1 INTRODUCTION 1.1 Poxviruses Viruses are extrem ely small, self-replicating, inf ectious agents which ar e parasitic in nature due to their dependence on cellular machinery for vi ral metabolism. Due to the simplistic nature of viruses, as compared to the complex nature of eukaryotic cells, viruses have been useful tools in the elucidation of basic aspects of mol ecular and cellular biology. Viruses have been successfully used to study eukaryotic gene regulation and much of what is known about the mechanism of eukaryotic transcription mach inery, mRNA processing and DNA replication is due to studies performed with viruses. One example of a viru s that has been used to elucidate these processes is vaccinia vi rus, the prototypical member of the poxviridae family. The poxviridae is a family of large, doublestranded DNA viruses that are capable of infecting either vertebrate cells (the Chordopoxviruses) or invertebrate cells (the Entomopoxviruses). Vaccinia virus is a memb er of the Chordopoxvirinae subfamily and the genus orthopoxvirus, and like a ll poxviruses, encode s all of the factor s required for viral transcription and viral DNA replication. As a result of encoding their own transcription and replication machinery, poxviruses do not require entry into the host cell nucleus for these processes and therefore, they replicate in the cytoplasm of the infected cells. This characteristic of vaccinia virus and poxviruses in general, make them extremely useful tools in understanding the cellular processes in eukaryotic cells. Vaccinia virus possesses a linear, double-stra nded DNA genome that is approximately 200 kilobases (kb) in length and which possesses covalently closed ends composed of inverted terminal repeats critical for viral DNA replica tion. The viral genome encodes approximately 200 genes and is encapsidated in the core of a complex brick-sh aped virion surrounded by a single

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14 lipid bilayer. The viral genes are arranged in such a fashion that the highly conserved genes as well as genes critical for the func tion of the virus are located in the center of the viral genome. The less conserved genes as well as genes involved with host interactions ar e located at the ends of the viral genome. The genes of orthopoxviru ses are named based on the HindIII restriction endonuclease map of the viral genome which give s each HindIII fragment a letter designation (16 HindIII fragments name d A-P, see Fig. 3-1 for schematic). Each HindIII fragment is further divided into open reading frames (ORFs) which are numbered from le ft to right and also labeled with a letter (R or L) to designate the transcriptional orienta tion of the gene. 1.2 Viral Life Cycle The vaccin ia virus life cycle consists of a number of processes which are temporally regulated. These processes include viral entry, viral gene transcription which is divided into early, intermediate, and late vi ral transcription, viral DNA rep lication, and virion morphogenesis and release (Figs. 1-1 and 1-2). These proces ses are complex in natu re and are understood in varying degrees with viral transcription and DNA replication being well -understood whereas the processes involved in viral entry and morphoge nesis are continuing to be worked out. 1.2.1 Viral Entry There are tw o primary forms of vaccinia virus that are recognized (reviewed in (Moss, 2006); mature virions (MV, formerly called in tracellular mature virion s (IMV)) are the most abundant form, and extracellular virions (EV, fo rmerly extracellular en veloped virions (EEV)) are simply MVs which possess one additional membra ne wrapper. Due to the difference in the number of membranes the two forms of virions possess, the protein co mposition of the outer membranes is different for each infectious form of the virus (Condit et al., 2006). Although the infectious forms of vaccinia virus have been de termined, the exact mechanism of viral entry is still a debatable topic in the fiel d. The current school of thought is that the virus enters through

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15 two distinct mechanisms; fusion of the virion with the plasma membrane or endosomal uptake of the virion followed by fusion of the virion with th e endosomal membranes, each of which will be discussed further below. Independent of the manner of viral entry, the ab ility of the virion membrane to fuse with a membrane that is cellular in origin is critical for successful entry of the infectious particle into the cell. Recently, a numb er of viral proteins have been show n to be involved in the entry of virions into the cell. These pr oteins are A16 (Ojeda et al., 2006b), A21 (Townsley et al., 2005b), A28 (Senkevich et al., 2004), F9 (Brown et al., 2006), G3 (Izmailyan et al., 2006), G9 (Ojeda et al., 2006a), H2 (Senkevich and Moss, 2005), I2 (Nichols et al., 2008), J5 (Senkevich et al., 2005), and L5 (Townsley et al., 2005a). At least nine of these proteins have been shown to interact and form an entry/fu sion complex (Brown et al., 2006; Senkevich et al., 2005) and inducible mutants in most of the proteins have shown that these proteins are required for successful entry of the virus into the cell by fusi on, be it fusion of the virion with the plasma membrane or fusion of the virion with the endosomal membrane. Mature virions are thought to enter the cell ei ther through direct fusion with the plasma membrane or through fusion with an endosomal membrane following endosomal uptake of the virion into the cell. Fusion of the virion with the plasma memb rane will be addressed here followed by discussion about viri on fusion with the endosomal membrane. The simplest manner in which a virion can enter the cell would be for the outer virion membrane to bind and fuse to the plasma membrane of the cell allowing for the vi rus core to be released into the cytoplasm of the cell. Electron microscopic evidence has show n that MVs are capable of this direct fusion with the plasma membrane and subsequent entry in to the cell (Carter et al., 2005). Analysis of EVs by electron microscopy has also provided ad ditional evidence of infectious particles

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16 entering cells through direct fusion with the plasma membrane. Af ter interaction with the cell, the EVs were found to lose their additional out er membrane by a ligand-induced non-fusogenic dissolution process after which the resulting virus particle, now a MV is able to fuse with the plasma membrane and the viral is core is ab le to enter the cell (L aw et al., 2006). This mechanism of virion entry makes it possible for the double-membraned EVs as well as the single-membraned MVs to successfu lly enter and infect cells. The other mechanism of virion entry is thr ough endosomal uptake and subsequent fusion of the virion membrane with the endosomal memb rane. Recent studies have shed more light on the exact mechanism that vaccinia virions use to enter the cell through endosomes. MVs have been shown to be endocytosed into the cell by macropinocytosis (Mercer and Helenius, 2008) and fluid phase endocytosis (Hua ng et al., 2008), two processes th at consist of non-selective bulk fluid-phase uptake of extr acellular material into the endosomes However, there is controversy on how exactly the virion induces the endocytic process; one school of thought is that the virions mimic apoptotic bodies which are similar in size to MVs (Mercer and He lenius, 2008) ,whereas, the second school of thought is that the virus exploits a novel cellular protein, vaccinia virus penetration factor (Huang et al., 2008). Successful endocytosis of vaccinia virions is dependent on two distinct low pH processes; the first step promotes the activation of entry/fusion complex and the second low pH step induces the fusion of the virion membrane with the endosomal membrane (Townsley et al., 2006; Townsley and Moss, 2007). Much like fusion of the virion with the plasma membrane, fusion of the virion with the endosomal membrane allows the viral core to enter the cytoplasm of the cell. 1.2.2 Viral Transcription Vaccinia v irus transcription is temporally regulated with each class of genes (early, intermediate, and late) having a distinct promot er and trans-acting factors. The genes are

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17 expressed in a cascade such that each class of genes encodes for f actors that are necessary for the transcription of the subsequent class. Early genes also en code factors required for DNA replication and late genes, in addition to encoding transcripti on factors for early transcription during the next round of viral infection, also en code factors required fo r virion morphogenesis. Viral DNA replication (discussed fu rther in section 1.2.4) separates the transcription cascade into early transcription and post-replicative transcription which includes both the intermediate and late classes of viral transcripts. This division of the transcript ion cascade necessitates that the virus possess two forms of the RNA polymerase. However, independent of the form of the RNA polymerase utilized in transcri ption, all viral transcripts are capped at their 5 end and polyadenylated at their 3 ends. 1.2.2.1 Viral RNA polymerase Vaccinia v irus possesses two multi-subunit form s of the viral RNA polymerase; one is specific for early transcription a nd the second is specific for post-r eplicative transcription. Both forms of the viral polymerase have eight subunits in common including three subunits, rpo147, rpo132, and rpo7, which share homology to eukaryo tic and prokaryotic polymerase subunits (Amegadzie et al., 1992; Broyles and Moss, 1986 ; Patel and Pickup, 1989). However, the early polymerase has an additional polypeptide associated with it, the RNA polymerase-associated protein (RAP94) encoded by the viral gene H4 L (Ahn and Moss, 1992). RAP94 is responsible for the recognition of early viral promoters (A hn et al., 1994; Deng and Shuman, 1994) and plays a role in early transcription te rmination (Mohamed and Niles, 2001) and at late times during viral infection, RAP94 is responsible for the targeting of the viri on components into the forming virions (Zhang et al., 1994). The ability of RA P94 to only recognize early promoters, which are distinct from both intermediate and late promoter s, through its association with the vaccinia early transcription factor (VETF) confer s specificity to the early form of the viral RNA polymerase.

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18 1.2.2.2 Early transcription There are several d ifferences be tween early and post-replicativ e transcription; mainly the cellular location of the tr anscription process, promoter struct ure, and transcription termination. Early viral transcription takes place in the viral core upon entry into the cytoplasm of the cell. As such, the infectious virus particle possesses all the factors that ar e necessary for early transcription including but not limited to the early RNA polymerase, the vaccinia early transcription factor (VETF), the mRNA capping enzyme, and the RNA poly(A) polymerase. The promoter structure of early transcription co nsist of a 16 base pair (bp) critical region separated from a 7-bp region of initiation by a 11-bp thymidine-rich, less cr itical region (Davison and Moss, 1989a). The VETF, a heterodimer comp rised of the products of the viral genes, A7L and D6R (Gershon and Moss, 1990), recognizes and bi nds to the early prom oter and confers the ATPase activity required for transcription (Broyles and Fesler, 1990). Transcription termination is another distincti on of early viral transc ription. The factors that are required for early transcription term ination have been determined. The vaccinia termination factor (VTF) is the mRNA capping enzyme, a multifunctional viral enzyme which will be discussed in further detail in section 1.4 (Luo et al., 1991; Shuman et al., 1987). The transcription termination occurs approximately 50 bps downstream of the termination sequence, TTTTTNT, in the non-template strand (Yuen and Moss, 1987). The recognition of the complementary sequence, UUUUUNU in the nascent mRNA transcript, by VTF signals for transcription termination (Shuman and Moss, 1988). In additional to VTF, several other viral factors are required for early transcription termination. The RAP94 subunit of the RNA polymerase as well as the nucleoside triphos phate phosphohydrolase I (NPH I), encoded by viral gene D11L, are essential for early transcripti on termination (Christen et al., 1998; Mohamed and Niles, 2001). The currently proposed mechanism for early transcrip tion termination is a two step

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19 model: in the first step VTF and RAP94 recognize the UUUUUNU sequence in the nascent mRNA and in the subsequent step the nascent transcript is released in the presence of NPH I and ATP (Piacente et al., 2008). 1.2.2.3 Intermediate and late transcription Interm ediate and late transcription differs more from early transcription than simply in the form of RNA polymerase that is utilized; post -replicative transcripts possess poly(A) heads at their 5 ends which are due to slippage of th e RNA polymerase during in itiation (Schwer et al., 1987; Schwer and Stunnenberg, 1988). This char acteristic contributes to the heterogeneous length of post-replicative transcript s. Intermediate and late tran scription differ from one another in the structure of their promoters as well as the factors that are re quired for transcription initiation. Intermediate promoters are thirty nuc leotides in length and contain a 14 bp critical region found 10-11 nucleotides upstream of the TAAA initiator element (Baldick, Jr. et al., 1992). Late promoters also are approximately th irty nucleotides in length and have a 20 bp thymidine and adenosine rich region separa ted from the highly conserved TAAAT initiator element by a 6 bp spacer (Davison and Moss, 198 9b). The factors that are required for transcription initiation are also unique to each cla ss of post-replicative transc ripts. There are four vaccinia intermediate transcription factors (V ITF-1, 2, 3, and A) which include both virally encoded and cellular factors. VITF-1 is rpo30, on e of the subunits of th e viral RNA polymerase (Rosales et al., 1994) and VITF-3 is a heterodime r of viral proteins A8 and A23 (Sanz and Moss, 1999). VITF-2 is a cellular heterodimer consis ting of p137 and G3BP proteins and is thought to be involved in the regulation of the switch from ea rly transcription to inte rmediate transcription (Katsafanas and Moss, 2004). The last intermedia te transcription factor VITF-A, is the viral mRNA capping enzyme which plays a structural role in the formation of the transcription initiation complex (Vos et al., 1991b). There are five vaccinia late transcription factors (VLTF-

PAGE 20

20 1, 2, 3, 4, and X) which like the intermediate fact ors are both virally (1-4) and host (X) encoded. VLTF-1 is encoded by G8R (Zhang et al., 1992), VLTF-2 is encoded by A1L (Keck et al., 1993), VLTF-3 is encoded by A2L (Passarelli et al., 1996), and VLTF-4 is encoded by H5R (Kovacs and Moss, 1996). The fina l late transcription factor is host-encoded and is comprised of the ribonucleoproteins A2/B1 a nd RBM3 (Wright et al., 2001). Another difference between early and post-replicative transc ription is, whereas, there is a known early termination signal, there is no known post-rep licative termination sequence and this lack of a consensus termination sequence leads to post-replicative transcripts being heterogeneous at the 3 ends. It is important to note that the early tr anscription termination si gnal is not recognized as a termination signal in post-replicative transcription. Although there is no known consensus termination sequence for post-replicative transcription, it is known that se veral virally encoded f actors are required for the 3end formation of post-replicative transcripts. These factors are A 18, G2, J3, and H5, encoded by A18R, G2R, J3R, and H5R, respectively. A18 has been shown to possess DNA helicase (Simpson and Condit, 1995) and DNA-dependent ATPase (Bay liss and Condit, 1995) ac tivities. The A18 protein has also been shown to be required fo r the release of nascent RNA from transcription elongation complexes in vitro (Lackner and Condit, 2000) and mu tants in A18 have been shown to synthesize longer than normal post-replicative transcripts (Xiang et al ., 1998). These results are consistent with A18 being the post-replicative transcription termination factor. The G2 protein was shown to be a positive post-replicative transcription elongation factor because temperature sensitive mutants in the G2 protein s ynthesized post-replicative transcripts that were shorter than normal (Black and Condit, 1996). The roles of the A18 and G2 proteins in transcription elongation were furt her confirmed when it was show n that a mutation in the G2R

PAGE 21

21 gene is able to compensate for a mutation in the A18R gene (Condit et al., 1996b). An additional viral protein, J3, was identified through two independent genetic se lections to also be a positive post-replicative transcription el ongation factor (Latner et al., 2000). Further analysis with mutants in the J3 protein, which synthesized shor ter than normal post-replicative transcripts, confirmed the role of the J3 pr otein in transcription elongation. The final viral protein with a role in post-replicative transcrip tion is H5. H5 has been identifi ed as a late gene transcription factor (Kovacs and Moss, 1996) and has been shown to interact with G2 through yeast twohybrid analysis and in vivo immunoprecipitation (Black et al., 1998). In the same analysis, the H5 and A18 proteins were al so shown to interact during in vivo immunoprecipitation. More recently, H5 has been identified as a positive post -replicative elongation factor when an isatin thiosemicarbazone (IBT)-resistant mutant was iden tified (Cresawn and Cond it, 2007). IBT is an anti-poxviral drug that is known to promote read-through of post-replicative transcripts. The H5 protein has also been implicated as the endoribonuclease involved in the formation of the 3 end of post-replicative tran scripts by a cleavage even t (D'Costa et al., 2008). 1.2.3 Viral DNA Replication Viral DNA replication occurs af ter early transcription but be fore interm ediate and late transcription. Presumably a second uncoating even t occurs after early tran scription which allows the release of the viral DNA molecu le from the viral core into the cytoplasm of the cell (Joklik, 1964a; Joklik, 1964b; Mallardo et al., 2002). Rep lication of the viral genome by the viral DNA polymerase, encoded by the gene E9L, (Traktma n et al., 1984) and othe r associated factors results in the formation of an area of electrode nse material that is free of cellular organelles known as viroplasm, where DNA replication and virion morphogenesis occur. The additional viral factors required for viral DNA replication are the processtiv ity factor, A20 (Punjabi et al.,

PAGE 22

22 2001); the uracil DNA glycosylase, D4 (Stanitsa et al., 2006); and a viral ATPase/DNA primase capable of oligomerization, D5 (B oyle et al., 2007; De Silva et al., 2007; Evans et al., 1995). 1.2.4 Virion Morphogenesis and Trafficking Virion m orphogenesis is the process of viri on assembly and maturation. The steps of virion morphogenesis have been determined by electron microscopic examination of vaccinia virus infections (reviewed in (Condit et al., 2006)). The location of virion morphogenesis is the viroplasm where viral DNA replication is occu rring as discussed in section 1.2.3. The first discernable structure in morphogene sis is the appearance of cresce nts which are comprised of at least one lipid bilayer (Fig. 1-2). The crescent s continue to grow until they become closed circles forming an immature virion (IV) which is filled with viroplasm. At nearly the same time, immature virions with nucleiod (IVN) appear. The nucleiod is a round region of electrodense material which contains DNA. It has been hypothesized that IVs and IVNs are the same structures that have been s ectioned through different planes during sample processing. The proteolytic processing of several virion protei n precursors is required for the morphogenesis from IVN to MV and extrusion from the viroplas m into the cytoplasm of the infected cell. After the MV exits the viroplasm, it is trafficked through the cell to the plasma membrane in order to exit the cell (reviewe d in (Smith and Law, 2004)). Th e MV is transported through the Golgi apparatus on microtubules which results in the formation of wrapped virions (WV) which are MVs that possess two Golgi-derived membra ne wrappers (Fig 1-2). The microtubules continue the transport of the WVs to the plas ma membrane where the outer membrane wrapper of the WV fuses with the plasma membrane resulti ng in an EV sitting on the cell surface. The EVs can mediate cell-to-cell spr ead by protrusion of the EV to adjacent cells as actin tail formation drives the EV away from the cell or the EV can be released from the cell surface and mediate long distance disse mination of the virus.

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23 1.3 Capping of mRNA Eukaryotic and viral m essenger ribonucle ic acid (mRNA) transcripts possess a cap structure at their 5-ends that is the result of the first post-translation modification of the nascent transcript. The structure and function of the mRNA cap as well as the enzymatic reactions required for the formation of the cap structure ha ve been elucidated and will be discussed in further detail in the following sections. 1.3.1 Structure and Function of the mRNA Cap The mRNA cap is a 7-methylguanosine (m7G) that is linked through an inverted triphosphate bridge (5-5 linkage ) to the initiat ing nucleoside of the nascent mRNA transcript. There are several different forms of the cap stru cture depending on the methylation status of the cap (Fig. 1-3); these structures are designated cap 0, cap 1, and cap 2 with the cap 1 structure being the most prevalent structure found in eukar yotic cells. The methylation status of each structure is as follows: the cap 0 structure is methylated at the N7 position on the guanosine residue the cap; the cap 1 stru cture in addition to the methyl ated guanosine residue is also methylated at the O2 position of the in itiating nucleoside; and the cap 2 structure in addition to having the same groups methylated as the cap 1 structure is also methylated at the O2 position of the second nucleoside of the nascent transcript The function of the mRNA cap structure in eukaryotic cells is to confer stability to the mRNA transcript as well as aid in the translatability of the transcript. The guanosine residue of the cap structure contributes to the transcript stability; it has been previously shown th at non-guanylylated reovirus RNA is less stable in wheat germ extracts, L cell protein synthesizing extracts as well as in X. laevis oocytes (Furuichi et al., 1977). Additionally, it has been demonstrated th rough the use of a guanylyltransferase mutant in yeast that the inactivation of the mRNA capping activity caused a decrease in the levels of mRNA and protein synthesis. The methylation status of the guanosine residue of the cap

PAGE 24

24 structure contributes to the translatability of the transcript. Methylation of the guanosine is required for efficient translation of the mRNA tr anscript as was observed in experiments with reovirus and vesicular stomatitis virus (VSV) RNA. Methylated RNA from either virus stimulated protein synthesis in wheat germ extracts better than unmethylated viral RNA (Both et al., 1975). An additional study with RNA from reovirus and VSV removed the methylated guanosine residue from the cap structure by -elimination and observed that the RNA transcripts were no longer translated (Muthukrishnan et al., 1975). Further studies w ith vaccinia virus RNA examined the interaction of the methylated guan osine residue of the cap with the ribosome as well as its role in translation. After -elimination, viral RNA was no longer able to bind to ribosomes or be translated; how ever, after adding back the m7G (but not an unmethylated guanosine residue) to the RNA, ribosome binding and translation activities were restored (Muthukrishnan et al., 1978). The formation of the mRNA cap 1 structure will be discussed in the next section. 1.3.2 Formation of the mRNA Cap The mRNA cap is form ed in a series of five enzymatic reactions (Equations 1-1 through 15) that are catalyzed by f our different enzymes. pppN(pN)n ppN(pN)n + Pi (1-1) GTP + Enz Enz-pG + PPi (1-2) Enz-pG + ppN(pN)n GpppN(pN)n + Enz (1-3) GpppN(pN)n + AdoMet 7mGpppN(pN)n + AdoHcy (1-4) 7mGpppN(pN)n + AdoMet 7mGpppNm(pN)n (1-5) The first step in the formation of the cap structure (Equation 1-1) is the removal of a gamma phosphate from the 5 end of the initiatin g nucleoside of the nascent transcript by RNA

PAGE 25

25 triphosphatase. The product of this reaction is an mRNA transcript with a diphosphate 5terminus. The second step in the formation of the cap structure is the transfer of a monophosphate guanosine residue to the di-phosphorylated 5-ter minus of the nascent mRNA transcript. This step, which is catalyzed by mRNA guanylyltransferase, has been shown to be reversible and proceeds in a series of two reac tions (Equations 1-2 and 1-3) (Roth and Hurwitz, 1984; Shuman and Hurwitz, 1981). The first guanyl yltransferase reaction involves the formation of a covalent capping enzyme-GMP comple x intermediate and the release of PPi in the presence of a divalent cation. The second guanylyltransferas e reaction is the attack and transfer of the guanosine residue to the beta phos phate of the nascent mRNA tr anscript to yield an mRNA transcript with a guanosine attached to it through a 5-5 triphosphate br idge (Roth and Hurwitz, 1984; Shuman and Hurwitz, 1981). The third reaction involved in the fo rmation of the mRNA cap structure is the transfer of a methyl group from S-adenosyl methionine (AdoMet) to the N7 position on the guanylylate residue of the nas cent mRNA transcript (Equation 1-4). This reaction is irreversible and is catalyzed by mR NA (guanine-N7) methyl transferase (Martin and Moss, 1975; Martin and Moss, 1976). The fourth and final reaction in the formation of the mRNA cap structure is the transfer of a methyl group from AdoMet to O2 position on the initiating nucleoside of the mRNA transcript (Equation 1-5). This reaction is catalyzed by mRNA (nucleoside-O2) methyltransferase and the product of the reaction is an mRNA transcript that possesses a cap 1 structure. 1.4 Vaccinia Virus mRNA Capping Enzyme Vaccinia v irus possesses a multifunctional, virally encoded mRNA capping enzyme. The viral capping enzyme is a heterodimer compri sed of a large and sma ll subunit (Guo and Moss, 1990; Martin and Moss, 1975) whic h are encoded by the D1R gene (97 kilodalton (kDa)) and the D12L gene (33 kDa) respectively (Morgan et al., 1984; Niles et al ., 1989). The vaccinia

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26 virus mRNA capping enzyme is responsible for the first three enzymatic steps in the formation of the cap structure; th e fourth reaction is catalyzed by a different virally encoded protein, the product of the gene J3R. In addition to its role in mRNA capping, bi ochemical analysis has demonstrated that the viral mRNA capping enzyme is also a factor required for early gene transcription termination (Luo et al., 1991; Shum an et al., 1987; Shuman and Moss, 1988) as well as a factor required for intermediate gene tr anscription initiation (Har ris et al., 1993; Vos et al., 1991b). These functions of th e viral mRNA capping enzyme will be further discussed in the following sections. 1.4.1 Enzymatic Activities of mRNA Capping The activ e sites for the three enzymatic activit ies catalyzed by the vaccinia virus mRNA capping enzyme are found on the large subunit of th e capping enzyme. The active sites for the RNA triphosphatase and the mRNA guanylyltransferase activities are both located on the aminoterminus (amino acids 1-545) of the large s ubunit (Guo and Moss, 1990; Higman et al., 1992; Shuman, 1990; Shuman and Morham, 1990); howev er it has been shown by isolating mutants that are defective in one enzymatic activity but no t the other, that the active sites for these two enzymatic activities are distinct from one anot her (Myette and Niles, 1996; Yu and Shuman, 1996). The active site of the R NA triphosphatase activity is comp rised of nine charged amino acids: Glu37, Glu39, Arg77, Lys107, Glu126, Asp159, Lys161, Glu192, and Glu194, which are all required for activity (Gong a nd Shuman, 2003; Yu et al., 1997; Yu and Shuman, 1996). The active site of the mRNA guanylyltr ansferase is Lys260 which is the residue to which the GMP covalently attaches to during catalysis (N iles and Christen, 1993) ; however, other amino residues, namely Glu375, Lys392, and Asp400, also play a role in the guanylyltransferase catalysis (Cong and Shuman, 1995). The mRNA (guanine-N7) methyltransferase active site is located in the carboxyl-terminus (amino acids 54 0-844) of the D1 protein (Higman et al., 1994;

PAGE 27

27 Mao and Shuman, 1994); however, the small subunit of the capping enzyme must be present and interacting with the larg e subunit to stimulate the intrinsic methyltransferase activ ity of the large subunit (Higman et al., 1992; Mao and Shuman, 1994). The methyltransferase active site contains amino acid residues Gly600 and His682 and the aromatic ring of Tyr683 has been shown to be critical for the methyltransfer ase activity (Mao a nd Shuman, 1994; Mao and Shuman, 1996). It is critical to note that althoug h the subunit association of the D1 and D12 proteins is required for the me thyltransferase activity of the viral mRNA capping enzyme, this subunit association is not requ ired for the RNA triphosphatase and mRNA guanylyltransferase activities (Shuman, 1989; Shuman and Morham, 1990). 1.4.2 Early Viral Transcription Termination The role of the vaccin ia virus mRNA capping in early viral transcription termination is based on biochemical analysis of the virus. Th e early viral transcription termination factor activity were first shown to co-purify with th e mRNA capping activity (Shuman et al., 1987) and further analysis showed that the capping enzyme and the terminator factor were one and the same (Luo et al., 1991). It is hypothesized that the capping enzyme recognizes the early transcription termination signal U5NU in the nascent mRNA tr anscript and signals for termination (Shuman and Moss, 1988). The early transcription terminati on function of the viral mRNA capping is independent of the capping f unctions of the enzyme; however, both subunits of the viral mRNA capping enzyme are required fo r the early transcription termination function (Condit et al., 1996a; Luo et al ., 1995; Yu et al., 1997). 1.4.3 Intermediate Viral Transcription Initiation Much lik e the early viral transcription termin ation function, the role of the vaccinia virus mRNA capping enzyme in intermediate viral tran scription is based on bi ochemical analysis of the virus. Intermediate-specific cytoplasmic extracts from vaccinia infected cells were

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28 fractionated into three components: VITF-A, VITF-B, and the viral RNA polymerase (Vos et al., 1991a). The most purified fractions of each of the three components were used to reconstitute intermediate transcription in an in vitro transcription assay. VITF -B was shown to confer promoter specificity and further studies on VITF-A have shown that this component of the intermediate transcription initiation complex is the vaccinia virus mRNA capping enzyme (Vos et al., 1991b). The intermediate transcription initiation function of th e capping enzyme also requires both subunits of the capping enzyme (Condit et al., 1996a; Vos et al., 1991b) and much like the early termination activity is independent of the mRNA guanylyltransferase activity of the viral mRNA capping enzyme (Harris et al., 1993). 1.5 Previous Mutants in Capping Enzyme Subunits Two tem perature sensitive mutants in both subunits of the vaccinia virus mRNA capping enzyme have been identified and characterized previously. The mutant in the large subunit of the capping enzyme was characterized both in vivo and in vitro (Hassett et al., 1997). The in vivo analysis showed that the mutant virus s ynthesized normal amount of proteins and had normal expression of RNA; however, the mutant was defective in telomere resolution, an intregral part of DNA replication, as well as in virion morphogenesis. In the biochemical analysis carried out in vitro the mutant D1 viral protein was found to be defective in two of the enzymatic activities of the capping enzyme, guany lyltransferase and methyltransferase. The second capping enzyme mutant possessed a mu tation in the small subunit of the capping enzymes and was characterized in vivo (Carpenter and DeLange, 1991) and in vitro (Condit et al., 1996a). Similar to what was observed with the mutant in the larg e subunit of the capping enzyme, the mutant in the small subunit of th e capping enzyme synthesized normal amounts of viral proteins and had normal expression of vira l RNA but was defective in telomere resolution. The biochemical analysis of the mutant D12 prot ein examined the role of the protein in early

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29 transcription termination and inte rmediate transcription initiati on; both of which were found to be absent in the mutant protein. 1.6 Study Objectives As explained in the p revious section of this introduction, in addition to its role in the capping of viral transcripts, the vaccinia mRNA capping enzyme has also been implicated by biochemical analysis to be involve d in early viral transc ription termination a nd intermediate viral transcription initiation. Two prev ious temperature sensitive (ts) mutants in both subunits of the capping enzyme have been characterized by in vivo and in vitro analyses as described in section 1.5 but these analyses have been unable to conf irm the role of the capping enzyme in these additional activities (Carpenter and DeLange, 1991; Condit et al ., 1996a; Hassett et al., 1997). During a complementation study that integrated the Dales collection of ts mutants with the Condit collection of ts mutants, a new mutant, Dts36, possessing a mutation in the large subunit of the viral capping enzyme wa s identified (Lackner et al., 2003). A complete phenotypic characterization of Dts36 was undertaken to furthe r examine the role of the viral capping enzyme in the vaccinia virus life cycle in an attempt to confirm the role of the viral capping enzyme in early transcription termination and intermediate transcription initiation in vivo The results from the phenotypic characterization wi ll be presented in Chapter 3. A biochemical characterization of Dts36 was also undertaken to examine the mR NA capping enzyme activities of the mutant D1 protein from Dts36 as well the early terminati on and intermediate ini tiation activities of the mutant protein by in vitro analysis. The results from the biochemical characterization will be discussed in Chapter 4. A unified mechanism that explains the results se en in both the in vivo and in vitro analyses, a defect in the association of the capping enzyme subunits, will be discussed in Chapter 5.

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30 Figure 1-1. Vaccinia virus life cycle. Infectious virus particles in the fo rm of MV or EV enter the cell through fusion; either fusion with the plasma membrane or fusion with an endosomal membrane. This fusion event resu lts in partial uncoa ting of the virus to produce a viral core in which early transcrip tion (early txn) occurs Early transcripts encode factors that are required for DNA rep lication and intermediate transcription. Following early transcription, a seconda ry uncoating occurs and viral DNA is extruded into the cytoplasm where it can be replicated by the viral DNA polymerase. Following DNA replication, intermediate then la te transcription occu rs. Intermediate transcripts encode factors that are required for late transc ription and late transcripts encode factors that are packaged in the vi rion for the subsequent infection as well as factors required for assembly and matura tion of the infectious particle.

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31 Figure 1-2. Overview of vaccinia virus morphogenes is. Crescents form in the viroplasm of the infected cell and continue to grow until they form closed circles which are IVs containing viroplasm. IVNs appear at nearly the same time that IVs appear; proteolytic processing of several viral protei ns occurs and leads to the maturation of the virus particle to MV. MVs are extruded from the vir oplasm into the cytoplasm of the infected cell and are transported by microtubules through the Golgi apparatus and to the plasma membrane. The outer membra ne wrapper of the MV will fuse with the plasma membrane resulting in EV sitting on the surface of the cell. EV-1 is the form of EV that aids in cell -to-cell spread of the virus as actin tail formation drives the EV-1 towards the adjacent cell and EV-2 is the form of EV that is used for long distance dissemination of the virus.

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32 Figure 1-3. The mRNA cap 1 structure. Th e initiating nucleoside of the nascent mRNA transcript is shown on the righ t side and is linked to the guanylylate residue of the cap structure by a 5-5 triphospha te bridge. The guanylylate re sidue is methylated at the N7 position as indicated by the arrow labeled, present in all caps. The second site of methlylation, the O2 position of the initiating nucleo side, is indicated by the arrow labeled, present in cap 1.

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33 CHAPTER 2 MATERIALS AND METHODS 2.1 Methods for Phenotypic Characterization 2.1.1 Cells and Viruses A continuous line of green m onkey kidney cells BSC40 cells, were grown as previously described (Condit et al., 1983; Condit and Motyczka 1981). Growth and propagation of the wild type vaccinia virus strain, IHDW, and the temper ature sensitive mutant, Dts36, was performed as previously described (Lackner et al., 2003). One-step growth and plaque titration protocols were performed as described previously (Condit et al., 1983; Condit and Motyczka, 1981). A multiplicity of infection (m.o.i) of 10 was used for all experiments unless otherwise noted. The permissive temperature for viral infections wa s 31C and the non-permissive temperature was 39.7C. The non-permissive temper ature is the temperature at wh ich the wild type virus grows as expected by the ts viruses do not. Experi ments were performed in force draft Hotpack incubators (SP Industries, Warmin ster, PA) with quick temperatur e recovery and the temperature inside of the incubator was closely monitore d throughout the experimental time course. 2.1.2 Viral DNA Isolation and DNA Sequencing Isolation of viral DNA from BS C40-infected cells incubate d at 31C was performed as previously described (Lackner et al., 2003) using a Qiagen DNeasy tissue kit (Qiagen, Valencia, CA). The D1R gene was amplified by PCR using primers outside of the open reading frame (ORF) of the gene to yield a 2750 bp-product. An additional eight internal primers were used for the sequencing reactions on the D1R product. The D12L gene was amplified by PCR using primers outside of the ORF of the gene to yield a 1000 bp-product. An additional two internal primers were used in the sequencing reactions on the D12L product. The sequencing reactions were performed by the University of Florid a ICBR DNA Sequencing Core Laboratory. The

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34 resulting DNA sequences were assembled and analyzed using the Vector NTI software program (Invitrogen, Carlsbad, CA). 2.1.3 Marker Rescue One-step m arker rescue mapping of Dts36 was performed as previously described (Kato et al., 2004b). BSC40 cells on 60-mm dish es were infected at 31C w ith an appropriate m.o.i. as determined by terminal dilution. The infected ce lls were then transfec ted with 1.5 g of DNA PCR-amplified from wild type viral DNA using Lipofectin reagent (Invitrogen). The infected and transfected cells were incuba ted at 39.7 C for four days at which time they were stained with crystal violet and the numbe r of wt plaques were counted. 2.1.4 Virion Thermostability The therm ostability of the wild type and mutant virions was assayed as described previously (Fathi and Condit, 1991). Wild type and mutant virions at a concentration of 1 104 pfu/mL were incubated at 45C. At various tim es, aliquots were removed and frozen at -80C until all samples were collected. In order to de termine if the virions were thermolabile, the samples were analyzed by plaque titration at 31C. 2.1.5 Metabolic Labeling of Prot eins an d Gel Electrophoresis Monolayers of infected BSC40 cells on 35-mm dishes were metabolically labeled and samples were analyzed as described previously with slight modifications (Condit and Motyczka, 1981). The infected cells were labeled w ith 0.5 mL of 100Ci/mL of Redivue PromixL-[35S] in vitro cell labeling mix (1000 Ci/mmo l) (GE Healthcare, Piscataway, NJ) for 30 minutes. The labeled cells were lysed with Laemmli sample buffer and samples were analyzed by 11% sodium dodecyl sulfate-polyac rylamide gel electrophoresis (SDS-PAGE ). The gels were stained with Coomassie blue, destained, dried, and exposed to film.

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35 2.1.6 Isolation of Viral RNA Isolation of viral RNA fr om infected cells was performed as described previously (Cresawn et al., 2007). Monolayer s of BSC40 cells were infected with an m.o.i. of 10 and incubated at either 31C or 39.7C until various times post-infection when RNA was isolated from the infected cells. Total cellular RNA was isolated using the RNeasy RNA isolation kit (Qiagen) following the directions of the manuf acturer. The RNA was eluted from the RNeasy column by with 50 L of RNase-free water. Th e concentration of the RNA was determined by measuring the optical density (OD) at 260 nm. 2.1.7 Synthesis of Riboprobes Riboprobes used in Northern blot analysis were synthesized in several dif ferent manners. Antisense riboprobes specific for C11R, G8R, an d F17R mRNA transcripts were prepared as described previously (Hassett et al., 1997). The template for the F11R riboprobe was amplified by PCR from a pGEM plasmid that contained the open reading frame of the F11R gene linked to the bacteriophage SP6 promoter. The M13 forw ard and reverse primers were used for the amplification. Riboprobes specific for approxima tely 500 bp of A20R, D5R, and E9L mRNA transcripts were transcribed fr om PCR products as previously described with the following modifications (Cresawn et al., 2007). Transc ription templates were PCR amplified using a forward primer that was complementary to the 5 end of the ORF and which was tagged with the sequence CG TAATACGACTCACTATAGG GAGA containing the bact eriophage T7 promoter and a reverse primer complementary for a region 500 bp downstream of the 5 end of the ORF and tagged with the sequence CG ATTTAGGTGACACTATAGA AGCG containing the bacteriophage SP6 promoter. (The essential promoter regions are shown in italics). The riboprobes used in the F11 primer walk experiment were synthesized in the same manner except the regions of complementary were not restricted to the 5 end of the ORF. All riboprobes were

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36 transcribed using the MAXIscript in vitro transcription system (Ambion, Austin, TX) and purified using NucAway Spin Columns (Ambion) according to the manufacturers protocol. 2.1.8 Northern Blot Analysis Northern blot analysis was performed as described previously with the following modifications (Cresawn et al., 2007 ). Denaturing for maldehyde gels were run at 80 volts for 2 hours. Following transfer of the RNA to a Ge neScreen neutral charge membranes (Perkin Elmer, Waltham, MA), the membranes were preh ybridized for at leas t 6 hours. Following overnight hybridization, the membranes were washed one time at room temperature with 0.1 X SSC/0.1% SDS followed by three washes at 65C with 0.1 X SSC/1.0% SDS. After all washes were completed, the membranes were exposed to film and to phosphor screen (GE Healthcare) for quantification of viral RNA transcripts. Th e quantification data was analyzed using a Storm phosphorimager (GE Healthcare) and the ImageQ uant software program (GE Healthcare). 2.1.9 Western Blot Analysis Whole cell, infected cell lyastes were el ectrophoresed on 11% SDS-PAGE and transferred to nitrocellulose m embranes as described previo usly (Kato et al., 2004b). After transfer, the membranes were blocked overnight in TBS-T/NFDM (0.15 M NaCl, 0.05 M Tris-HCl, pH 7.5, 0.1% Tween-20, and 5% nonfat dry milk). The me mbranes were incubated with an appropriate dilution of primary antibody in TBS-T/NFDM for at least 1.5 hours after which the membranes were rinsed with TBS-T. Following the rinse step, the membranes were incubated for 1.5 hours with anti-rabbit Ig-conjugated to horseradish peroxidase (GE H ealthcare) at a dilution of 1:5000 in TBS-T/NFDM. The membranes were then rins ed six times with TBS-T and developed with the Enhanced chemiluminescence detection kit (G E Healthcare) according to the manufacturers protocol. The primary antisera used were as foll ows: anti-E9 (1:500), anti-D5 (1:1000), and antiA20 (1:1000) rabbit sera supplied by Dr. Paula Traktman (Medical College of Wisconsin). Anti-

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37 D1 (1:500) and anti-D12 (1:500 or 1:6000 dependi ng on the experiment) rabbit antisera were supplied by Dr. Ed Niles (SUNY-Buffalo). The anti-F11 (1:2000) rabbit serum was previously described (Kato et al., 2004a). Western blot autoradiograms were quantified using Image J (Abramoff et al., 2004). 2.1.10 Isolation of Viral DNA Replication Samples Viral DNA was isolated and viral DNA replicati on was analyzed as previously described (Traktm an and Boyle, 2004). The DNA was isolated from virally infected cells by scraping the infected cell monolayer into the culture media and recovering the cells by cen trifugation. The cells were washed once with phosphate buffered saline (PBS) and after washing, the cells were resuspended in a solution of 10 X SSC (1.5 M Na Cl, 0.15 M sodium citrate) and 1 M ammonium acetate. Samples were subject to three cycles of freeze/thawing to disrupt the cells and were subsequently stored at -20C. 2.1.11 Slot Blot Hybridization for Viral DNA Replication Isolated DNA replication sam ples were analy zed as previously described (Traktman and Boyle, 2004). The samples were applied to a hydrated and equilibrated Nytran Supercharge nylon transfer membrane (GE Healthcare) on a Minifold II Slot-Blotter apparatus (GE Healthcare). While the membra ne was still on the slot blot apparatus, the DNA was denatured with a solution containing 0.5 M NaOH and 1.5 M Na Cl and then neutralized by two washes of 10 X SSC. After removal of the membrane from the slot blot apparatus, the membrane was prehybridized at 42C in a hybridi zation oven (Labnet International, Inc., Edison, NJ) for at least 2 hours in a solution of 6 X SSC, 50% formamid e, 0.5% SDS, 5 X Denhardts solution (0.1% BSA, 0.1% polyvinylpyrolidone, 0.1% Ficoll), and 100 g/mL denatured salmon sperm DNA. After the prehybridization, 2.25 106 cpm of the randomly 32P-labeled (DECAprime II kit (Ambion)) HindIII E fragment was added to fresh hybridization solution and incubated with the

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38 membrane overnight at 42C. After the overnig ht incubation, the membrane was washed three times with 2 X SSC at room temperature followed by two washes at 55C with 0.2 X SSC/0.1%SDS. Following the washes, the membrane was exposed to film and was quantified using a phosphor screen (GE H ealthcare) and analyzed usi ng a Storm phosphorimager (GE Healthcare) and the ImageQuant so ftware program (GE Healthcare). 2.1.12 AraC Experiment Confluent monolayers of BSC40 cells were infect ed with an m.o.i. of 10 in the presence of 40 g/mL of cytosine -D-arbinofuranoside (araC). At va rious times post-infection, metabolic labeling of proteins, RNA isol ation, and DNA isolation were pr eformed as described above. 2.1.13 Shift-Up Experiment Confluent monolayers of BSC40 cells were inf ected with an m.o.i of 10 and incubated at 31C for eight hours. At 8 hours post-infection (hpi), the dishes of infected cells were moved to 39.7C. At various times post-infection, metabolic labeling of proteins, RNA isolation, and DNA isolation were preforme d as described above. 2.2 Methods for Biochemical Characterization 2.2.1 Virus Purification W ild type and mutant viruses were purified as previously described (Kato et al., 2004b). Briefly, confluent monolayers of BSC40 cells in 150 mm dishes were infected with an m.o.i. of 0.01 and were incubated at 31C until a complete CPE was observed. Cell-associated virus was purified by differential centrifuga tion through a 36% sucrose cushi on followed by banding of the virus on a 24-40% sucrose gradient. The purif ied virions were quantified by OD at 260 nm (1 OD260 = 68 g protein) and by the Bradford assay (Bio-Rad protein assay). Infectivity of the purified virus was measured by plaque titration of the virus on BSC40 cell monolayers at 31C.

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39 2.2.2 Preparation of Virion Extracts Virion extracts were prepared essentially as previously described but with the following modifications (Gross and Shum an, 1996). Purifi ed virus, 5 OD units, was incubated for 10 minutes at room temperature in 0.8 mL of a solution containing 50 mM Tris-HCl, pH 8, 10 mM dithiothreitol (DTT), and 0.05% nonidet P40 (NP-40). Virus cores were recovered by centrifugation and the resulting pellet was resu spended in 150 L of buffer N (300 mM TrisHCl, pH 8, 250 mM KCl, and 50 mM DTT). Th e samples were adjusted to 0.15% sodium deoxycholate and incubated on ice for 30 minutes with occasional mixing. After the 30 minute incubation, the samples were adjusted to 10% gl ycerol and insoluble ma terial was removed by centrifugation. The supernatants were applied to a 150 L DEAE column equilibrated with buffer M (50 mM Tris-HCl, pH 8, 250 mM KCl, 1 mM EDTA, 0.1% Triton X-100, 3 mM DTT, and 10% glycerol) and eluted with buffer M. Th e protein concentrations of the eluted fractions were measured by Bradford assay and the peak frac tions were combined and stored in aliquots at -80C. 2.2.3 In vitro Tra nscription by Permeabilized Virions Permeabilized virion transcription reactions were essentially performed as previously described (Kato et al., 2004b; Ka to et al., 2007). In order to determine the amount of incorporation into RNA, reaction mixtures containing 60 mM Tris-HCl, pH 8, 0.05% NP-40, 10 mM DTT, 10 mM MgCl2, 5 mM ATP, 1 mM GTP, 1 mM UTP, 0.2 mM CTP, 40 nM -32P-CTP (3000 Ci/nmole), and 2 OD/mL of purified virus were incubated at 31 or 37C. At various times, 50 L aliquots were removed, precipitated with 5% trichloroacetic acid (TCA), filtered on glass microfibre filters (934-AH, GE Healthcare), and TCA-precipita ble radioactivity was determined by liquid scintillation counting. In order to determine virion core-associated RNA versus released RNA, reactions were performed as above except that the 50 L aliquots were

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40 added to 150 L of a solution containing 50 mM Tris-HCl, pH 8, 10 mM EDTA, 10 mM DTT, and 0.05% NP-40 and centrifuged at full speed for 3 minutes. The resulting supernatants were removed and were precipitated with 5% TCA. The pellets were resuspended in 200 L of a solution of 50 mM Tris-HCl, pH 8 and 0.1% SDS and precipitated with 5% TCA. 2.2.4 Enzyme-GMP Complex Formation in Permeabilized Virions and Virion Extracts The for mation of the covalent intermed iate between the D1R subunit and the GMP molecule was analyzed as described previously (Hassett et al., 1997; Shuman and Moss, 1990). Various amounts of either purified virus or solu ble virion extracts were incubated for 10 minutes at 31C in a solution containing 50 mM Tris-HCl, pH 8.2, 0.05% NP-40, 5 mM MgCl2, 10 mM DTT, and 21 nM -32P-GTP (3000 Ci/nmole). The reaction was stopped by the addition of 5 X Laemmli sample buffer and the samples were analyzed by 10% SDS-PAGE. The gels were fixed, stained and destained, drie d, and exposed to film and the amount of labeled D1 protein was quantified by exposing the dried gel to a phos phor screen (GE Healthcare). The data was quantified using a Storm phosphorimager (GE Healthcare) and the ImageQuant software program (GE Healthcare). 2.2.5 Coupled Transcription and Methyltransf erase Assay in Permeabiliz ed Virions The methyltransferase activity of the cappi ng enzyme was analyzed by measuring the transfer of a radiolabeled met hyl group from S-adenosylmethioni ne (SAM) to newly synthesized and guanylylated virion transcript s as described previously wi th the following modifications (Gershowitz and Moss, 1979; Hassett et al., 1997). Equal amounts of either a 2 X transcription mixture (10 mM ATP, 2 mM GTP, 2 mM UTP, 0.4 mM CTP, and 80 nM -32P-CTP (3000 Ci/nmole)) or a 2 X methyltransferase mixture (10 mM ATP, 2 mM CTP, 2 mM GTP, 2 mM UTP, and 4 M 3H-SAM (10 Ci/nmole)) were combined w ith 0.5 OD/mL of purified virus in a solution of 60 mM Tris-HCl, pH 8, 0.05% NP-40, 10 mM DTT, 10 mM MgCl2. The samples

PAGE 41

41 were incubated at either 31 or 37C and at various times, 20 L aliquots were removed. The transcription samples were processed as described above in the in vitro transcription section. The methyltransferase samples were spotted onto DE81 filters (GE Healthcare) and unincorporated radioactivity was removed by wa shing two times with 0.5 M sodium phosphate, once with water, once with ethanol, and air dried. All filters were analyzed by liquid scintillation counting. 2.2.6 Uncoupled Methyltransferase Assay in P ermeabiliz ed Virion and Virion Extracts The transfer of a radiol abeled methyl group from 3H-SAM to GTP was measured in permeabilized virions and soluble virion extracts esse ntially as previously described (Hassett et al., 1997; Shuman and Moss, 1990). Purified vi rions at a concentration of 5 OD/mL were incubated at 31 or 37C in a solution contai ning 60 mM Tris-HCl, pH 8, 0.05% NP-40, 10 mM DTT, 10 mM MgCl2, 10 mM GTP, and 4 M 3H-SAM (10 Ci/nmole). At various times, 20 L aliquots were removed and spotted on DE81 filte rs. Various amounts of soluble virion extracts were incubated at 31 or 37C for 30 minutes in 25 L reactions containing 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 10 mM GTP, and 4 M 3H-SAM (10 Ci/nmole). At various times, 20 L aliquots were removed and spotted on DE81 filters. Filters were washed four times with 25 mM ammonium formate, once with water, twice with ethanol, dried, and analyzed by liquid scintillation counting. 2.2.7 Non-Specific RNA Polymerase Activity The non-specific RNA polym erase activity in soluble virion extracts was assayed as previously described with the following modificati ons (Hassett et al., 1997 ; Kato et al., 2007). Various amounts of soluble virion extracts were incubated for 30 minutes at 31 or 37C in 100 L reactions containing 60 mM Tris-HCl, pH 8, 10 mM DTT, 3 mM MnCl2, 1 mM ATP, 1 mM GTP, 1 mM UTP, 0.1 mM CTP, 10 nM -32P-CTP (3000 Ci/nmole), and 1g single-stranded

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42 M13 DNA. At the end of the incubation period, th e samples were precipitated with 5% TCA, filtered on glass microfibre filters (934-AH, GE Healthcare), and TCA-precipitable radioactivity was determined by liquid scintillation counting.

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43 CHAPTER 3 PHENOTYPIC ANALYSIS OF A TEMPERATU RE SENSITIVE MUTANT IN THE LARGE SUBUNIT OF THE VACCINIA VIRUS MR NA CAPPING ENZYME 3.1 Introduction The vaccin ia virus mRNA capping enzyme is a multifunctional enzyme involved in several aspects of viral transcription in addition to the capping of newly transcribed mRNA transcripts. These additional functions of th e capping enzyme, as an early transcription termination factor and an intermediate transcription initiation factor, have been h ypothesized as functions of the capping enzyme based on previous biochemical analyses as discussed in the introduction (Chapter 1). As mentioned in the introduction (Section 1.5), alt hough there has been a ts mutant in each of the capping enzyme subunits that has b een previously character ized, neither of these mutants have shown defects in early termina tion or intermediate initiation functions in vivo (Carpenter and DeLange, 1991; Hassett et al., 1997). In this chapter, the role of the viral mRNA capping enzyme in early transcription termination and intermediate transcription initiation in vivo will be explored through th e phenotypic characterization of two temperature sensitive vaccinia virus mutants which both have a mutation in the large subunit of the vaccinia virus capping enzyme. Characterization of one of th e two viruses, Dts36, shows that this virus possesses an early phenotype. Specifically, in Dt s36 at the non-permissive temperature, early mRNA metabolism is defective, early protein expression is ir regular, and both DNA replication and post-replicative gene expres sion are absent. Furthermore, temperature shift up experiments reveal a defect in intermediate mRNA metabolism in Dts36 infected cells. The results presented in this study indicate that at the non-permissive temperat ure, the mRNA capping, the early transcription termination, and the intermediate transcription initiation act ivities of the vaccinia virus capping enzyme are affected in Dts36.

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44 3.2 Results 3.2.1 Marker Rescue and DNA Sequencing Dts36 and Dts50 tem perature sensitive v accinia virus mutants comprise a newly discovered complementation group (L ackner et al., 2003). In order to determine the site of the mutation in these viruses, three rounds of a one-step marker resc ue were utilized (Kato et al., 2008). Briefly, confluent monolayers of BSC40 cells were infected with either the Dts36 or the Dts50 virus and transfected with DNA fragments that were PCR-amplified from wild type vaccinia virus DNA. The infected and transfecte d cells were incubated at 39.7C for four days and subsequently stained with crystal violet. A successful rescue of the ts virus is evident from the appearance of plaques on the dishes that were infected and transfecte d. The first round of marker rescue used PCR products comprising over lapping 20 kilobase (k B) fragments spanning the vaccinia virus genome (Yao and Evans, 2003) The second round of marker rescue used PCR products that used overl apping 5kB PCR fragments spa nning the vaccinia virus genome (Luttge and Moyer, 2005) and the third round used PCR products for individual open reading frames of vaccinia virus genes. Marker rescue of Dts36 (Fig. 3-1) and Dts50 (data not shown) shows that each mutant was rescued by a PCR pr oduct amplified from the wild type D1R gene which indicates that both viruses possess a mutati on in D1R, the large su bunit of the viral mRNA capping enzyme. The D1R gene from Dts36, Dts50, and two pare ntal strains of vaccinia virus, IHDW and WR, was sequenced as described in materials an d methods. IHDW was sequenced because it is the parental strain from which the ts mutants in the study were made and WR was sequenced to determine if there are any differences in the D1R gene between the two parental strains of vaccinia virus. The sequence of D1R in the WR strain was found to be identical to the previously published sequence. By contrast, there are 24 polymorphisms in D1R comparing the

PAGE 45

45 WR and IHDW parental strains. Of these 24 po lymorphisms, only three re sult in changes in the protein sequence of D1 (V5I, T202K, and R812K). Both Dts36 and Dts50 were found to have an identical single point mutation at nucleotide residue 2114 which cause s a glycine to aspartic acid coding change at amino acid residue 705 in th e protein sequence (G705D) (Fig. 3-2). Thus, Dts36 and Dts50 are sibling viruses and therefore, for the rema inder of this study, only Dts36 was used. The D12L gene, which encodes the small s ubunit of the viral mRNA capping enzyme, of Dts36, IHDW, and WR was also sequenced as descri bed above. The D12L gene from all viruses sequenced were found to have iden tical sequences to each other as well as to the previously published sequence. 3.2.2 Growth Properties and Virion Thermostability of Dts36 A one-step growth experim ent was used to determine the growth properties of Dts36 and IHDW. Confluent monolayers of BSC40 cells were infected with a high m.o.i. and incubated at 31 or 39.7C and infected cells and media were ha rvested and used to measure viral yield in plaque assays at 31C (Fig. 3-3). At 24 hours post-infecti on, IHDW grown at either the permissive or the non-permissive temperature produced between 16 and 29 pfu per cell. Dts36 grown at 31C was virtually i ndistinguishable from IHDW grow n at either temperature. However, Dts36 grown at 39.7C produced no detect able infectious progeny when analyzed by plaque assays at 31C. The viral mRNA capping enzyme is a virion protei n so it was critical to determine if the Dts36 virions were thermolabile; a characteristic th at could impact further experiments. A virion thermostability assay was used to determine the stability of the Dts36 and the IHDW viruses at 45C. A known amount of virus, 1 104 pfu/mL, was incubated for up to 4 hours and the stability of the viruses was measured by plaque titration (Fig. 3-4). Throughout the entire time

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46 course, both the wild type virus, IHDW, and the mutant virus, Dt s36, are equally stable with only a slight decrease in thermostability seen as s oon as two hours into the incubation with either virus. 3.2.3 Basic Phenotypic Characterization of Dts36 The basic ph enotypic characterization of Dts36 included examining viral protein synthesis, viral DNA replication, and viral mRNA synthesi s at the permissive and non-permissive temperatures. 3.2.3.1 Viral protein synthesis Viral protein synthesis was used as a m easure of overall vi ral gene expression in virus infected cells. Cells infected with either Dts36 or IHDW at 31 or 39.7C were metabolically labeled with 35-S methionine, samples were analyzed by SDS-PAGE, and the radiolabeled proteins were detected by autoradiography (Fig. 3-5A). The normal temporal expression of viral genes is observed in the wild type infection. Early vira l proteins are visible at two hpi and their synthesis peaks at 4 hpi when in fected cells are incubated at 31 C and at 2 hpi when infected cells are incubated at 39.7C. Th e shut-off of host cell protein synthesis is also evident in wild type infections as early as 2 hpi and extends th roughout the remainder of the infection. Postreplicative protein synthesis is marked by th e appearance two viral proteins, p4a and p4b, beginning at 6 hpi in infected cells incubated at 31C and at 4 hpi in infected cells incubated at 39.7C and continuing throughout the remainder of the experiment. The pattern of protein expression in Dts36 infections at 31C is indistinguishable from IHDW at either temperature. By contrast, non-permissive Dts36 infections sh owed extended early viral protein synthesis, decreased shut-off of host protein synthesis, and no post-replicativ e viral protein synthesis. The synthesis of the D1 protein was verified by West ern blot analysis and quantified using Image J (Fig. 3-5B, C). Dts36 synthesized 50-60% less D1 protein compared to IHDW at both

PAGE 47

47 temperatures; however, at both the permissive and the non-permissive temperatures, Dts36 synthesized similar amounts of the D1 protein, as did IHDW (Fig. 3-5C). 3.2.3.2 Viral DNA synthesis Viral DNA synthesis in Dts36 a nd IHDW infected cells at bo th the permissive and the nonperm issive temperatures was also examined. DNA was isolated from infected cells, applied to a nylon membrane, probed with a radiolabeled prob e specific for a region of the vaccinia virus genome, and quantified using a phosphorimager (Fig. 3-5D). The kinetics of DNA replication in IHDW infected cells at both 31 and 39.7C and in Dts36 infected cells at 31C were virtually indistinguishable. In non-permissive infectio ns, no viral DNA replication was observed in Dts36 infected cells. 3.2.3.3 Viral RNA synthesis The synthesis of viral m RNA in Dts36 infec tions at the non-permissive temperature was analyzed by northern blot. RNA wa s isolated from infected cells incubated at either 31 or 39.7C, separated by gel electrophoresis, transferred to a neutral membrane and probed for viral gene expression. The riboprobes used were specific for a gene from each of the three classes of viral genes. Autoradiograms of northerns for the early gene (C11R, the vaccinia growth factor), the intermediate gene (G8R, a vaccinia late tran scription factor) and the late gene (F17R, a DNA binding phosphoprotein) are shown in Fig. 3-6A. In infections with the WR strain of vaccinia virus, early gene expression peak s and then declines for the rema inder of the infection (Baldick, Jr. and Moss, 1993). The current study is the first time that gene expression has been examined in IHDW infections and much to our surprise in wild type IHDW infections, early gene expression peaks, decreases and then resumes at ti mes late in infection. Early gene expression as analyzed by northern analysis for IHDW infected cells at 31 and 39.7C and for Dts36 infected cells at 31C are virtually indistinguishable from one another. The peak of synthesis of the

PAGE 48

48 C11R transcript occurs at 6 hpi at 31C for both viruses and at 3 hpi at 39.7C for IHDW. The peak of synthesis of C11R transcript in Dts36 at 39.7C occurs at 3 hpi as in IHDW at this temperature; however, the mutant produces only 35% the amount of C11R transcript compared to wild type virus infections at this time. The C11R transcript in the Dt s36 infection incubated at 39.7C also does not reappear at times late in inf ection. It should also be noted that the C11R transcript in both viruses at bot h temperatures is of the correct size, approximately 0.5kb (Fig. 36B). Much like the analysis of C11R gene e xpression, the intermediate and late gene expression for IHDW infections at 31 and 39.7C and fo r Dts36 infections at 31C are virtually indistinguishable from one anothe r; however, the analysis of postreplicative gene expression in mutant infections at 39.7C showed no intermediate or late viral transcript s, consistent with the absence of viral DNA replication and post-replicative proteins di scussed earlier. These results also suggest that the resumption of early gene expression observ ed in permissive infections requires late gene expression. 3.2.3.4 Viral protein and RNA synthesi s in the presence of araC Dts36 appeared to synthesize the sam e amount of early viral proteins as IHDW at the nonpermissive temperature when measured by protein pulse labeling (Fig. 3-5A). However, the lack of host cell protein synthesis shut-off in Dts36 infections incubated at 39.7C and the earlier switch to post-replicative protei n synthesis in IHDW infections incubated at 39.7C made it difficult to accurately compare pr otein synthesis levels in the two viruses. Therefore, an additional pulse labeling and RNA isolation was performed in the presence of cytosine -Darabinofuranoside (araC), an i nhibitor of DNA synthesis that w ould allow accumulation of early viral message and protein (Fig. 3-7). The pattern of ear ly viral protein synthesis in infections with Dts36 and IHDW at both temperatures was i ndistinguishable with bo th viruses apparently synthesizing the same proteins in the same amounts (Fig. 3-7A). In the analysis of early viral

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49 mRNA synthesis, cells infected w ith IHDW at both temperatures and cells infected with Dts36 at 31C were indistinguishable; how ever, the amount of C11R me ssage at 3 hpi in the Dts36 infections was 15% of that measured in the IHDW infections (Fig. 3-7B). 3.2.4 Early gene transcription termination is defective in Dts36-infected cells Analysis of other early viral genes by nor thern and western blots was perf ormed to investigate whether the observed decrease in steady state early viral RNA is a global phenomenon or if it is specific for the C11R gene. The early viral genes examined were F11L (a gene involved in cell signaling (Valderrama et al., 2006)), E9L (the DNA polymerase), A20R (a DNA polymerase processivity factor (Klemper er et al., 2001)), and D5R (an ATPase/DNA primase (Boyle et al., 2007; De Silva et al., 2007)). 3.2.4.1 Analysis of F11L transcript in Dts36 non-permissive infections Analysis of the F11L transcript showed that sim ilar patterns of expression were evident in infections with IHDW at both temperatures and Dt s36 at the permissive te mperature (Fig. 3-8A). In the wild type infections, the major F11L transcript migrates at 1 kb. A second, slightly slower migrating band is seen at 1.35 kb (band 4 in Fig. 3-8A) and most likely repr esents termination at the next downstream termination signal past the F11L termination signal. Both bands peak at 3 hpi, decrease, and reappear at late times during th e wild type infection at both the permissive and the non-permissive temperatures. At late times during the wild t ype infection, read through from an upstream late gene, most likely F13L, is evid ent as a smear from 1 kb to greater than 4 kb. The Dts36 infections at 31C are similar to wild type infections; however, there are two differences. First, by visual inspection, the ra tio of the 1.35 kb band to the 1 kb band (band 4 to the F11L band in Fig. 3-8A) appear s to be greater in the Dts36 31C infections than in the wild type infections. Second, there is an additional band at 2.4 kb in the mutant infections at the permissive temperature indicating that the mutant is slightly leaky at the pe rmissive temperature.

PAGE 50

50 The pattern of F11L transcript expression was different in Dts 36 infected cells incubated at 39.7C. The F11L transcript was present but wa s no longer the predominant transcript. There were four additional transcripts: band 1 ( 3.6 kb), band 2 (2.4 kb), band 3 (1.65 kb) and band 4 (1.35 kb) (Fig 3-8A). The F11L transcript and the additional transcript s all peaked at 3 hpi, decreased slowly, and did not reap pear at times late during infec tion. The read through from the upstream late genes was also not apparent in mutant inf ections at the non-permissive temperature. The size of these additional bands corresponds to the predicte d length of transcripts that initiate at the F11L prom oter and terminate at terminatio n sequences downstream suggesting that these bands result from read-through of term ination sequences (Fig. 3-8B). In order to confirm that these bands were indeed longer-th an-expected F11L transcripts, further northern analyses were performed using riboprobes designed to detect the l ongest two additional transcripts seen in the initial F11L northern analysis (Fig. 3-9). One riboprobe, designated F10, was capable of detecting the two longest of the additional transcripts (bands 1 and 2) and a second riboprobe, designated F8/F7, was capable of detecting only the longest additional transcript (band 1). The results showed that when the F10 riboprobe was utilized only the two largest transcripts were seen and when the F8/F7 riboprobe was utilized only the largest transcript was seen in Dts36 at 39.7C. The leakiness seen in permissive infections with Dts36 in Fig. 3-9 is also apparent in this experiment. In addition to detecting band 1 in non-permissive Dts36 infections, the F8/F7 riboprobe was also able to detect the F7L transcript. This transcript behaved much like the C11R transcript in cells infected with Dts36 and incubated at 39.7C: it was present in decreased amounts co mpared to the wild type infec tions at either temperature and the mutant at the permissive temperature.

PAGE 51

51 3.2.4.2 Analysis of the E9L, A20R, and D5R trans cripts in Dts36 non-permissive in fections The analysis of the E9L transcript yielded resu lts similar to those seen in the analysis of the F11L transcript (Fig. 3-10A). The E9L transcript is visible at 3 hpi in wild type infections at both the permissive and the non-permissive temperat ures; however, it is di fficult to determine if the transcript reappears late dur ing infections because of the la rge amount of read through from an upstream late gene (most likely E11L). In infe ctions with Dts36 at 31C, the E9L transcript is visible as is an additional, slow er migrating band at 4.7 kb (band 2 in Fig. 3-10A). Both of these transcripts peak at 3 hpi, decr ease, and reappear at times late during infection. Much like the wild type infections, there is re ad through of a late gene that is located upstream of E9L in the Dts36 infections incubated at 31C. In mutant infections incubated at 39.7C, the amount of the E9L transcript was decreased at 3 hpi, expressi on of E9L did not resume late during infection, and longer-than-expected E9L tran scripts appeared. As with F 11L, the size of the additional transcripts corresponded to termination sequences downstream of the E9L termination sequence (Fig. 3-10B). Northern analyses were also us ed to examine the viral transcripts encoded by A20R and D5R (data not shown). In Dts36 infect ed cells at 39.7C, the A20R transcript was terminated correctly but was pr esent in decreased amounts and did not reappear late during infections. The D5R transcript in mutant inf ections at the non-permissive temperature was present in decreased amounts, di d not reappear late during viral infection, and did not terminate correctly. 3.2.4.3 Analysis of the F11, E9, A20, and D5 pr otein synthesis in D ts36 non-permissive infections It was of interest to determin e the effect that the longer-th an-expected transcripts and the decreased amount of transcripts had on viral prot ein synthesis. Therefore, the synthesis of proteins encoded by these transcri pts was analyzed by western blot (Fig. 3-11). For all proteins

PAGE 52

52 examined, the protein of interest was expressed in similar amounts in the wild type infections at both temperatures and the mutant infections at 31 C. When F11 was examined in Dts36 infected cells at 39.7C, the mutant expres sed at least the same amount if not more of the protein than IHDW at the same temperature. On the other ha nd, the E9, A20, and D5 proteins were expressed in decreased amounts in Dts36 infected cells at 39.7C compared to IHDW at 39.7C. 3.2.5 Intermediate Gene Transcription is Compromised in Dts36-infected Cells The m ultifunctional vaccinia virus capping enzyme has been implicated in intermediate gene transcription initiati on; therefore, it was of interest to determine whether intermediate gene transcription was defective in Dts36 infections at the non-permissive temperature. Because Dts36-infected cells are defec tive in DNA replication and post -replicative gene expression, a typical time course of infecti on does not provide any insight into the impact of the mutation on intermediate gene expression. However, we were able to circumvent this problem by performing a shift-up experiment in which the infection was initiated and incubated at 31C for 8 hours and then shifted to 39.7C for the remainder of the ex periment (Fig. 3-12A). Viral protein synthesis was analyzed by pulse labeling (F ig. 3-12B) and viral DNA replicati on was assayed by slot blot analysis (Fig. 3-12C). Before and after the shift to 39.7C, similar amounts of post-replicative viral proteins and of DNA replication were seen for both IHDW and Dts36 infections. It is important to note that Dts36 continues bot h post-replicative protein synthesis and DNA replication. Viral RNA synthesis was also examined in th e shift-up experiment by northern analysis in which autoradiograms were used to qualitati vely assess RNA synthesis (Fig. 3-13A) and phosphorimager analysis was used to quantitativel y assess RNA synthesis (Fig. 3-13B-D). Early and late transcripts were synthe sized in similar amounts by both viru ses after the shift to 39.7C. When an intermediate transcript was analyzed, the IHDW infections showed a constant amount

PAGE 53

53 of intermediate synthesis whereas the Dts36 infections showed a striking decrease in intermediate synthesis af ter the shift to the non-pe rmissive temperature. 3.3 Summary In this chapter, the ph enotypic characterizati on of a temperature sens itive mutant with a mutation in the large subunit of the vaccinia virus mRNA capping enzyme is discussed. The detailed analysis of Dts36 showed that this mutant possesses an early phenotype. At the nonpermissive temperature, Dts36 infections s how aberrant early gene expression, are DNA negative, and show no post-replicative gene expression. Analysis of various early mRNA transcripts in Dts36 infections incubated at the non-permissive temperature reveals that some but not all early viral mRNAs are synthesized in reduced amounts. This phenomenon extends to early viral protein synthesis as well. It is important to note that we have not directly measured the cap status of viral mRNAs in mutant infected cells. However, given the role of the D1R gene in mRNA capping, the reduction in amounts of both early mRNA and proteins in Dts36 infected cells is consistent with a loss of stability of th e nascent mRNA transcripts resulting from a defect in mRNA capping. Additional analysis of early viral mRNA transcripts from non-permissive Dts36 infections also reveals that some early mR NA transcripts are longer than expected. This phenomenon indicates that at the non-permi ssive temperature the mutant capping enzyme encoded by Dts36 is defective in early viral tran scription termination. Finally, analysis of postreplicative viral transcripts produ ced during an infection that was initiated at the permissive temperature and then shifted to the non-permissive temperature reveals that the intermediate transcript examined in this study decreases in amount after the temperature shift while the late viral transcript examined is not affected. This observation is consistent with a defect in the intermediate transc ription initiation function of the mutant capping enzyme encoded by Dts36.

PAGE 54

54 Figure 3-1. Marker rescue of Dt s36. A) Hind III restriction map of the vaccinia virus genome. The area of the genome that comprises LM 23, the 5 kb PCR product that successfully rescued the mutant infection at the non-perm issive temperature, is shown. B) Dishes stained with crystal violet are shown labeled with the ge ne-specific PCR product used in the transfection.

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55 Figure 3-2. The D1 protein. The enzymatic domains of the vaccinia virus mRNA capping enzyme are shown. The location of the mutati on in the D1 protein from Dts36 is also indicated by the arrow and *.

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56 Figure 3-3. One-step growth of IH DW and Dts36 viruses. Cells were infected with an m.o.i. of 10 and incubated at either the permissi ve (31C) or non-permissive (39.7C) temperature. Samples were collected at va rious times post-infection (x-axis) and viral yields were determined by plaque titration at 31C (y-axis).

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57 Figure 3-4. Virion thermostability of Dts36 and IHDW at 45C. Wild type or mutant virus was incubated at 45C for various amounts of tim e (x-axis), aliquots were removed, and the amount of virus titer remaining (y-axis) was determined by plaque titration at 31C.

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58 Figure 3-5. Basic phenotypic characterization of Dts36: protein synthesis, D1 synthesis, and DNA replication. A) Autoradiograms of vira l protein synthesis in Dts36 and IHDWinfected cells. The virus with which the cells were infected is listed to the right of the autoradiograms and the temperature at whic h the infected cells were incubated is listed above the autoradiograms. The molecular weights in kD are listed to the left of the autoradiograms. Examples of the different classes of viral proteins are labeled as follows: = host protein, # = early viral prot ein, ^ = post-replicativ e viral protein. B) Western blot analysis of the amount of D1 protein present in Dts36 and IHDWinfected cells. Listed above each autoradiogr am is the virus used to infect the cells, the temperature at which the infected cells were incubated, and the hour postinfection at which the sample was isolate d. The molecular wei ghts in kDa are listed to the left of the autoradiogram. C) Quan tification of the autoradiograms of the D1 western blots. The hour post-infection when the sample was isolated is on the x-axis and the y-axis shows the re lative intensity. D) Synthe sis of viral DNA in cells infected with Dts36 or IHDW. Samples were analyzed in triplicate and the resulting plot of the quantified data represents an av erage of these values. Error bars represent the standard deviations based on the data.

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59 Figure 3-5. Continued

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60 Figure 3-6. Analysis of early, intermediate and la te RNA synthesis in cells infected with Dts36 or IHDW. A) Autoradiograms of northern blot analysis of tota l RNA isolated from cells infected with Dts36 or IHDW. Ribopr obes used in this analysis were specific for early (C11R), intermediate (G8R), and late (F17R) viral transcripts. The virus with which the cells were infected, the temp erature at which the infected cells were incubated, and the hour post-infection at wh ich the RNA was isolated are listed above the autoradiograms. The riboprobe which was used for each northern is listed to the right and the approximate molecular weights in kb is listed to the left of the autoradiograms. B) A map of the region of the vaccinia virus genome which contains the C11R gene. The C11R gene and the downstream C10L gene are shown as arrows which point in the direction in which each gene is transcribed. The early transcription termination signals are shown as black boxe s. Below the genome map are arrows which represent viral transcripts that initiate at the C11R initiation site and terminate at each of the shown termination signals. The length of each of these transcripts is listed to the right of each transcript.

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61 Figure 3-7. Viral protein and mRNA synthesis in cells infected Dts36 or IHDW in the presence of araC. A) Autoradiograms of protein synthesis in cells infected with Dts36 or IHDW in the presence of araC. Labeled a bove the autoradiograms are the virus with which the cells were infected, the temper ature at which the infected cells were incubated, and the hour post-infection at which the samples were isolated. The molecular weights in kDa are listed to the left of the autoradiograms. B) Synthesis of the early viral transcript, C11R, in cells in fected with Dts36 or IHDW as determined by Northern blot analysis. The riboprobe used in this analysis was specific for C11R. Labeled above the autoradiograms are the virus with which the cells were infected, the temperature at which the infected cells were incubated, and the hour postinfection at which the RNA was isolated. The molecular weights in kb are listed to the left of the autoradiograms.

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62 Figure 3-8. Analysis of the early viral transcript, F11L, in cells infected with Dts36 or IHDW. A) Autoradiograms of northern blot anal ysis using a riboprobe specific for F11L. Labeled above the autoradiogram are the viru s with which the cells were infected, the temperature at which the infected cells we re incubated, and the hour post-infection at which the viral RNA was isolated. The appr oximate molecular weight in kb is found to the left of the autoradiogram. The F 11L transcript, as well as the longer than expected F11L transcripts, ar e labeled to the right of the autoradiogram. B) A map of the region of the vaccinia virus genome which contains the F11L gene. The F11L gene and the downstream genes are shown as arrows which point in the direction in which each gene is transcribed. The early transcription termination signals are shown as black boxes. Below the genome map are arrows which represent viral transcripts that initiate at the F11L initiation site and terminate at each of the shown termination signals. The length of each of these transcri pts is listed to the right of each transcript and those transcripts which correspond to bands 1-4 in part A are labeled at the left end of the transcript. The regions of the genome where the F10 and F8/F7 riboprobes hybridize are shown in boxes above the transcripts.

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63 Figure 3-9. Primer walk experiment with various riboprobes for the analysis of the longer than expected F11L transcripts. The RNA analy zed in the northern blot was isolated at 3 hpi. The riboprobe used in the analysis and the virus with which the cells were infected is listed above the autoradi ograms. I31 = IHDW, 31C; I40 = IHDW, 39.7C; D31 = Dts36, 31C; D40 = Dts36, 39.7C. The molecular weights in kb are listed to the right of autora diograms and bands 1-4, the F 11L transcript, and the F7L transcript are labeled to the right of the autoradiogram.

PAGE 64

64 Figure 3-10.Analysis of the early viral transcript, E9L, in cells infected with Dts36 or IHDW. A) Autoradiograms of northern blot analysis using a riboprobe specifi c for E9L. Labeled above the autoradiogram are the virus w ith which the cells were infected, the temperature at which the infected cells we re incubated, and the hour post-infection at which the viral RNA was isolated. The appr oximate molecular weight in kb is found to the left of the autoradiogram. The E9L transcript, as well as the longer than expected E9L transcripts, are labeled to th e right of the autoradiogram. B) A map of the region of the vaccinia vi rus genome which contains the E9L gene. The E9L gene and the downstream genes are shown as arrows which point in the direction in which each gene is transcribed. The early tran scription termination signals are shown as black boxes. Below the genome map are arrows which represent viral transcripts that initiate at the E9L initiation site and te rminate at each of the shown termination signals. The length of each of these transcri pts is listed to the right of each transcript and those transcripts which correspond to ba nds 1 and 2 in part A are labeled at the left end of the transcript.

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65 Figure 3-11. Synthesis of the F11, E9, A20, a nd D5 viral proteins during Dts36 and IHDW infections. Samples were analyzed and blots were prepared and probed. The virus with which the cells were infected, the temp erature at which the infected cells were incubated, and the hour post-infection at whic h the samples were isolated are listed above the autoradiograms. The primary antibody used to probe each membrane is listed to the right of the autoradiograms and the molecular weights in kDa are listed to the left.

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66 Figure 3-12. Analysis of protein synthesis a nd DNA replication in Dts36 and IHDW-infected cells during a shift-up experiment. A) Sche matic of the shift-up experiment in which cells were infected and inc ubated at 31C for 8 hours and then shifted to 39.7C. B) Autoradiogram of protein synthesis in wh ich the virus with which the cells were infected, the temperature at which the in fected cells were incubated, and the hour post-infection at which the samples were is olated are listed above. The molecular weights in kDa are labeled to the left of the autoradiogram. B) Synthesis of viral DNA in cells infected with Dts36 or IHDW. Samples were analyzed in triplicate and the resulting plot of the quantified data repr esents an average of these values. Error bars represent the standard deviations based on the data.

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67 Figure 3-13. Analysis of viral RNA synthesis in Dts36 and IHDW-infected cells during a shiftup experiment. A) Autoradiograms of norther n blot analysis of total RNA isolated from cells infected with Dts36 or IHDW. Riboprobes used in this analysis were specific for C11R, G8R, and F 17R viral transcripts. The virus with which the cells were infected, the temperature at which th e infected cells were incubated, and the hour post-infection at which the RNA was isolated are listed above the autoradiograms. The riboprobe which was used for each northern is listed to the right and the approximate molecular weights in kb is listed to the left of the autoradiograms. B-D) Quantificatio n of the viral mRNA synthesis by phorphorimager analysis. B) C11R, C) G8R, and D) F17R.

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68 Figure 3-13. Continued

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69 CHAPTER 4 BIOCHEMICAL ANALYSIS OF A TEMPERATURE SENSITIVE MUTANT IN THE LARGE SUBUNIT OF THE VACCIN IA VIRUS MRNA C APPING ENZYME 4.1 Introduction As discussed in Chapter 3, the tem perature se nsitive virus Dts36 has an early phenotype and is defective in both early transcription termination and inte rmediate transcription. Based on the phenotypic analysis, it was possible that Dts3 6 had a defect in one of the three enzymatic activities that are required for the formation of a cap structure on the nascent mRNA transcript. The decreased message stability observed in the phenotypic characterization (Figs. 3-5 3-9) is consistent with a defect in the guanylyltransferase activity and the decreased translation efficiency of some viral proteins (Fig. 3-10) is consistent with a defect in the methyltransferase activity. In this chapter, the mRNA guanyl yltransferase and the mRNA (guanine-N7)methyltransferase activities of Dts36 will be explored through biochemical assays in vitro Characterization of Dts36 shows that the muta nt capping enzyme has a slight defect in guanylyltransferase activity as measured by the formation of the enzyme-GMP complex and that the mutant capping enzyme is completely defective in methyltransferase as measured by two separate assays. The results presented in th is chapter show that the Dts36 mRNA capping enzyme is not able to properly cap ne wly synthesized viral transcripts. 4.2 Results 4.2.1 Analysis of D1 Protein in Virions and Virion Extracts Af ter purification of the IHDW and Dts36 viruses, it was of gr eat interest to determine the protein composition of the purified virions as well as the amount of D1 and D12 proteins present in the purified virions (Fig. 4-1). Analysis of a coomassie-stained SDS-polyacrylamide gel determined that the protein composition of the purified virions as well as the amount of protein present in the purified virions were indistinguishable between IHDW and Dts36 virions (Fig. 4-

PAGE 70

70 1A). The relative amount of D1 and D12 protei ns, as compared to the amount of F17, present in the purified virions was determined by western bl ot analysis (Fig. 4-1B) and quantified using Image J (Fig. 4-1C). Purified virions from both viruses were shown to synthesize similar amounts of both viral capping enzyme subunits and the core protein, F17. The amount of D1 and D12 proteins present in the extracts of IHDW or Dts36 virions was also determined by western blot analysis (Fig 4-2) and it was revealed that equivalent amounts of extracts contained equivalent amounts of both subunits of the viral capping enzyme. 4.2.2 Analysis of mRNA Cappin g Enz yme Activities in Permeabilized Virions The purified vaccinia virus virions can be tr eated with NP-40, a non-ionic detergent, and DTT, a reducing agent, which will permeabilize the virus by removing the outer membrane but the virus core is left intact. The resulting pe rmeabilized virions are functionally active and when incubated with nucleotide triphosphates, the perm eabilized virions are capa ble of all stages of early viral transcription incl uding modification of the nascen t transcripts. In this study, permeabilized virions were used to assess two of the enzymatic functions of the D1 protein, guanylyltransferase and methyltransferase activitie s, as well as a preliminary assessment on the termination function of the vacci nia virus mRNA capping enzyme. 4.2.2.1 In vitro tra nscription in permeabilized virions In this study, preliminary in vitro transcription experiments were performed to determine the thermal-inactivation profile of the wild type and mutant virions. From these preliminary experiments, it was determined that the wild type and mutant virions were producing similar amounts of transcripts at all temperatures analyzed (31, 37, and 42C); however, the amount of transcripts synthesized at 42C by either wild type or mutant vi rions plateaued very quickly and therefore, this temperature was not used in further experiments. The initial in vitro transcription reactions were used as controls for the coupled methyltransferase assay that will be discussed in

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71 further detail subsequently (in section 4.2.2.3). Additional in vitro transcription experiments were performed to determine if the RNA that wa s being transcribed in the permeabilized virions was being released or if it was being retained in the viral core due to a lack of termination (Shuman and Moss, 1989). In vitro transcription reactions were in cubated at 31 or 37C for various amounts of time, core and released fractions were separa ted by centrifugation, and incorporation of radiolabeled CMP was measured by TCA precipitation (Fig.4-3). Both the wild type and the mutant virions showed the same pattern of transcription at all temperatures assayed; the synthesized viral transcripts were released from the core as expected if termination is occurring. 4.2.2.2 Enzyme-GMP complex formation In perm eabilized virions, guanylyltransferase ac tivity can be assessed through the use of an enzyme-GMP complex formation protocol. Th e guanylyltransferase re action in the mRNA cap formation proceeds in two steps; the first of which involves the formati on of a covalent capping enzyme-GMP intermediate. It is the form ation of the covalent capping enzyme-GMP intermediate which is used as a measure of guanyl yltransferase activity in permeabilized virions. The successful formation of the D1-GMP complex implies that the second step of the guanylyltransferase reacti on, the transfer of the guanylylate re sidue to the beta phosphate of the 5 end of the nascent RNA transcript, has occurr ed allowing the formation of a suitable substrate for the subsequent methyltransferase assay th at will be discussed in section 4.2.2.3. Varying amounts of IHDW and Dts36 permeabilized virions were incubated with [ -32P]-GTP at 31C and the radiolabeled D1-GMP complexes were analyzed by SDS-PAGE, autoradiography and phosphorimager analysis (Fig. 4-4). The au toradiographic analysis, as well as the phosphorimager analysis in which the amount of enzyme-GMP complex formation was compared to the amount of D1 protein present in the purified virions as determined by western

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72 blot (Fig. 4-1), showed that there was a slig ht difference in the formation of the D1-GMP complex between the wild type and mutant virion s, with the Dts36 D1 protein only expressing approximately 80% of the activity of the wild type protein. 4.2.2.3 Analysis of (guanine-N7 )-methyltransferase a ctivity The (guanine-N7)-methyltransferase activity of the vaccinia virus mRNA capping enzyme can be measured in one of two ways: by a coupl ed transcription, methyltransferase assay or by an uncoupled methyltransferase assay. The coupled methyltransferase assay uses newly synthesized and guanylylated transc ripts from the virion cores as the substrate for the (guanineN7)-methyltransferase reaction (G ershowitz and Moss, 1979). The wild type or Dts36 purified virions were permeabilized with NP-40 and DTT and either analyzed for methyltransferase activity or in vitro transcription (Fig. 4-5) The methyltransferase activity was assayed by following the incorporation of a radiolabeled methyl group from SAM into acid insoluble material and the transcription activity was assaye d by following the incorpor ation of radiolabeled CMP into nascent transcripts. The transcripti on activities for both the wild type and mutant virions at both temperatures examined, 31 and 37C, were virtually indi stinguishable from one another. This results sh ows that virions from both viruses were able to synthesize substrates that were suitable for the methyltransferase reactions that were being examined in parallel reactions. In stark contrast to the transcription results, the mutant virions have no me thyltransferase activity at either temperature tested whereas the wild type virions have comparable amounts of methyltransferase activity at both temperatures. The second assay that is used to examine th e (guanine-N7)-methyltransferase activity of the viral mRNA capping enzyme is an uncoupled assa y. In this assay, instead of using a newly synthesized transcript as the s ubstrate for the methylation reacti on, a GTP residue is used as the methyl acceptor which allows for the direct meas urement of the (guanine-N7)-methyltransferase

PAGE 73

73 activity (Fig. 4-6). Much like th e coupled assay, the methyltransferase activity was determined by following the incorporation of radiolabeled methyl group from SAM to a GTP molecule (Fig. 4-7). In agreement with the re sults from the coupled assay, mutant virions had no appreciable (guanine-N7)-methyltransferase activ ity compared to the wild type virions. The results from the two methyltransferase assays clear ly show that the capping enzyme found in the Dts36 virions is defective in the (guanine-N7)-met hyltransferase activity which is also consistent with data from the phenotypic characterization of Dts36. 4.2.3 Analysis of mRNA Capping Enzyme Activities in Virion Extracts Permeabilized virions can be further solubi lized upon treatment with deoxycholate, an ionic detergent. The resulting sol uble extracts are less stable than permeabilized virions but still contain the functional viral protei ns involved in the transcripti on of early genes including both subunits of the mRNA capping enzyme (reviewed in (Condit and Niles, 2002)). In the study presented in this chapter, so luble virion extracts were used to further assess the mRNA guanylyltransferase and the mRNA (guanine-N7)-methyltransferas e activities of the mutant capping enzyme. 4.2.3.1 Non-specific RNA polymerase activity The solubilization of the enzymes from the wild type and mutant virions was determined to be equivalent by testing the RNA polymerase acti vity of the extracts in a non-specific RNA polymerase assay. Equivalent amounts of soluble extracts were tested for incorporation of [ -32P]-CTP into RNA transcripts programmed with single stranded DNA in the presence of manganese (Fig. 4-8). The non-specific RNA polymerase activity was found to be equivalent in the wild type and mutant extracts over a range on extract concentrations as well as at different temperature. These data, as well as the western blots analysis of the capping enzyme proteins (Fig. 4-2), confirm that the protein composition of the wild type a nd mutant extracts was similar,

PAGE 74

74 and that viral proteins not affect ed by the mutation in Dts36 app ear to function normally in the soluble extracts. 4.2.3.2 Enzyme-GMP complex formation The for mation of D1-GMP complexes were anal yzed in soluble virion extracts using the same assay as described for the permeabilized virions. Autoradiographs and phosphorimager analysis showed that when equivalent amounts of IHDW or Dts36 soluble extracts were tested for formation of D1-GMP complexes, the D1 protein from Dts36 possesses approximately 60% of the guanylyltransferase activity of the D1 protein from IHDW (F ig. 4-9). This is a moderate decrease in the formation of the D1-GMP co mplex in the mutant and is a more pronounced defect then what was seen in the permeabilized virions. 4.2.3.3 Analysis of methyltransferase activity The IHDW and Dts36 soluble extracts were te sted for methyltransferase activity using the uncoupled assay which measures the transfer of a radiolabeled methyl group to a GTP acceptor (Fig. 4-6). Both wild type and mutant extracts were examined for methyltransferase activity over a range of extract concentrations at 31C and 37C (Fig. 4-10). The Dts36 extracts had no measurable methyltransferase activ ity above the negative controls. These results were consistent with the results seen when permeabilized virions were used in the assay. 4.3 Summary In this chapter, the b iochemical characterization of a temperature sensitive mutant in the large subunit of the vaccinia virus mRNA capping en zyme is presented. The detailed analysis of the D1 protein from Dts36 showed that this pr otein is defective in several of the enzymatic activities involved in the formation of the mRNA cap structure. The mutant D1 protein has reduced guanylyltransferase activity in both permeabilized virions and virion extracts as measured by the formation of the covalent enzy me-GMP intermediate as well as no measurable

PAGE 75

75 methyltransferase activity in either permeablized virions or vi rion extracts. As was the case in the phenotypic study, the cap structure in vitro has not yet been directly determined. However, based on the results presented in this chapter, the mutant D1 protein is de fective in the (guanineN7)-methyltransferase activity which would resu lt in the formation of a guanylylated but unmethylated cap structure. These results c onfirm the results from the phenotypic analysis of Dts36 (chapter 3) which pointed to a defect in the mRNA capping of viral transcripts as well as the possibility that the capping defect may be a result of inefficient subunit association in the vaccinia virus mRNA capping enzyme which will be discussed in more detail in chapter 5.

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76 Figure 4-1. SDS-PAGE and wester n blot analysis of purified IH DW and Dts36 virions. A) The protein composition of purified virions was analyzed by electrophoresis through an 11% gel which was subsequently stained with coomassie blue. The amount of protein in g that was loaded is labeled above each lane as is the identity of the purified virion. B) Purified virions were analyzed and blots were prepared and probed. The source of the purified virions and the amount of protein in g are listed above the autoradiograms. The primary antibody used to probe each membrane is listed to the right of the autoradiograms and the molecular weights in kDa are listed to the left. C) Quantification of the western blots by Im age J analysis comparing the amount of either capping enzyme subunit to amount of F17 protein in purified virus.

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77 Figure 4-2. Western blot analysis of IHDW and Dts36 virion ex tracts. Virion extracts were electrophoresed and blots were prepared and probed. The identity of the virion extracts and the amount of protein an alyzed in g are listed above the autoradiograms. The membrane was probe d first with the D1 antibody and then probed with the D12 antibody. The identity of the D1 and D12 proteins are shown to the right of the autoradiogram and the molecula r weight in kDa are listed to the left of the autoradiograms.

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78 Figure 4-3. In vitro transcription in permeabilized IHDW an d Dts36 virions. The incorporation of [ 32P]-CMP into viral transcripts was meas ured for 60 minutes at either 31C or 37C. At the times indicated, the transcrip tion reactions were separated into core and released fractions and the acid precipi table radioactivity was measured.

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79 Figure 4-4. Enzyme guanylylate complex formation in purified virions. A) Varying amounts of purified virions were incubated with [32P]-GTP and analyzed by SDS-PAGE. The autoradiogram shows the radiolabeled D1 prot ein. The source of purified virions and the amount of protein in g analyzed are both listed above the autoradiogram. The molecular weights in kDa are listed to the le ft of the autoradiogr am. B) The relative amount of enzyme-GMP complex formation in purified virions as determined by comparing the amount of complex formation as determined by phosphorimager analysis to the amount of D1 protein in th e purified virions as determined by western blot analysis.

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80 Figure 4-5. Coupled transcrip tion and methyltransferase assa y in permeabilized IHDW and Dts36 virions. Purified virions were permeabilized by treatment with NP-40 and DTT and split in two; one sample was assayed for in vitro transcription (A) and the other was assayed for methyltransferase activity (B). A) Incorporation of [ 32P]CMP into viral transcripts was measured by TCA precipitation. B) The transfer of a radiolabeled methyl group to in vitro synthesized and guanylyl ated transcripts was measured.

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81 Figure 4-6. Uncoupled methyltran sferase assay schematic. In th e presence of an enzyme that possesses methyltransferase activity, a guanylylate residue (top left) and [3H]-Sadenosylmethionine (top right) interact a nd the radiolabeled methyl group from the SAM (shown in red box) is transferred to the guanylylate residue. The products of this reaction are a guanylylate residue po ssessing a tritium labeled methyl group at the N7 position (bottom left) and S-ad enosylhomocysteine (bottom right).

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82 Figure 4-7. Uncoupled methyltransferase assay in IHDW and Dts36 permeabilized virions. The transfer of a radiolabeled methyl group from SAM to a GTP molecule was measured in permeabilized virions.

PAGE 83

83 Figure 4-8. Non-specific RNA polymerase activity in IHDW and Dts36 viri on extracts. Varying amounts of wild type and mutant virion extr acts were assayed for the incorporation of [ 32P]-CMP into acid-insoluble ma terial in the presence of Mn2+ and using singlestranded DNA as a template.

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84 Figure 4-9. Enzyme-guanylylate complex formati on in IHDW and Dts36 virion extracts. A) Varying amounts of virion extrac ts were incubated with [ 32P]-GTP and analyzed by SDS-PAGE. The autoradiogram shows the ra diolabeled D1 protein. The source of virion extracts and the amount of protein in g analyzed in the reaction are both listed above the autoradiogram. The molecular weight s in kDa are listed to the left of the autoradiogram and the location of the D1 protein is labeled to the right of the autoradiogram. B) Quantification of en zyme-GMP complex formation in virion extracts by phosphorimager analysis.

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85 Figure 4-10. Uncoupled methyltransferase assa y in IHDW and Dts36 virion extracts. The transfer of a radiolabeled methyl group from SAM to a GTP molecule was measured in wild type and mutant virion extracts.

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86 CHAPTER 5 DISCUSSION 5.1 Introduction The role of the vaccin ia virus mRNA cappi ng enzyme during viral infection has been analyzed both phenotypically and biochemically using a temperature sensitive mutant that encodes a mutated D1 protein (chapters 3 and 4). The in vivo analysis showed that in Dts36 infections early gene transcription termination is compromised, early genes are expressed at late times during infection, DNA replication is abse nt, and intermediate gene transcription is compromised. These findings will be disc ussed further in Sections 5.3-5.6. The in vivo analyses also showed that in vivo the mutant possessed defects that we re consistent with a defect in mRNA capping; the observed instability of the viral mRNA transcripts could signify a defect in the guanylyltransferase activity of the capping enzy me and the decreased translation efficiency of some viral proteins could si gnify a defect in the methyltransferase activity of the capping enzyme. The biochemical analysis of the D1 pr otein encoded by Dts36 showed that the mutant protein was defective in the methyltran sferase activity of the viral capping in vitro Together these results point to a specific defect in mRNA me thyltransferase activity of the mutant protein; however, it is not known if this defect is due to a change in the enzyme active site or if the mutant D1 protein is unable to efficiently inte ract with the small subuni t of the viral capping enzyme. Both of these possibilities will be discussed further in Sections 5.7 and 5.8. 5.2 Temperature Sensitivity A tem perature sensitive virus is a virus that can grow at a permissive temperature but is not able to grow at a non-permissive temperature. In the study presented in Chapter 3, Dts36 was determined to be temperature sensitive in vivo by a one-step growth e xperiment (Fig. 3-3) and these results were confirmed with the rema inder of the phenotypic characterization which

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87 showed that Dts36 possessed transcription def ects at the non-permissive temperature. An intriguing development in the biochemical characterization of the mutant virus (Chapter 4) showed that the mutant virus is not temperature sensitive in vitro but instead shows defects in the enzymatic activities at all temperat ures examined in the course of this study. This is not an unprecedented phenomenon; in a study examining a a nother temperature sensitive mutant in the large subunit of the vaccinia virus mRNA capping enzyme, the virus was shown to be ts in vivo but possessed defects in the enzymatic activit ies at low temperatures as well as higher temperatures in vitro (Hassett et al., 1997). The differences in the temperature sensitivities can most likely be explained by a decrease in the stability of the samples. The virus particle that is analyzed in the phenotypic studies is the most stable form of the viru s. This is most likely due to the stabilizing environment of th e infected cell. As the virus becomes more and more soluble, such as in permeablized virions followed by the soluble virus extracts, the stabilization that was present in the environment of th e infected cell is lost which ma y lead to the phenomenon of the defects being observed at lower temperatures. 5.3 Early Viral Gene Transcription Termination Early gene transcription term ination is comp romised in Dts36 mutant infections. This represents the first in vivo evidence supporting a role for th e mRNA capping enzyme in early gene transcription terminati on, a phenomenon well established in vitro (Condit and Niles, 2002). The data presented in Chapter 3 re veals that termination occurs w ith varying efficiency at some but not all predicted downstream termination signals in Dts36 mutant infections. These observations are consistent with in vitro experiments which demonstrate that early viral transcription termination signals are only 75-80% efficient (Earl et al., 1990; Yuen and Moss, 1987). Interestingly the C11R tran script appeared to be terminated properly in Dts36 mutant infections. Analysis of the termination signals of this transcript showed that there are two

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88 additional termination signals present within twel ve nucleotides of the first termination signal (Fig. 3-6B). This is consistent with previous in vitro studies which predicted that multiple termination signals within a small region enhanc e termination (Rohrmann et al., 1986; Yuen and Moss, 1986). There has been no direct in vitro analysis of the early termination function encoded by the mutant D1 protein. However, ba sed on the phenotypic char acterization of Dts36, in which termination occurs but seems to be less efficient at certain termination sites, it is believed that the in vitro transcription experiments described in Chapter 4 are showing that termination is occurring but these experiments are not able to show if the synthesized transcripts are properly terminated. Further experimental evidence is required to determine if the in vitro synthesized viral transcripts are longer than expected which would show that the mutant D1 protein is also less efficient at early transcription termination in vitro 5.4 The Reactivation of Early Viral Messages L ate during Viral Infection The reappearance of early transcripts late du ring viral infections with IHDW and Dts36 could be due to genetic differences between the commonly used wild type strain of vaccinia virus, WR, and the wild type used in this study, IHDW. In vaccinia viru s, early transcription factors are encoded by late gene s (Gershon and Moss, 1990) and are packaged into newly formed virions late during infection. A lthough the factors required for early transcription are present late in infection (Wright and Moss, 1989), vaccinia ap parently possesses a mechanism that inhibits transcription from early promot ers late during infection. It has been hypothesized that this repression of transcription results either from formation of DNA s uperhelical structures in newly formed virions or association of the viral genome with DNA-binding prot eins (Masternak and Wittek, 1996). However, previous studies have s hown that a small subset of early promoters, including the vaccinia virus 7.5k promoter, are reactiv ated at times late during infection with the WR strain of virus (Garces et al., 1993). In the study presented here, both IHDW and permissive

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89 Dts36 infections were shown to express early mRNA transcripts at times late in infection for all transcripts analyzed. Numerous IHDW gene s have been sequenced and no significant differences have been found in the promoter regi ons relative to the WR strain. Therefore, one possibility is that the repression mechanism that is present in infections with the WR strain of vaccinia is not present in the IHDW strain of vaccinia virus. Another possible cause of the r eactivation of early transcripts late during infections with IHDW and Dts36 is superinfection. Superinfec tion occurs when virions are able to enter previously infected cells. Ther e are several examples of viruse s encoding factors that prevent superinfection; these include the neuraminidase of the influenza virus (Palese et al., 1974), the G protein of VSV (Whitaker-Dowling et al., 1983), and glycoprotein D of herpes simplex virus (Johnson and Spear, 1989). Prevention of superinfec tion is also present in vaccinia virus based on experiments using the WR wild type vaccini a virus strain that showed that although a superinfecting virus could enter a previously infected cell, the s uperinfecting virus was incapable of early gene transcription (Chr isten et al., 1990). This is in dicative of a block somewhere between virus adsorption and early transcripti on and recent advances in experimental technology has allowed the determination of the process that is blocked during superinfection as discussed further below. Although this initial study did not address the mechanis m of prevention of superinfection in vaccinia virus, other studies with vaccin ia virus have implicated the nonessential, virally-encoded hemagglutinin (HA) in the inhibition of fusion and superinfection (Ichihashi and Dales, 1971; Seki et al., 1990). Recent work using cowpox virus has shown that SPI-3, a viral serine proteinase inhibitor, and HA colocalize on the surface of infected cells and prevent superinfection (Brum et al., 2003; Turn er and Moyer, 2006). A recently published study has shown that HA and SPI-3 also interact on the surface of vaccinia virus infected cells and that

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90 the resulting complex reduces superinfection by slowing the entry-fusion process (Turner and Moyer, 2008). The wild type vaccinia virus st rain used in this study, IHDW, has been shown previously to be HA-negative and to show extens ive cellular fusion as early as 12 hpi (Ichihashi and Dales, 1971). Therefore, it is more conceiva ble that the early transc ripts seen late during infections with IHDW and Dts36 are the result of superinfection as opposed to the reactivation of early genes late during infection. 5.5 DNA Negativity of Dts36 We believe that the defect in DNA replicati on observed during Dts36 m utant infections results from the observed generalized decrease in early viral protein synthesis and does not reflect a direct role for the mRNA capping enzyme in DNA repl ication. As discussed above, western analysis of several early viral protei ns involved in DNA replication, A20, D5 and E9, showed that these proteins were present in de creased amounts in Dts36 infected cells incubated at the non-permissive temperatur e. A previous study has shown that D5 interacts with A20 by yeast 2-hybrid analysis (McCraith et al., 2000). Additional studies of D5 have shown that D5 is capable of oligomerization and more importantly that this oligomerization is required for ATPase activity (Boyle et al., 2007). The A20 and E9 proteins have also been shown to interact with each other by co-immunoprecipitation (Klemperer et al., 2001). Additional analysis of the A20/E9 interactions have shown that A20 inte racts with D4, the uracil DNA glycosylase, and that this dimeric complex interacts with E9 to form a complex which possesses processive DNA polymerase activity (Stanitsa et al ., 2006). The reduced amounts of the A20, D5 and E9 proteins in non-permissive Dts36 infec tions could prohibit the forma tion of viable DNA replication complexes which would therefore explain the lack of DNA replic ation in these infections. Alternatively, it may be that one or another of these or another unidentif ied early viral protein

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91 required for DNA replication are normally present in critically limiting amounts, such that a moderate decrease in protein synthesis cau ses a profound defect in DNA replication. An intriguing alternative explanation for the DNA negative phenotype of Dts36 relates to the early stages of virion uncoa ting. Previous studies have show n that vaccinia virus uncoating proceeds in two steps; an init ial rapid uncoating after which vi ral DNA is DNase-resistant and a secondary uncoating after which the viral DNA is no longer DNase-resistant. The second stage of uncoating requires early viral protein synthesis and presumably releases the viral DNA from the core, making it available as a template for DNA replication (Joklik, 1964a; Joklik, 1964b; Mallardo et al., 2002). The proteins required for secondary uncoating have not been identified. The observed decrease in early viral protein synt hesis could theoretically affect the secondary uncoating of the virus particle in non-permissive infections with Dts36. A defect in secondary uncoating would prevent release of viral DNA from the core and a halt of the virus life cycle at the DNA replication stage. Although it is not possible to determine which of the two possibilities hypothesized here is the correct explanation for the DNA negative phe notype of Dts36, it has been shown that the capping enzyme is not involved in the process. If the defect in the capping enzyme encoded by Dts36 did cause the defect in DNA replication ob served in the non-permissive infections, when the shift-up experiment (Fig. 3-12) was perfor med, DNA replication in the mutant infections should have terminated. This was not the cas e; DNA replication in the Dts36 infections continued after the shift to the non-permissive temperature. Th ese results confirm that viral mRNA capping enzyme is not involved in the process of viral DNA replication. 5.6 Intermediate Viral Gene Transcription in Dts36 Biochem ical experiments have shown that the vaccinia mRNA capping enzyme is an intermediate gene transcription initiation factor (Vos et al., 1 991b). In the shift-up experiment

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92 presented in this study, the steady state levels of the G8R intermediate transcript decreased drastically in Dts36-infected ce lls after a shift to the non-permi ssive temperature, whereas the steady state levels of the early and late viral transcripts ex amined did not show a similar decrease. The decrease in steady state levels of mRNA could theoretically result either from a decrease in mRNA synthesis or an increase in mRNA degradati on. While the cap status of postreplicative mRNAs in mutant infections is not known, and while a defect in capping of postreplicative mRNAs could obviously destabili ze the RNAs, it is expected that such a destabilization would affect all postreplicative RNAs rather than a specific subclass. Since the decrease observed is specific to the intermediate transcript it is believed that the intermediate gene transcription initiation f unction of the capping enzyme is defective in Dts36. This represents the first in vivo evidence in support of a role for the vaccinia capping enzyme in intermediate gene tr anscription initiation. 5.7 mRNA Capping In the phenotypic study presented in chapte r 2, Dts36 infected cells were found to synthesize decreased amounts of seve ral early viral proteins at th e non-perm issive temperature as analyzed by western blot. By c ontrast, no differences were observ ed between early viral protein synthesis in wild type and mutant infections as assayed by metabolic labeling. This discrepancy is most likely due to the specificity of each experiment: metabolic labeling focuses predominantly on the most abundantly synthesi zed viral proteins wh ile western analysis examines steady state levels of specific proteins, regardless of abundance. It may be that for the most actively transcribed genes, mRNA is synthe sized in amounts in exce ss of the translation capacity of the infected cell, so that decreases in the steady state levels of mRNA due to defects in the capping of the mRNA transcripts would not a ffect the levels of s ynthesis of the cognate proteins, as assayed by pulse labeli ng. Several of the proteins that we have analyzed by western

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93 blot, notably E9, D5 and A20, may be from genes e xpressed at relatively lo wer levels in infected cells. The mRNA for these genes may be more limiting during infection so that decreases in steady state levels of active mRNA could have a larger impact on accumulation of the cognate proteins. In addition to a decrease in amount of so me early viral proteins, some early viral transcripts were also reduced in amount in non-permissive Dts36 infections. As noted above, we have not measured directly th e cap status of viral mRNAs in mu tant infected cells. However, both the decrease in the amount of early viral RNA a nd protein are consistent with a defect in the ability of the capping enzyme to cap newly synthesized viral tran scripts. The defect in the capping activity could represent a specific defect in either the guanyl yltransferase or the methyltransferase activity of the mutant enzyme. A defect in th e guanylyltransferase activity of the capping enzyme would result in nascent mRNA transcripts that are not guanylylated and therefore, not capped. It has been shown pr eviously that unblocked, i.e. non-guanylylated, reovirus mRNA is less stable in X. laevis oocytes, wheat germ extracts and L cell protein synthesizing extracts (Furuich i et al., 1977). Furthermore it has been demonstrated that a decrease in mRNA levels and protein synthe sis is observed when mRNA capping activity is inactivated in a yeast guanylyltransferase mutant (Schwer et al., 1998). A defect in methyltransferase activity could affect mRNA function or stability either directly by affecting translation efficiency or indirect ly by affecting cap stability. Previous studies in vaccinia virus, reovirus, and vesicular stomatitis virus have shown that methylation of the guanylylate residue of the cap structure of viral transcri pts is critical for translation (Both et al., 1975; Muthukrishnan et al., 1975; Muthukrishnan et al., 1978). Thus a de fect in the methyltran sferase function of the Dts36 enzyme could result in synthesis of viral mRNA with decreased translation efficiency. A

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94 defect in methyltransferase activity could indirect ly affect the guanylylated state of the nascent transcript. The guanylyltransferas e reaction is reversible in the absence of methylation (Martin and Moss, 1975). Thus a defect in the methyltran sferase activity in Dts36 could destabilize the mRNA cap which in turn could le ad to degradation of the viral mRNA transcripts and a decrease in viral protein synthesis. During the phenotypic analysis, it had not been possible to distinguish which, if any, of the two enzy matic activities is defective in vivo in the mutant capping enzyme encoded by Dts36. However, the favored hypot hesis was that the mutant capping enzyme encoded by Dts36 is defective in the methyltransferase activity. This hypothesis was confirmed upon biochemical analysis of Dts36 when the mutant D1 protein was found to be defective in methyltransferase activity. The methyltransferase defect and th e role that the mRNA capping enzyme subunit association may play in this def ect are discussed further in the following section. 5.8 mRNA Capping Enzyme Subunit Association In Dts36, the large subunit of the mRNA cappi ng enzym e contains a glycine to aspartic acid substitution at residue 705. G705 is c onserved in all poxviru ses and cellular cap methyltransferases (Schwer and Shuman, 2006) as well as those from Sh ope fibroma virus and African swine fever virus (Mao and Shuman, 1994). Recent X-ray crystallographic data has shown that G705 is located in st rand B5 of a seven stranded beta sheet that comprises the conserved core of the enzyme (Fig. 5-1) (De la et al., 2007). Thus G705 is not directly involved in the D1-D12 subunit interaction (Fig. 5-2), as was previously suggested based on biochemical data (Schwer and Shuman, 2006). Nevertheless, a triple mutant of the large subunit of the vaccinia virus capping enzyme (G704, G705A, V707A ) was shown to have no interaction with the small subunit and no methyltransferase activ ity when both proteins were synthesized in reticulocyte lysates (Mao and Shuman, 1994). A yeast complementation assay which assesses vaccinia virus methyltransferase activity (Saha et al., 2003) has shown that the G705D mutation

PAGE 95

95 is temperature sensitive in yeast (S. Shuman, personal communication). Based on these studies, it is possible that the G705D muta tion found in Dts36 affects the st ructure of the D1 protein in such a way that the two subun its of the capping enzyme are no longer able to associate efficiently at the non-permissive temperature. The lack of efficient subunit association in the vaccinia viru s capping enzyme could affect the methyltransferase activity as well as the early transcription termination and intermediate transcription initiation functions of the enzyme. Inefficient subunit asso ciation would directly affect the methyltransferase ac tivity of the enzyme since the small subunit is required to stimulate the intrinsic methyltr ansferase activity of the larg e subunit by causing a predicted confirmation change in the methyltransferase active site of the large subunit (Higman et al., 1992; Niles et al., 1994). Both subunits of the capping enzyme ha ve been shown previously to be required for early gene transcription termination (Luo et al., 1995) and intermediate gene transcription initiation (Condit et al., 1996a). Howe ver, it is not known if these activities simply require the presence of both subunits or if subunit association is also required. If in fact subunit association is required for both early gene transcription termination and intermediate gene transcription initiation, then a mechanistic e xplanation that could account for all of the phenotypic features of non-permissive Dts36 infectio ns, as well as the lack of methyltransferase activity in vitro is a defect in association of the capping enzyme subunits. 5.9 Future Studies The phenotypic and biochem ical analysis of the mutant D1 protein from Dts36 has provided additional insights into the function of vaccinia virus mRNA capping enzyme. The mutant protein was thought to be defective in the methyltransferase activity based on in vivo analysis and this was confirmed by in vitro analysis. However, the other results from the in vivo analysis have yet to be substantiated by in vitro analysis. Future work with Dts36 will attempt to

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96 determine if the mutant D1 protein possesses gu anylyltransferase activit y in soluble enzyme extracts. This experiment utilizes a synt hetic RNA molecule as the substrate of the guanylyltransferase reaction and can be used to examine the enzymatic activity of the protein and with a slight modifi cation of the assay, the cap status of in vitro synthesized transcripts can be determined. Work will also be performed in order to confirm that the mutant D1 protein is defective in the early transcription termination an d intermediate transcrip tion initiation functions of the capping enzyme. The early transcription termination function will be analyzed by two distinct methods; the visualization of in vitro transcription products on an agarose-formaldehyde gel or through use of cell-free extracts in a well-described termination assay. The intermediate transcription initiation function will be analyz ed through use of cell-free extracts in a welldescribed initiation assay. Finally, the associa tion of the vaccinia virus capping enzyme subunits in the mutant virus will be determined by co-precipitations which will allow it to be determined if the subunit interaction of the D1 and D12 proteins from Dts36 is less efficient, leading to the defects that have been observed th roughout the course of this study.

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97 Figure 5-1. Structure of the carboxyl-terminus (a a 545-844) of the large s ubunit of the vaccinia virus mRNA capping enzyme. The Swiss-Pdb Viewer, version 4.0.1, was used to view the protein structure (Protein Data Bank code 2vdw) and export the figure. The location of amino acid residue 705 is highlighted in green.

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98 Figure 5-2. Structure of the in teraction of the carboxyl-terminus of the large subunit with the entire small subunit of the vaccinia virus mRNA capping enzyme. The Swiss-Pdb Viewer, version 4.0.1, was used to view the pr otein structure (Protein Data Bank code 2vdw) and export the figure. The D1 subunit is colored blue and the D12 subunit is colored orange. The location of amino aci d residue 705 is highlighted in green.

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99 LIST OF REFERENCES Abram off, M.D., Magelhaes, P.J., Ram, S.J ., 2004. Image Processing with ImageJ. Biophotonics International 11, 36-42. Ahn, B.Y., Gershon, P.D., Moss, B., 1994. RNA pol ymerase-associated protein Rap94 confers promoter specificity for initiating transcrip tion of vaccinia virus early stage genes. J.Biol.Chem. 269, 7552-7557. Ahn, B.Y., Moss, B., 1992. RNA polymerase-associa ted transcription specif icity factor encoded by vaccinia virus. Proc Natl.Acad Sci U.S.A 89, 3536-3540. Amegadzie, B.Y., Ahn, B.Y., Moss, B., 1992. Ch aracterization of a 7kilodalton subunit of vaccinia virus DNA-dependent RNA polymerase with structural similarities to the smallest subunit of eukaryotic R NA polymerase II. J.Virol. 66, 3003-3010. Baldick, C.J., Jr., Keck, J.G., Moss, B., 1992. Mu tational analysis of the core, spacer, and initiator regions of vaccinia virus interm ediate-class promoters. J.Virol. 66, 4710-4719. Baldick, C.J., Jr., Moss, B., 1993. Characterizati on and temporal regulation of mRNAs encoded by vaccinia virus intermediate-sta ge genes. J.Virol. 67, 3515-3527. Bayliss, C.D., Condit, R.C., 1995. The vaccini a virus A18R gene product is a DNA-dependent ATPase. J.Biol.Chem. 270, 1550-1556. Black, E.P., Condit, R.C., 1996. Phenotypic character ization of mutants in vaccinia virus gene G2R, a putative transcription elonga tion factor. J.Virol. 70, 47-54. Black, E.P., Moussatche, N., Condit, R.C., 1998. Characterization of the interactions among vaccinia virus transcription factors G2 R, A18R, and H5R. Virology 245, 313-322. Both, G.W., Banerjee, A.K., Sh atkin, A.J., 1975. Methylation-de pendent translation of viral messenger RNAs in vitro. Proc Natl.Acad Sci U.S.A 72, 1189-1193. Boyle, K.A., Arps, L., Traktman, P., 2007. Bioc hemical and genetic analysis of the vaccinia virus d5 protein: Multimeriza tion-dependent ATPase activity is required to support viral DNA replication. J.Virol. 81, 844-859. Brown, E., Senkevich, T.G., Moss, B., 2006. Vacci nia virus F9 virion membrane protein is required for entry but not virus assembly, in co ntrast to the related L1 protein. J.Virol. 80, 9455-9464. Broyles, S.S., Fesler, B.S., 1990. Vaccinia virus gene encoding a component of the viral early transcription factor. J.Virol. 64, 1523-1529.

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100 Broyles, S.S., Moss, B., 1986. Homology between RNA polymerases of poxviruses, prokaryotes, and eukaryotes: nucleotide sequence and transcriptional analysis of vaccinia virus genes encoding 147-kDa and 22-kDa s ubunits. Proc Natl.Acad Sci U.S.A 83, 31413145. Brum, L.M., Turner, P.C., Devick, H., Baquero, M.T., Moyer, R.W., 2003. Plasma membrane localization and fusion inhibito ry activity of the cowpox virus serpin SPI-3 require a functional signal sequence and the virus en coded hemagglutinin. Virology 306, 289-302. Carpenter, M.S., DeLange, A.M., 1991. A temper ature-sensitive lesion in the small subunit of the vaccinia virus-encoded mRNA capping en zyme causes a defect in viral telomere resolution. J.Virol. 65, 4042-4050. Carter, G.C., Law, M., Hollinshead, M., Smith, G.L., 2005. Entry of the vaccinia virus intracellular mature virion and its interactions with glycosaminoglycans. J.Gen.Virol. 86, 1279-1290. Christen, L., Seto, J., Niles, E.G., 1990. Superinfection exclusion of vaccinia virus in virusinfected cell culture s. Virology 174, 35-42. Christen, L.M., Sanders, M., Wiler, C., N iles, E.G., 1998. Vaccinia virus nucleoside triphosphate phosphohydrolase I is an essential viral early gene transcription termination factor. Virology 245, 360-371. Condit, R.C., Lewis, J.I., Quinn, M., Christen, L.M., Niles, E.G., 1996a. Use of lysolecithinpermeabilized infected-cell extracts to investigate the in vitro biochemical phenotypes of poxvirus ts mutations altered in viral tr anscription activity. Virology 218, 169-180. Condit, R.C., Motyczka, A., 1981. Isolation and preliminary characterization of temperaturesensitive mutants of vaccinia virus. Virology 113, 224-241. Condit, R.C., Motyczka, A., Spizz, G., 1983. Isolation, characterizat ion, and physical mapping of temperature-sensitive mutants of vaccinia virus. Virology 128, 429-443. Condit, R.C., Moussatche, N., Traktman, P., 2006. In a nutshell: structure and assembly of the vaccinia virion. Adv.Virus Res. 66, 31-124. Condit, R.C., Niles, E.G., 2002. Regulation of viral transcription elongation and termination during vaccinia virus infecti on. Biochim.Biophys.Acta 1577, 325-336. Condit, R.C., Xiang, Y., Lewis, J.I., 1996b. Mu tation of vaccinia virus gene G2R causes suppression of gene A18R ts mutants: implicat ions for control of transcription. Virology 220, 10-19. Cong, P., Shuman, S., 1995. Mutational analys is of mRNA capping enzyme identifies amino acids involved in GTP binding, en zyme-guanylate formation, and GMP transfer to RNA. Mol.Cell Biol. 15, 6222-6231.

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101 Cresawn, S.G., Condit, R.C., 2007. A targeted a pproach to identification of vaccinia virus postreplicative transcription el ongation factors: genetic evidence for a role of the H5R gene in vaccinia transc ription. Virology 363, 333-341. Cresawn, S.G., Prins, C., Latner, D.R., Condit, R.C., 2007. Mapping and phenotypic analysis of spontaneous isatin-beta-thiosemicarbazone resi stant mutants of vaccinia virus. Virology 363, 319-332. D'Costa, S.M., Bainbridge, T.W., Condit, R.C., 2008. Purification and properties of the vaccinia virus mRNA processing fact or. J.Biol.Chem. 283, 5267-5275. Davison, A.J., Moss, B., 1989a. Structure of vacc inia virus early promoters. J.Mol.Biol. 210, 749-769. Davison, A.J., Moss, B., 1989b. Structure of vacci nia virus late promoters. J.Mol.Biol. 210, 771-784. De la, P.M., Kyrieleis, O.J., Cusack, S., 2007. Structural insights into the mechanism and evolution of the vaccinia virus mRNA cap N7 methyl-transferase. EMBO J. 26, 49134925. De Silva, F.S., Lewis, W., Berglund, P., Koonin, E.V., Moss, B., 2007. Poxvirus DNA primase. Proc Natl.Acad Sci U.S.A 104, 18724-18729. Deng, L., Shuman, S., 1994. A role for the H4 subunit of vaccinia RNA polymerase in transcription initiation at a viral early promoter J.Biol.Chem. 269, 14323-14328. Earl, P.L., Hugin, A.W., Moss, B., 1990. Removal of cryptic poxvirus tran scription termination signals from the human immunodeficiency virus type 1 envelope gene enhances expression and immunogenicity of a recomb inant vaccinia virus. J.Virol. 64, 2448-2451. Evans, E., Klemperer, N., Ghosh, R., Traktma n, P., 1995. The vaccinia virus D5 protein, which is required for DNA replication, is a nucleic acid-independent nucleos ide triphosphatase. J.Virol. 69, 5353-5361. Fathi, Z., Condit, R.C., 1991. Phenotypic charact erization of a vaccin ia virus temperaturesensitive complementation group affecti ng a virion component. Virology 181, 273-276. Furuichi, Y., LaFiandra, A., Sh atkin, A.J., 1977. 5'-Terminal structure and mRNA stability. Nature 266, 235-239. Garces, J., Masternak, K., Kunz B., Wittek, R., 1993. Reactivati on of transcription from a vaccinia virus early promoter late in infection. J.Virol. 67, 5394-5401. Gershon, P.D., Moss, B., 1990. Early transcription factor subunits are enc oded by vaccinia virus late genes. Proc Natl.Acad Sci U.S.A 87, 4401-4405.

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102 Gershowitz, A., Moss, B., 1979. Abortive tran scription products of vaccinia virus are guanylylated, methylated, and polya denylylated. J.Virol. 31, 849-853. Gong, C., Shuman, S., 2003. Mapping the active site of vaccinia virus RNA triphosphatase. Virology 309, 125-134. Gross, C.H., Shuman, S., 1996. Vaccinia virions lacking the RNA helicase nucleoside triphosphate phosphohydrolase II are defective in early transcripti on. J.Virol. 70, 85498557. Guo, P.X., Moss, B., 1990. Interaction and mutual stabilization of the two subunits of vaccinia virus mRNA capping enzyme coexpressed in Es cherichia coli. Proc Natl.Acad Sci U.S.A 87, 4023-4027. Harris, N., Rosales, R., Moss, B., 1993. Transcription initiation factor activ ity of vaccinia virus capping enzyme is independent of mRNA guanyl ylation. Proc Natl.Acad Sci U.S.A 90, 2860-2864. Hassett, D.E., Lewis, J.I., Xing, X., DeLange, L., Condit, R.C., 1997. Analysis of a temperaturesensitive vaccinia virus mutant in the viral mRNA capping en zyme isolated by clustered charge-to-alanine mutagenesis and transi ent dominant selection. Virology 238, 391-409. Higman, M.A., Bourgeois, N., Niles, E.G., 1992. The vaccinia virus mRNA (guanine-N7-)methyltransferase requires both subunits of the mRNA capping enzyme for activity. J.Biol.Chem. 267, 16430-16437. Higman, M.A., Christen, L.A., Niles, E.G., 1994. The mRNA (guanine-7-)methyltransferase domain of the vaccinia virus mRNA capping enzyme. Expression in Escherichia coli and structural and kinetic comparison to the intact capping enzyme J.Biol.Chem. 269, 14974-14981. Huang, C.Y., Lu, T.Y., Bair, C.H., Chang, Y.S., Jwo, J.K., Chang, W., 2008. A novel cellular protein, VPEF, facilitates vaccinia virus penetration into HeLa cells through fluid phase endocytosis. J.Virol. 82, 7988-7999. Ichihashi, Y., Dales, S., 1971. Biogenesis of poxviruses: inte rrelationship between hemagglutinin production and polyk aryocytosis. Virology 46, 533-543. Izmailyan, R.A., Huang, C.Y., Mohammad, S., Isaacs, S.N., Chang, W., 2006. The envelope G3L protein is essential for entry of vacci nia virus into host cells. J.Virol. 80, 84028410. Johnson, R.M., Spear, P.G., 1989. Herpes simplex virus glycoprotein D mediates interference with herpes simplex virus infection. J.Virol. 63, 819-827. Joklik, W.K., 1964a. The intracellular uncoating of poxvirus DNA. I. The fate of the radioactively-labeled rabbitpox virus. J.Mol.Biol. 8, 263-276.

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105 Muthukrishnan, S., Both, G.W., Furuichi, Y., Shatkin, A.J., 1975. 5'-Terminal 7methylguanosine in eukaryotic mRNA is re quired for translation. Nature 255, 33-37. Muthukrishnan, S., Moss, B., Cooper, J.A., Maxwell, E.S., 1978. Influence of 5'-terminal cap structure on the initiation of translation of vaccinia virus mRNA. J.Biol.Chem. 253, 1710-1715. Myette, J.R., Niles, E.G., 1996. Domain structur e of the vaccinia virus mRNA capping enzyme. Expression in Escherichia coli of a subdoma in possessing the RNA 5'-triphosphatase and guanylyltransferase activities and a kinetic comparison to the full-size enzyme. J.Biol.Chem. 271, 11936-11944. Nichols, R.J., Stanitsa, E., Unger, B., Traktma n, P., 2008. The vaccinia virus gene I2L encodes a membrane protein with an essential ro le in virion entry. J.Virol. 82, 10247-10261. Niles, E.G., Christen, L., 1993. Identification of the vaccinia virus mRNA guanyltransferase active site lysine. J.Biol.Chem. 268, 24986-24989. Niles, E.G., Christen, L., Higman, M.A., 1994. Dire ct photolinkage of GTP to the vaccinia virus mRNA (guanine-7-) methyltransferase GT P methyl acceptor site. Biochemistry 33, 9898-9903. Niles, E.G., Lee-Chen, G.J., Shuman, S., Moss, B., Broyles, S.S., 1989. Vaccinia virus gene D12L encodes the small subunit of the vi ral mRNA capping enzyme. Virology 172, 513522. Ojeda, S., Domi, A., Moss, B., 2006a. Vaccinia vi rus G9 protein is an essential component of the poxvirus entry-fusion complex. J.Virol. 80, 9822-9830. Ojeda, S., Senkevich, T.G., Moss, B., 2006b. Entr y of vaccinia virus and cell-cell fusion require a highly conserved cysteine-rich membrane pr otein encoded by the A16L gene. J.Virol. 80, 51-61. Palese, P., Tobita, K., Ueda, M., Compans, R.W., 1974. Characterization of temperature sensitive influenza virus mutants defectiv e in neuraminidase. Virology 61, 397-410. Passarelli, A.L., Kovacs, G.R., Moss, B., 1996. Tr anscription of a vaccinia virus late promoter template: requirement for the product of the A2L intermediate-stage gene. J.Virol. 70, 4444-4450. Patel, D.D., Pickup, D.J., 1989. The second-larg est subunit of the poxvirus RNA polymerase is similar to the corresponding subunits of pro caryotic and eucaryotic RNA polymerases. J.Virol. 63, 1076-1086. Piacente, S., Christen, L., Dickerman, B., Mohamed, M.R., Niles, E.G., 2008. Determinants of vaccinia virus early gene transcription termination. Virology 376, 211-224.

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106 Punjabi, A., Boyle, K., DeMasi, J., Grubisha, O., Unger, B., Khanna, M., Traktman, P., 2001. Clustered charge-to-alanine mutagenesis of the vaccinia virus A 20 gene: temperaturesensitive mutants have a DNA-minus phenotype and are defective in the production of processive DNA polymerase act ivity. J.Virol. 75, 12308-12318. Rohrmann, G., Yuen, L., Moss, B., 1986. Transc ription of vaccinia virus early genes by enzymes isolated from vaccinia virions terminates downstream of a regulatory sequence. Cell 46, 1029-1035. Rosales, R., Harris, N., Ahn, B.Y., Moss, B., 1994. Purification and identif ication of a vaccinia virus-encoded intermediate stage promote r-specific transcription factor that has homology to eukaryotic transcri ption factor SII (TFIIS) and an additional role as a viral RNA polymerase subunit. J.Biol.Chem. 269, 14260-14267. Roth, M.J., Hurwitz, J., 1984. RNA capping by the vaccinia virus guanylyltransferase. Structure of enzyme-guanylate intermed iate. J.Biol.Chem. 259, 13488-13494. Saha, N., Shuman, S., Schwer, B., 2003. Yeast-base d genetic system for functional analysis of poxvirus mRNA cap methyltransf erase. J.Virol. 77, 7300-7307. Sanz, P., Moss, B., 1999. Identification of a transcription factor, encode d by two vaccinia virus early genes, that regulates the intermedia te stage of viral ge ne expression. Proc Natl.Acad Sci U.S.A 96, 2692-2697. Schwer, B., Mao, X., Shuman, S., 1998. Acceler ated mRNA decay in conditional mutants of yeast mRNA capping enzyme. Nucleic Acids Res. 26, 2050-2057. Schwer, B., Shuman, S., 2006. Ge netic analysis of poxvirus mR NA cap methyltransferase: suppression of conditional mutations in th e stimulatory D12 subunit by second-site mutations in the ca talytic D1 subunit. Virology 352, 145-156. Schwer, B., Stunnenberg, H.G., 1988. Vaccinia virus late transcripts generated in vitro have a poly(A) head. EMBO J. 7, 1183-1190. Schwer, B., Visca, P., Vos, J.C., Stunnenberg, H.G., 1987. Discontinuous transcription or RNA processing of vaccinia virus late messengers results in a 5' poly(A) leader. Cell 50, 163169. Seki, M., Oie, M., Ichihashi, Y., Shida, H., 1990. Hemadsorption and fusion inhibition activities of hemagglutinin analyzed by vaccinia virus mutants. Virology 175, 372-384. Senkevich, T.G., Moss, B., 2005. Vaccinia virus H2 protein is an essential component of a complex involved in virus entry and ce ll-cell fusion. J.Virol. 79, 4744-4754. Senkevich, T.G., Ojeda, S., Townsley, A., Ne lson, G.E., Moss, B., 2005. Poxvirus multiprotein entry-fusion complex. Proc .Natl.Acad.Sci.U.S.A 102, 18572-18577.

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107 Senkevich, T.G., Ward, B.M., Moss, B., 2004. Vacci nia virus entry into cells is dependent on a virion surface protein encoded by th e A28L gene. J.Virol. 78, 2357-2366. Shuman, S., 1989. Functional domains of vacci nia virus mRNA capping enzyme. Analysis by limited tryptic digestion. J.Biol.Chem. 264, 9690-9695. Shuman, S., 1990. Catalytic activity of vaccini a mRNA capping enzyme subunits coexpressed in Escherichia coli. J.Biol.Chem. 265, 11960-11966. Shuman, S., Broyles, S.S., Moss, B., 1987. Purifi cation and characterizati on of a transcription termination factor from vaccini a virions. J.Biol.Chem. 262, 12372-12380. Shuman, S., Hurwitz, J., 1981. Mechan ism of mRNA capping by vaccinia virus guanylyltransferase: character ization of an enzyme--guany late intermediate. Proc Natl.Acad Sci U.S.A 78, 187-191. Shuman, S., Morham, S.G., 1990. Domain stru cture of vaccinia virus mRNA capping enzyme. Activity of the Mr 95,000 subunit expressed in Escherichia coli. J.Biol.Chem. 265, 11967-11972. Shuman, S., Moss, B., 1988. Factor-dependent tr anscription termination by vaccinia virus RNA polymerase. Evidence that the cis-acting te rmination signal is in nascent RNA. J.Biol.Chem. 263, 6220-6225. Shuman, S., Moss, B., 1989. Bromouridine triphos phate inhibits transcription termination and mRNA release by vaccinia vi rions. J.Biol.Chem. 264, 21356-21360. Shuman, S., Moss, B., 1990. Purification and use of vaccinia virus messenger RNA capping enzyme. Methods Enzymol. 181, 170-180. Simpson, D.A., Condit, R.C., 1995. Vaccinia vi rus gene A18R encodes an essential DNA helicase. J.Virol. 69, 6131-6139. Smith, G.L., Law, M., 2004. The exit of vaccinia vi rus from infected cells. Virus Res. 106, 189197. Stanitsa, E.S., Arps, L., Traktman, P., 2006. Vaccinia virus uracil DNA glycosylase interacts with the A20 protein to form a heterodimeric processivity fact or for the viral DNA polymerase. J.Biol.Chem. 281, 3439-3451. Townsley, A.C., Moss, B., 2007. Two distinct lowpH steps promote entry of vaccinia virus. J.Virol. 81, 8613-8620. Townsley, A.C., Senkevich, T.G., Moss, B., 2005a. The product of the vaccinia virus L5R gene is a fourth membrane protein encoded by all poxviruses that is requi red for cell entry and cell-cell fusion. J.Virol. 79, 10988-10998.

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108 Townsley, A.C., Senkevich, T.G., Moss, B., 2005b. Vaccinia virus A21 virion membrane protein is required for cell entr y and fusion. J.Virol. 79, 9458-9469. Townsley, A.C., Weisberg, A.S., Wagenaar, T. R., Moss, B., 2006. Vaccinia virus entry into cells via a low-pH-dependent endos omal pathway. J.Virol. 80, 8899-8908. Traktman, P., Boyle, K., 2004. Methods for an alysis of poxvirus DNA replication. Methods Mol.Biol. 269, 169-186. Traktman, P., Sridhar, P., Condit, R.C., Robe rts, B.E., 1984. Transcriptional mapping of the DNA polymerase gene of vaccinia virus. J.Virol. 49, 125-131. Turner, P.C., Moyer, R.W., 2006. The cowpox virus fusion regulator proteins SPI-3 and hemagglutinin interact in infected and uninfected cells. Virology 347, 88-99. Turner, P.C., Moyer, R.W., 2008. The vaccinia virus fusion inhibitor proteins SPI-3 (K2) and HA (A56) expressed by infected cells reduce the entry of supe rinfecting virus. Virology Valderrama, F., Cordeiro, J.V., Schleich, S., Frischknecht, F., Way, M., 2006. Vaccinia virusinduced cell motility requires F11L-mediate d inhibition of RhoA signaling. Science 311, 377-381. Vos, J.C., Sasker, M., Stunnenberg, H.G., 1991a. Promoter melting by a stage-specific vaccinia virus transcription factor is independent of the presence of RNA polymerase. Cell 65, 105-113. Vos, J.C., Sasker, M., Stunnenberg, H.G., 1991b. Vaccinia virus capping enzyme is a transcription initiation factor. EMBO J. 10, 2553-2558. Whitaker-Dowling, P., Youngner, J.S., Widnell, C.C., Wilcox, D.K., 1983. Superinfection exclusion by vesicular stomatitis virus. Virology 131, 137-143. Wright, C.F., Moss, B., 1989. Identification of fact ors specific for transcrip tion of the late class of vaccinia virus genes. J.Virol. 63, 4224-4233. Wright, C.F., Oswald, B.W., Dellis S., 2001. Vaccinia virus late tr anscription is activated in vitro by cellular heterogeneous nuclear ribonucleoproteins. J.Biol.Chem. 276, 4068040686. Xiang, Y., Simpson, D.A., Spiegel, J., Zhou, A ., Silverman, R.H., Condit, R.C., 1998. The vaccinia virus A18R DNA helicase is a postr eplicative negative transcription elongation factor. J.Virol. 72, 7012-7023. Yao, X.D., Evans, D.H., 2003. High-frequency genetic recomb ination and reactivation of orthopoxviruses from DNA fragme nts transfected into leporipoxvirus-infected cells. J.Virol. 77, 7281-7290.

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109 Yu, L., Martins, A., Deng, L., Shuman, S., 1997. Structure-function analysis of the triphosphatase component of vaccinia virus mRNA capping enzyme. J.Virol. 71, 98379843. Yu, L., Shuman, S., 1996. Mutational analys is of the RNA triphosphatase component of vaccinia virus mRNA capping enzyme. J.Virol. 70, 6162-6168. Yuen, L., Moss, B., 1986. Multiple 3' ends of mRNA encoding vaccinia virus growth factor occur within a series of rep eated sequences downstream of T clusters. J.Virol. 60, 320323. Yuen, L., Moss, B., 1987. Oligonucleotide sequen ce signaling transcriptional termination of vaccinia virus early genes. Proc Natl.Acad Sci U.S.A 84, 6417-6421. Zhang, Y., Ahn, B.Y., Moss, B., 1994. Targeting of a multicomponent transcription apparatus into assembling vaccinia virus particles re quires RAP94, an RNA polymerase-associated protein. J.Virol. 68, 1360-1370. Zhang, Y., Keck, J.G., Moss, B., 1992. Transcription of viral late genes is dependent on expression of the viral intermediate gene G8R in cells infected with an inducible conditional-lethal mutant vacci nia virus. J.Virol. 66, 6470-6479.

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110 BIOGRAPHICAL SKETCH Am ber Nicole Shatzer was born in 1980, in Chambersburg, Pennsylvania. She attended Ursinus College in Collegeville, Pennsylvania, where she earned her Bachelor of Science in biochemistry and molecular biology, with a minor in sociology, in May 2003. In the fall of 2003, she started graduate school in the Interdisciplinary Program in Biomedical Sciences at the University of Florida. Upon the completion of her Ph.D. in December 2008, she plans on continuing her career in scientific research as a postdoctoral associate.