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Open Thesis.Pdf The Pennsylvania State University The Graduate School College of Medicine UTILIZATION OF CELLULAR PROTEINS BY ROUS SARCOMA VIRUS DURING REPLICATION A Thesis in Microbiology and Immunology by Jared Lynn Spidel © 2005 Jared Lynn Spidel Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2005 The thesis of Jared Lynn Spidel was reviewed and approved* by the following: John W. Wills Professor of Microbiology and Immunology Thesis Adviser Chair of Committee Richard J. Courtney Professor of Microbiology and Immunology Head of the Department of Microbiology and Immunology Leslie J. Parent Associate Professor of Medicine Associate Professor of Microbiology and Immunology David J. Spector Associate Professor of Microbiology and Immunology Vincent Chau Professor of Cellular and Molecular Physiology *Signatures are on file in the Graduate School ABSTRACT The goal of this dissertation was to analyze the role of cellular proteins during the final stages of budding leading up to membrane fission. Studies have already identified some of the cellular proteins which interact with various late (L) domains. The L domain of Rous sarcoma virus (RSV) interacts with a ubiquitin (Ub) ligase, a cellular enzyme which conjugates Ub to a target lysine. Indeed, all retroviruses examined so far contain ~100 molecules of Ub, suggesting Ub might have a role during budding. However, unlike all other retroviruses, Ub conjugated to RSV Gag has never been detected. This observation seems inconsistent with the observation that budding of this virus (and many others) is dependent on the presence of free cellular Ub. Therefore, the role of Ub during RSV budding was examined. If transient ubiquitination of RSV Gag is required during budding, the five lysines located just upstream of the RSV L domain in matrix (MA) would be the most likely targets of ubiquitination based on known sites of ubiquitination of Gag in other viruses. These residues were changed to arginines to eliminate the potential for ubiquitination. As predicted, substitution of these five residues with arginine (mutant 1-5KR) reduced budding by 80-90%. The block to budding was found to be on the plasma membrane, and the few virions released had normal size, morphology, and infectivity. Budding was restored when any one of the residues was changed back to lysine or when lysines were inserted in novel positions within the region of the original five substituted residues or C- terminal in the p10 region. Similar to an L domain mutant, the 1-5KR mutant could be rescued into particles by coexpression of budding-competent Gag molecules. These data suggest that ubiquitination of Gag is likely important for budding. iii Through examination of lysines involved in RSV budding, a lysine (K244) in capsid (CA) was found to be required for efficient replication. Analysis of the amino acids flanking K244 revealed it resides in a sumoylation consensus sequence ΨKxE, where Ψ is a hydrophobic residue and x is any residue (243IKTE246 in RSV CA). Small Ub-like MOdifier (SUMO)-1 is a member of the Ub-like protein family that transiently modifies lysines of various target proteins in a manner similar to Ub. Sumoylation of target proteins is important for nuclear events such as nuclear entry, subnuclear structure formation, and modulation of transcriptional activity. The enzyme Ubc9 is capable of conjugating SUMO-1 to a target lysine within the sumoylation consensus sequence. Indeed, sumoylation has been hypothesized to be involved in the replication of Mason- Pfizer monkey virus where CA was shown to interact and colocalize with Ubc9 in vivo. The RSV mutants K244R and E246A were found to be normal for budding and assembly but were reduced in infectivity, revealing a potential role for sumoylation of Gag or CA in replication. The virions contained normal amounts of Pol, Env, and RNA and were normal in core morphology. The defect was during reverse transcription and possibly nuclear import of the preintegration complex (PIC). Revertant viruses of the mutants K224R and E246A were isolated, and the genomes sequenced to identify second-site suppressors. The K244R phenotype was suppressed by the double mutation R325C/C431R; the E246A phenotype was suppressed by a N343D mutation. C431 is thought to be involved in CA-CA interactions based on its position in the predicted dimerization helix. The involvement of C431 in the suppression of the K244R phenotype suggests the K244R and E246A substitutions may disrupt intra- or intermolecular interactions in CA which is repaired in the suppressors. iv Further experimentation is required to determine if the K244 and E246 are important in the structural stability of CA or if the suppressors allow for SUMO-independent replication. Whatever the function of K244 and E246, these data support a model whereby CA plays an active role during reverse transcription and/or nuclear import of the PIC. The exact function of Ub in retrovirus budding is unknown. Ub has many cellular functions, including involvement in cellular budding events that produce multivesicular bodies (MVBs), and, interestingly, viral budding is similar in topology to this budding. Therefore, the cellular proteins which interact with Ub to mediate MVB formation could feasibly be involved in viral release. Ubiquitination of a target protein acts to recruit complexes known as ESCRT-I, -II, and -III to facilitate sorting of the target protein into the budding vesicles. To determine the role of the ESCRT complexes during RSV budding, dominant-negative forms of these proteins were coexpressed with RSV Gag. ESCRT-I protein Tsg101, the ESCRT-III proteins CHMP3, CHMP4A, CHMP4B, CHMP4C, and CHMP6, and Vps4A inhibit RSV budding implicating a role for these proteins during budding. During the course of this study it was noticed that RSV Gag also contains a YPxL sequence that may allow binding to AIP1/ALIX which itself interacts with ESCRT-III and is important for HIV and EIAV budding. Substituting this sequence with alanines had a modest effect on budding suggesting this sequence may have a slight role during budding. This site may interact with AIP1/ALIX to stabilize ESCRT complexes with RSV Gag, and failure to do so results in inefficient budding. Therefore, from these data it appears that RSV and HIV utilize a similar mechanism and cellular proteins for their release. v TABLE OF CONTENTS LIST OF FIGURES ..........................................................................................................x LIST OF TABLES......................................................................................................... xii LIST OF ABBREVIATIONS....................................................................................... xiii ACKNOWLEDGEMENTS......................................................................................... xvii CHAPTER I: LITERATURE REVIEW...........................................................................1 Introduction.................................................................................................................2 Organization of the RSV Genome ..............................................................................6 Particle Morphology and Structure...........................................................................10 The Immature Virion ..........................................................................................11 Maturation...........................................................................................................12 The Mature Virion ..............................................................................................15 Entry..........................................................................................................................21 Attachment..........................................................................................................21 Fusion and Uncoating .........................................................................................24 Reverse Transcription.........................................................................................25 Nuclear Targeting and Entry...............................................................................34 Integration...........................................................................................................36 Transcription.............................................................................................................37 Translation ................................................................................................................41 Virus Assembly.........................................................................................................42 Gag Trafficking and the M Domain....................................................................43 Gag-Gag Interactions and the I domain..............................................................49 RNA Packaging ..................................................................................................51 vRNA Used For Packaging...........................................................................52 RNA Dimerization........................................................................................53 Packaging of the tRNA Primer .....................................................................54 Pol Incorporation ................................................................................................55 Env Incorporation ...............................................................................................56
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