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Insights Into the Dynamic Properties of the Betaretroviral Gag Polyprotein

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Insights Into the Dynamic Properties of the Betaretroviral Gag Polyprotein The Pennsylvania State University The Graduate School College of Medicine INSIGHTS INTO THE DYNAMIC PROPERTIES OF THE BETARETROVIRAL GAG POLYPROTEIN A Dissertation in Microbiology and Immunology by Andrea Rae Beyer ©2011 Andrea Rae Beyer Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2011 The dissertation of Andrea Rae Beyer was reviewed and approved* by the following: Leslie J. Parent Professor of Medicine and Microbiology and Immunology Chief, Division of Infectious Diseases Dissertation Adviser Chair of Committee Richard J. Courtney Professor and Distinguished Educator of Microbiology and Immunology Chair of the Department of Microbiology and Immunology Neil D. Christensen Professor of Pathology, and Microbiology and Immunology Associate Chief, Division of Experimental Pathology John W. Wills Distinguished Professor of Microbiology and Immunology Sarah K. Bronson Associate Professor and Distinguished Educator of Cellular & Molecular Physiology *Signatures are on file in the Graduate School. ii Abstract Mouse mammary tumor virus (MMTV) is an oncogenic retrovirus that causes mammary carcinoma in infected mice. Unlike most retroviruses that assemble virus particles concurrent with budding at the plasma membrane, MMTV and other betaretroviruses first form immature capsids in the cell cytoplasm and subsequently target the immature particles to the plasma membrane for budding. The entire assembly process is driven by the viral structural protein, Gag, which is also responsible for the selective packaging of the viral genomic RNA. Additionally, MMTV Gag contributes to tumorigenesis in infected mice through an unidentified mechanism. Though MMTV has served as a classical model system for exploring oncogenes and signaling pathways involved in breast cancer, the molecular mechanisms of assembly for this betaretrovirus remain largely unexplored. Recent evidence from the Parent laboratory and others indicate that the trafficking of the retroviral Gag protein from translation on ribosomes to the plasma membrane for budding is not a direct linear pathway. Studies using the avian oncoretrovirus, Rous sarcoma virus (RSV), have shown that its Gag protein follows a complex pathway whereby targeting and export signals within Gag direct it to transiently traffic through the nucleus. As a Type C morphogenetic virus, RSV Gag is then targeted to the host membrane for simultaneous capsid assembly and egress. Despite evidence that Gag proteins of other retroviruses participate in nuclear trafficking, further studies have not determined whether mechanisms similar to RSV Gag nuclear import/export are employed. The existence of putative nuclear trafficking signals within MMTV Gag, as well as data from a collaborator that suggested MMTV Gag interacts with a cellular ribosomal protein, provided the impetus for me to characterize the MMTV Gag protein. The research presented in this dissertation focuses on the elucidation of signals within MMTV Gag that mediate the trafficking, localization, and budding of virus particles from infected cells. iii A GFP-tagged Gag system was employed to visualize the localization of Gag proteins in transfected mouse cells. Under steady state conditions, MMTV Gag exists primarily in the cytoplasm within bright foci with very little detectable Gag in the nucleus. However, fractionation of transfected cells indicates that a population of Gag is detectable in cell nuclei. Localization of Gag truncation mutants implies that a nuclear localization signal within the p8 domain of Gag plays a role in nuclear import. MMTV Gag was found to be insensitive to the drug Leptomycin B, which specifically inhibits nuclear export by interacting with the cellular factor CRM1. Additionally, data showing no effect of mutation on putative CRM1-export signals in Gag suggest that MMTV Gag nuclear export is mediated by alternative means. Further lines of evidence suggest that MMTV Gag participates in dynamic trafficking throughout the infected cell. Interestingly, overexpression of ribosomal protein L9 (RPL9), a host interacting protein of MMTV Gag, results in the relocalization of Gag to the nucleoli where RPL9 accumulates. Fluorescence resonance energy transfer (FRET) analysis and coimmunoprecipitation reveals the direct protein-protein interaction between Gag and RPL9. The NC domain of Gag contains nucleolar localization signals, though RPL9 appears to mediate the translocation of Gag to the nucleolus through interactions with the CA domain. With the knowledge that RPL9 serves as a tumor suppressor, it is tempting to speculate that Gag interferes with the activity of RPL9 in the nucleolus, leading to increased carcinogenesis. Additionally, the aggregation of Gag into cytoplasmic foci under steady- state conditions was shown to colocalize with protein components of cellular P-bodies and stress granules, repositories of ribonucleoprotein complexes. Collectively, these data reveal two novel steps in the MMTV Gag assembly process involving the cell nucleus and cytoplasmic RNA granules which may serve as assembly nucleation sites. With the established dogma that retroviral Gag proteins are sufficient for the production and release of virus-like particles (VLP), it was surprising that MMTV Gag-GFP expression did not lead to the production of VLPs. Further evaluation of the components iv required for Gag budding reveal that the Env protein, which aids the assembly of other intracellular-capsid-forming retroviruses, does not rescue VLP formation. Although studies have indicated that RNA transport elements within the viral RNA direct the proper assembly and budding of retroviral Gag proteins, the addition of various RNA transport signals to MMTV Gag expression vectors did not rescue Gag particle production. Thus the minimal components needed for Gag budding remain unknown, and efforts aimed to elucidate this information are ongoing. The observations described within this dissertation are the first to examine the localization of MMTV Gag in cells and show that MMTV Gag is insufficient to form detectable capsids in cell media. The collective data of this thesis confirms the uniqueness of MMTV in comparison to other retroviruses and supports the need to continue efforts in characterization and understanding of MMTV viral assembly. v Table of Contents List of Figures……………………………………………………………………………………..viii List of Tables………………………………………………………………………………………..x Abbreviations List………………………………………………………………………………....xi Preface……………………………………………………………………………………………...xiv Acknowledgements……………………………………………………………………………….xv Chapter 1: Introduction to Dissertation…………………………………………………….…..1 1.1 Introduction……………………………………………………………………………………...1 1.2 Development of the research presented in this dissertation……………………………….3 1.3 Rationale for these studies……………………………………………………………………5 Chapter 2: Literature Review…………………………………………………………………...6 2.1 Historical perspective of MMTV……………………………………………………………….6 2.2 Taxonomy of retroviruses………………………………………………………………………7 2.3 Virion morphology……………………………………………………………………………….8 2.3.1 Categories of particle morphology………………………………………………….8 2.3.2 MMTV particle morphology and general composition……………………………9 2.4 MMTV genome organization…………………………………………………………………..9 2.5 Proteins of MMTV………………………………………………………………………………11 2.5.1 MMTV mRNA and ribosomal frameshifting………………………………………11 2.5.2 Group specific antigen (Gag)………………………………………………………11 2.5.2.1 Matrix (MA)………………………………………………………………..14 2.5.2.2 In media res: The unique pp21, p3, p8, and n domains of Gag…….15 2.5.2.3 Capsid (CA)………………………………………………….……………16 2.5.2.4 Nucleocapsid (NC)……………………………………………………….16 2.5.3 Deoxyuridine triphosphatase (dUTPase or DU)………………………………….17 2.5.4 Protease (Pro or PR)………………………………………………………………..18 2.5.5 Reverse transcriptase (RT)…………………………………………………………19 2.5.6 Integrase (IN)………………………………………………………………………...20 2.5.7 Regulator of export/expression of MMTV mRNA (Rem)………………………..21 2.5.8 Envelope (Env)………………………………………………………………………23 2.5.9 Superantigen (Sag)………………………………………………………………….24 2.5.10 Negative acting factor (Naf)……………………………………………………….26 2.6 Spread of MMTV……………………………………………………………………………….27 2.6.1 Endogenous transmission of MMTV (Mtvs)………………………………………27 2.6.2 Exogenous transmission of MMTV………………………………………………..28 2.7 The MMTV replication cycle…………………………………………………………………..32 2.7.1 Adsorption and entry into host cells……………………………………………….33 2.7.2 Reverse transcription………………………………………………………………..34 2.7.3 Nuclear trafficking of the preintegration complex (PIC)………………………...39 2.7.4 Integration of the MMTV provirus………………………………………………….40 2.7.5 Viral transcription…………………………………………………………………....42 2.7.6 Translation of viral proteins…………………………………………………………45 2.7.7 MMTV virion assembly…………………………………………………………...…47 vi 2.7.8 Envelopment, budding, and release……………………………………………….51 2.8 Mechanisms of MMTV tumorigenesis………………………………………………………..53 2.9 The barriers to MMTV study…………………………………………………………………..56 2.10 Is there a human mammary tumor virus?......................................................................58 2.11 Ribosomal protein L9 (RPL9)………………………………………….……………………60 2.12 The dynamic nucleolus………………………………………………..……………………..63 2.13 P-bodies (PBs) and stress granules (SGs)………………………………………………..65 Chapter 3: Interaction of Mouse Mammary Tumor Virus Gag Protein with Ribosomal Protein L9 in the Nucleolus………………………………………….……………..……………67 3.1 Abstract………………………………………………………………………………………….67 3.2 Introduction……………………………………………………………………………………...67 3.3 Results ………………...………………………………………………………………………..70
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