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.

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Abstract

Mouse mammary tumor (MMTV) is an oncogenic that causes mammary carcinoma in infected mice. Unlike most that assemble virus particles concurrent with budding at the plasma membrane, MMTV and other betaretroviruses first form immature 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 , 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 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 assembly and egress. Despite evidence that Gag 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.

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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 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.

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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 (RT)…………………………………………………………19 2.5.6 (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 ………………………………………………………………..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

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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 3.4 Discussion..……………………………………………………………………………………..81 3.5 Materials and Methods…………………………………………………………………………85 3.6 Acknowledgements…………………………………………………………………………….89

Chapter 4: Trafficking and Budding Requirements of the Anomalous Mouse Mammary Tumor Virus Gag Protein……………...…………………………………………..121 4.1 Abstract………………………………………………………………………………………...121 4.2 Introduction…………………………………………………………………………………….122 4.3 Results…………………………………………………………………………………………125 4.4 Discussion..……………………………………………………………………………………137 4.5 Materials and Methods……………………………………………………………………….142 4.6 Acknowledgements…………………………………………………………………………...147

Chapter 5: Overall Dissertation Discussion…………………………………………..……172

References….....……………………………………………………………………………….…186

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List of Figures

Chapter 2 Figure 2.1 Features of the MMTV genome………………………………………………………12 Figure 2.2 The infection cycle of MMTV in mice………………………………………………...30 Figure 2.3 The MMTV replication pathway………………………………………………………35 Figure 2.4 Retroviral reverse transcription……………………………………………………….37 Figure 2.5 Organization of the immature and mature MMTV virus particles…………………49

Chapter 3 Figure 3.1 Yeast two hybrid data implicating RPL9 as a binding partner of MMTV Gag...…91 Figure 3.2 Localization of MMTV Gag in transfected and infected cells……………………...93 Figure 3.3 Localization of mouse RPL9 in context of nucleolar markers and MMTV Gag in NMuMG cells……………………...………………………………………………………..95 Figure 3.4 Examination of nucleolar cellular proteins with MMTV Gag and viral Gag proteins with mouse RPL9 in NMuMG cells……………………………………………………….97 Figure 3.5 RPL9-MMTV Gag interactions as shown by FRET analysis………………….....101 Figure 3.6 RPL9 immunoprecipitates with Gag from infected and transfected NMuMG cells …...…………………………………………………………………………………………103 Figure 3.7 C-terminal truncations of C3H Gag in context of RPL9-mCherry overexpression ..….……………………………………………………………………………...…………105 Figure 3.8 Localization of C3H Gag domains in context of RPL9 overexpression…………107 Figure 3.9 Examining the conservation of RPL9-Gag interactions across species with confocal microscopy………………..……………………………………………………109 Figure 3.10 Mapping the Gag interaction domain within RPL9………………………………111 Figure 3.11 Further detailed mapping of RPL9 sequences…………………………………..113 Figure 3.12 RPL9 is not detected in MMTV virions and equally expressed in infected and uninfected cells…..……………………………………………………………………….115 Figure 3.13 Model for downstream pathways of RPL9 that may be affected by interactions with MMTV Gag and lead to deregulation of tumor suppressive activities…………117

Chapter 4 Figure 4.1 The signals and putative motifs of MMTV Gag ………………………...…………148 Figure 4.2 Visualization of MMTV Gag in cells under stead-state conditions by confocal microscopy………………………...………………………………………………………150 Figure 4.3 Examination of the CRM1 nuclear export capabilities of MMTV Gag…………..152 Figure 4.4 Nuclear fractionation of NMuMG cells transfected with Gag-GFP constructs…154 Figure 4.5 Expression and localization of MMTV Gag truncations……….………………….156 Figure 4.6 Chart of P-body and stress granule proteins used in this study…………………158 Figure 4.7 Colocalization of MMTV Gag with PB and SG markers………………………….160 Figure 4.8 Examination of collected media from Gag-GFP transfected cells for the presence of virus-like particles (VLPs)……………………………………………………………..162 Figure 4.9 Expression of C3H Gag-GFP in selected Gag-GFP NMuMG cells……………..164 Figure 4.10 Transmission electron microscopy images of negative-stained NMuMG cells.166 Figure 4.11 Schematics and confocal images of C3H Gag constructs bearing RNA transport elements…………………………………………………………………………………...168 viii

Chapter 4 (continued) Figure 4.12 Examination of collected lysates and media from cells transfected with the Gag constructs bearing RNA transport signals……………………………………………..170

Chapter 5 Figure 5.1 Summary and model of MMTV Gag intracellular possibilities…………………...184

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List of Tables

Chapter 3 Table 3.1 Amino acid comparisons of different species to mouse RPL9 (NCBI: NM_011292) …….………………………………………………………………………………………. 99 Table 3.2 Potential RPL9 interacting proteins derived from internet protein databases…..119

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Abbreviations List

ALV avian leukosis virus bp base pair CA capsid CMV cytomegalovirus CTD C-terminal domain CTE constitutive transport element CTRS cytoplasmic targeting/retention signal DC dendritic cell DLS dimer linkage structure DNA deoxyribonucleic acid dUTPase deoxyuridine triphosphatase EM electron microscopy Env envelope ER ESCRT endosomal sorting complexes required for transport FRET fluorescence energy transfer FV foamy virus g gravity Gag group specific antigen GFP green fluorescent protein GRE glucocorticoid response element gRNA genomic RNA HERV-K human endogenous retrovirus-K HIV human immunodeficiency virus HRE hormone response element HTLV human T-cell leukemia IAP intracisternal type-A particle IN integrase IRES internal ribosome entry site ITAM immunoreceptortyrosine-based activation motif (Env) JSRV Jaagsiekte sheep retrovirus kb kilobase kD kilodalton

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L domain late domain LMB Leptomycin B LTR long terminal repeat MA matrix mCherry monomeric cherry fluorescent protein MHC major histocompatibility complex MHR major homology region min minute MLV murine leukemia virus mM millimolar MMTV mouse mammary tumor virus MPMV Mason Pfizer monkey virus mRNA messenger RNA Mtv Endogenous MMTV Naf Negative acting factor NC nucleocapsid NES nuclear export signal NLS nuclear localization signal nm nanometer NMR nuclear magnetic resonance NoLS nucleolar localization signal NRE negative regulatory element nt nucleotide NTD N-terminal domain ORF open reading frame PAGE polyacrylamide gel electrophoresis PBS primer binding site PIC preintegration complex Pol II RNA polymerase II Pol polymerase Poly(A) polyadenylated PPT polypurine tract Pr precursor Pro protease R repeat

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Rem regulator of export/expression of MMTV mRNA RemRE Rem response element (MMTV) Rev Regulator of virion (HIV) RevRE Rev response element RNA ribonucleic acid RP ribosomal protein rRNA ribosomal RNA RSV Rous sarcoma virus RT reverse transcriptase s second S Svedberg unit Sag superantigen SA splice acceptor SD splice donor SDS sodium dodecyl sulfate SIV simian immunodeficiency virus SP signal peptide ssDNA single stranded DNA SU surface unit (Env) TCR T-cell receptor TfR1 transferrin receptor 1 TLR4 Toll-like receptor 4 TM transmembrane (Env) tRNA transfer RNA U3 unique 3’ U5 unique 5’ UTR untranslated region VLP virus-like particle WB Western blot

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Preface

The intent of the work presented in this dissertation is to expand the breadth of knowledge about the mammalian retrovirus mouse mammary tumor virus (MMTV).

Specifically, dissection of the main MMTV structural protein, Gag, offers information on how like MMTV use the host cell for assembly of new particles and may possibly interfere with normal cellular activities to cause disease.

The data within this body of work represents work I performed in the laboratory of Dr.

Leslie Parent as a graduate student in her laboratory. With the exception of the yeast two hybrid analysis (sections 3.3, 3.5, Figure 3.1), which was performed by my collaborator, Dr.

Tatyana Golovkina, I performed all of the experiments included in this document.

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Acknowledgments

Looking through this dissertation after I have compiled all of my data from the past several years, I am amazed at how far I have come as a scientist and the volume of information I have learned. While earning a doctorate degree is no easy feat, the process was made less difficult with the love and support of my family and friends. To Momma,

Daddy, Brandon, Gram, and my friends “back home” and in Hershey, I thank you for tolerating all of my “science talk” and for your attention even when you had no idea what I was talking about. Your encouragement kept me going when I feel I could not.

I can honestly admit that I never saw myself as a scientist while I was growing up on a quaint farm in West-Central Pennsylvania. My personal expectations for a future were narrow-minded and local. With the intervention of some amazing teachers and scientists, though, my eyes were opened to the opportunities available for me to become educated in the sciences. This incredible journey began with my 12th grade AP Biology class taught by

Mrs. Patrice Stiffler. Her class was beyond the ordinary textbook didactics; she made science alive and hands-on, and I could not help but get caught up in her energy and enthusiasm. Mrs. Stiffler provided the impetus for me to apply to study biology at Cedar

Crest College, where I was accepted into the Genetic Engineering program.

As the first one “off the farm” to go to college, moving across the state to Cedar Crest was an eye-opening experience for me in terms of science, culture, and discovering the person I am. Early on in my time there, I was invited by biology professor Dr. Alan Hale to join his research team that studied the diversity of bacteriophage living in bacteria isolated from a local creek. With his guidance, Dr. Hale taught me how the scientific process works and assisted me in designing my first experiments, all with a touch of humor and fun. I was fortunate to have him as a mentor, as he encouraged me to think outside of the box and always consider the “big picture” of what science is trying to accomplish. At his suggestion, I participated in a summer internship at the New York State Department of Health, Wadsworth

Center, in Albany, New York. There I worked with the lively and energetic Dr. Jacques Izard xv on the genetic system of spirochete bacteria related to those that cause syphilis. Dr. Izard taught me not only several new laboratory techniques, time management, and how to prioritize tasks, but he taught me about all of the opportunities available to do research

(starting with graduate school) and highly encouraged me to continue with research science.

It was through my conversations with Dr. Izard that I decided I wanted to earn my Ph.D., and after my graduation from Cedar Crest I applied to and was accepted at the Penn State

College of Medicine in Hershey, Pennsylvania. Without the stepping stones of great mentors such as Mrs. Stiffler, Dr. Hale, and Dr. Izard, I would have never made it this far.

To them I am eternally grateful.

My time in the Department of Microbiology at the College of Medicine has been a very challenging and engaging period in my life, in terms of both science and my life outside of the lab. Under the direction of Dr. Leslie Parent, I feel I have developed into an independent and responsible scientist, and with her constructive criticism and encouragement I have honed my communication skills in writing and presentation. I appreciate her trust and respect in me as a colleague and her understanding when life threw me curves. I could not have asked for a better mentor and role model as I pursued my doctorate degree.

Last and certainly not least, I need to thank those that I live with on a daily basis: To my lab mates, who saw more of me than their own families, I am grateful to have had such wonderful and entertaining people to work with. Thank you to Scott and Eileen for training me and answering my hundreds of naïve questions, and to everyone that followed for all of the stimulating conversations, fun, and happy memories that made the time go faster. And then I need to thank Tim, who not only had to work with me but also live with me. How he has tolerated my antics these past few years I have no idea. I can never repay him for his thoughtfulness, generosity, support, and encouragement that has undoubtedly sustained me through graduate school. With him in my life, I am a better person, and a better scientist.

Tim, your Bear loves you… thank you. This dissertation is dedicated to you. xvi

Chapter 1: Introduction to Dissertation

1.1 Introduction

Retroviruses are ubiquitous pathogens that infect nearly every species from yeast to man and can cause a variety of cancers and immunodeficiency syndromes in animals. Among humans, the primary retroviral threats are human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome or AIDS, and human T-lymphotrophic virus (HTLV) which causes T-cell leukemia and lymphoma. The problem of HIV infection has manifested as an epidemic of global proportions with over

33.3 million people currently living with the virus, 22.5 million of which are living in Sub-

Saharan Africa (unaids.org). As we enter the fourth decade of the HIV epidemic, encouraging data show that the number of newly infected individuals has dropped by

19% since 1999. However, with statistics such as those for Swaziland where over 25% of the population is infected with HIV (unaids.org), the efforts to halt spread of the infection are still in their infancy.

Amazingly, researchers initially believed that human retroviruses did not exist.

This was based on data from over several decades showing that retroviruses were easily detectable in animal models but not in human specimens, were difficult to culture in human cells, and were readily lysed with human sera containing immune system proteins called complement (152). However, multiple other factors such as the identification of retroviruses in species closely-related to man and the observation of interspecies retroviral transmission, along with advances in technology, pointed to retroviruses as culprits for at least some human diseases. With the discovery of HTLV, the first human retrovirus identified in 1979, and the subsequent discovery of HIV in

1981, the field of retrovirology has exploded, with laboratories all over the world studying the different aspects and facets of retroviral replication and infection. More recent

1 studies have found evidence linking two other retroviruses to human disease: xenotropic murine leukemia virus-related virus (XMRV) has been associated with prostate cancer and chronic fatigue syndrome (CFS), while a human version of mouse mammary tumor virus (HMTV) is suspected of playing a role in human breast cancers.

Over the years, animal retroviruses have served as models to study infection and replication, providing an understanding of the mechanisms behind how the human pathogens HIV and HTLV function and cause disease. One of these classic model systems has been the avian oncoretrovirus, Rous sarcoma virus (RSV). Using RSV, several pivotal discoveries (leading to Nobel prizes) have been made including the finding that viruses can cause cancer, cellular genes can become dysregulated and cause cancer as oncogenes, and retroviruses encode an enzyme called reverse transcriptase which allows for conversion of their RNA genome into a DNA copy. The continued use of such model systems has delineated the classification of retroviruses into 7 genera based on sequence homology and two groups depending on how the viruses assemble new virions. Additionally, the generic retroviral life cycle has been defined, including the steps of viral attachment to the host, cell entry, reverse transcription, integration into the host genome, transcription of the viral genes, translation of the viral proteins, assembly of new virions, egress from the cell surface and maturation to allow infection of new hosts.

Current antiretroviral therapies target the well-studied steps of retroviral replication such as reverse transcription, maturation of the virus by protease, viral entry, and integration. Despite the variety of treatments available and used for highly active antiretroviral therapy or HAART, retroviruses readily mutate and develop drug resistance over time. The combined use of drugs that target a variety of virus replication steps is essential in suppressing the viral titres of the patient. It is therefore imperative that new drugs be sought to abrogate other steps of the virus replication cycle. The

2 oligomerization of viral proteins into nascent virus particles is an imperative part of productive retroviral infection, however, there are currently no drug inhibitors specifically made to negate assembly.

Retroviral assembly encompasses the stage after translation of viral proteins on host ribosomes and the subsequent trafficking of those proteins to a cellular site for oligomerization into immature virus particles. Along the way, viral proteins must coalesce with the viral genomic RNA for packaging into the developing virion. Viral proteins then interact with cellular machinery to enclose the genome within a spherical virus particle and again use host factors to mediate the release of the particles from the cell. These steps are known to be imperative for the production of infectious virus particles, yet the detailed choreography of this process and the signals responsible remain obscure for many retroviruses. Until further research fills the gaps of knowledge in how retroviruses interact with their host to achieve particle formation, development of drugs targeting this phase of viral replication is at a standstill.

1.2 Development of the research presented in this dissertation

Shortly after my entry into the Parent laboratory, I pursued experiments to understand host-viral interactions using RSV, which is the primary model system used in our studies of retroviral assembly. Previous investigations in our laboratory discovered novel nuclear trafficking steps of the RSV Gag protein, which is the main structural protein of retroviruses that mediates assembly and packaging of genomic RNA. Initially,

I was examining whether RSV Gag directly interacted with the cellular protein, CRM1, which mediates export of Gag from the nucleus. During my unsuccessful attempts at coimmunoprecipitation, Dr. Parent approached me with the possibility of collaboration with Dr. Tatyana Golovkina at the University of Chicago. Dr. Golovkina’s laboratory studies MMTV in a mouse model system and had discovered that a new strain of MMTV

3 was formed from recombination between a highly tumorigenic exogenous strain of

MMTV called C3H and an endogenous strain, Mtv1, that is inherited in the mouse genome. This new strain, HeJ, is less pathogenic in mice despite the presence of C3H sequences in the genome. Further mapping revealed that sequences within the gag

gene contributed to the degree of carcinogenesis produced in the mice, reflecting the

different strains of MMTV used in the infection. To further study these differences in the

Gag proteins of the C3H and HeJ strains, Dr. Golovkina conducted a yeast two hybrid

assay and identified a cellular factor, ribosomal protein L9 (RPL9) that appears to

interact with C3H Gag but not HeJ Gag. In the interest of learning more about Gag

interactions with RPL9, Dr. Golovkina offered our laboratory the initial tools to examine

MMTV Gag based on our previous experience with the molecular characterization of

RSV Gag. With the opportunity to examine the signaling and movements of MMTV Gag,

which forms immature particles in the cytoplasm as opposed to at the membrane like

RSV, we embarked upon this journey to further examine MMTV.

The first 2-3 years of my graduate research consisted of developing the tools,

assays, and skills to examine MMTV Gag in our cell culture system. During this time we

came to appreciate the paucity of information concerning the processes of assembly and

budding in MMTV, and realized the opportunity to study MMTV Gag in a way never

examined before. Thus, my research focus was divided into two main arms:

investigations of the interaction between RPL9 and Gag, and the characterization of the

intracellular trafficking, localization, and budding of Gag. Initially, I divided my time

between both projects, but the RPL9 story emerged as the primary focus and the data

from my efforts are presented in Chapter 3 of this dissertation. The accumulation of my

observations on how MMTV Gag localizes in the cell and aggregates to form/release

virus particles embodies Chapter 4, which represents both my earliest and my most

recent research enterprises.

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1.3 Rationale for these studies

As already mentioned, our laboratory discovered a novel intracellular trafficking pathway of the RSV Gag protein whereby Gag transiently passes through the nucleus prior to assembly at the membrane. Currently, it is not known if other retroviruses follow similar pathways. To expand upon these findings, MMTV serves as an opportunity to determine whether other retroviral Gag proteins use a parallel nuclear transport pathway. Additionally, in considering that MMTV forms immature particles in the cytoplasm as opposed to the plasma membrane, characterization of MMTV Gag will provide further understanding of the trafficking of retroviruses that follow a different morphogenetic pathway from RSV. Considering the data that implicate MMTV Gag in tumor progression, examination of the signals responsible for Gag movement and localization could also shed light on how this viral protein contributes to tumor progression. Should further deliberation support a role of MMTV or a related virus in human cancers and disease, we will be poised with information that may be invaluable for the development and use of therapeutic options.

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Chapter 2: Literature Review

2.1 Historical perspective of MMTV

The discovery of mouse mammary tumor virus (MMTV), otherwise known as the

Bittner virus, manifested from observations by cancer scientists spanning over a century

of time. The first report of mammary tumors in wild mice was as early as 1854, with the

brief commentary that a mammary “scirrhus-like tumor” was observed on a rodent

caught in a trap (80). A short time later, scientists began to specifically breed and use

mice for experimental cancer research (80). It was noted by Lathrop and Loeb in 1918

that in selective mouse breeding studies, mammary tumors appeared to have a genetic

component that was dominated by the tumor phenotype of the maternal parent (243).

However, a non-Mendelian mode of inheritance of mammary tumors in mice was

confirmed in 1933 (209) and the manner in which neoplasms were transmitted to

offspring continued to perplex scientists. Shortly thereafter in 1936, a scientist by the

name of J.J. Bittner at the Jackson Memorial Laboratory performed a landmark

experiment that, although simple in nature, changed the thinking of cancer biology

forever. By nursing newborn mouse pups on mothers of differing tumor incidence

strains, he showed that the mammary tumor agent was milk-transmitted and not in the

mouse genome (40). Through ultracentrifugation of tumor extracts and milk, and

inoculation of these materials into mice that subsequently developed tumors, it was

determined that the “active agent [was] a colloid of high molecular weight” (42) and most

likely a virus (62,444).

With data suggesting a viral etiology for mouse mammary cancer, the research

on Bittner’s tumor agent quickly expanded. In 1948, some of the first electron

microscope (EM) images of spherical, virus-like entities with dense centers were

captured within tumor cells from high tumor incidence mice (351). Additional electron

6 microscopy revealed that similar particles could be purified from mouse milk and were capable of causing mammary carcinomas upon inoculation into low tumor-bearing strains of mice (171). It was observed that two different types of virus-like particles existed within all tumor tissues of high tumor incidence mice: intracytoplasmic particles with a concentric double membrane (called A particles) and extracellular particles, which were larger in size, possessed an “eccentric nucleoid” (called B particles), and were derived from A particles (33). Characterization of purified B-type particles led to the discovery that they possessed RNA, consisted of lipids and proteins, were ether- sensitive and had distinguishing radial spikes protruding from their surfaces

(256,257,281,347,377,379).

By 1965 it was accepted that Bittner’s milk agent was an oncogenic RNA virus

(257,301), and around this same time evidence arose that the virus could become heritable (301,347). The work of Bentvelzen (1970) and Varmus et al. (1972) confirmed that in addition to an infectious exogenous form, the Bittner virus could be transmitted as an endogenous provirus within mouse germline DNA (31,441). With the discovery of reverse transcription in 1970 by Baltimore, Temin, and Mizutani (22,421), it was found shortly thereafter that MMTV possessed the RNA-dependent DNA polymerase, marking it as the first known mammalian retrovirus (43,388,389,394).

2.2 Taxonomy of retroviruses

Retroviruses comprise the virus family Retroviridae, whose members are enveloped, spherical viruses that possess two copies of single-stranded, positive-sense

RNA and undergo reverse transcription during their replication cycle (1,447). The family is divided into two subfamilies and seven genera based on conservation within the polymerase gene sequence and overall genomic organization: Orthoretrovirinae

(Genera: Alpha-, Beta-, Gamma-, Delta-, and Epsilonretrovirus, and Lentivirus) and

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Spumaretrovirinae (Genus Spumavirus). The Alpha-, Beta-, Gamma-, Delta- and

Epsilonretrovirus genera members were formerly known as oncoviruses due to their ability to cause cancer in infected organisms (447). Lentiviruses, such as HIV, primarily infect non-dividing cells which are killed or altered by the infection, resulting in chronic disease of the host (447). The least famous group of retroviruses is the spumaviruses, which are ubiquitous but not known to cause pathogenesis (447). Of these groups,

MMTV is categorized as a betaretrovirus and serves as the representative species for this genus; other betaretroviruses include Mason Pfizer monkey virus (MPMV) and

Jaagsiekte sheep retrovirus (JSRV) (3).

2.3 Virion morphology

2.3.1 Categories of particle morphology

Betaretroviruses are characterized by their ability to assemble immature virus particles in the cytoplasm of infected cells prior to transport to the plasma membrane for envelope acquisition and budding. Pre-assembly of particles in the cytoplasm prior to budding is known as Type B/D morphogenesis. This is opposed to virion assembly that takes place at the plasma membrane concurrent with budding and envelopment, which is more common among retroviruses and is called Type C. A fourth morphology, Type

A, can refer to immature particles in the cytoplasm that are precursors to Type B/D particles or to intracisternal type-A particles (IAPs) that bud primarily into and accumulate in the endoplasmic reticulum (ER) without a plasma membrane step (448).

MMTV represents the Type B morphogenetic pathway, which only differs from Type D in the mature virus form by the presence of prominent glycoprotein spikes around the envelope and an eccentrically placed round core; the prototypical Type D retrovirus,

MPMV, contains a bar-shaped core and less prominent envelope projections (448).

8

2.3.2 MMTV particle morphology and general composition

Under an electron microscope, mature MMTV virions appear as a roughly spherical particle with an eccentrically placed, irregularly-shaped angular nucleocapsid core (54,379). Virions are 120-140 nm in diameter and have icosahedral symmetry, with approximately 400 large prominent glycoprotein spikes protruding from the viral membrane (33,54,319,378). These 800-1000S particles consist of proteins (60%), lipids derived from the host cell membranes (35%), carbohydrates (3%), and RNA (2%)

(2,171,178,223,379).

Type A or immature particles are approximately 65-71 nm in diameter and are found in the cytoplasm of infected cells (33,418). Often, these doughnut-like double ring structures are found abundantly in tight clusters or associated with the cytoplasmic face of vacuoles/vesicles (33,285,325,406). Type A particles contain RNA but lack DNA and lipid, and are the precursors of Type B mature virions (418).

2.4 MMTV genome organization

Along with classification by genera and particle type, retroviruses are further subdivided based on the protein-coding content of their genome. Simple retroviruses contain little more than the basic gag (group specific antigen), pro (protease), pol

(polymerase), and env (envelope) genes that all retroviruses share in the same 5’ to 3’

order. For example, MPMV is classified as a simple retrovirus, by virtue of its basic

retroviral gene repertoire and only one additional accessory gene product (dUTPase).

The “simple lifestyle” reflects the lack of virally-encoded trans genes that influence viral

RNA synthesis or processing, and the minimal use of splicing for viral gene products (i.e.

only for env) (95). Conversely, the genomes of complex retroviruses encode additional

nonstructural regulatory proteins that are derived from a variety of spliced RNA

messages. At least one accessory gene product of complex retroviruses possesses

9 virus-specific trans-activating functions, such as the Rev protein of HIV (95). HIV is the champion of complex retroviruses, encoding six gene products in addition to the basic

Gag, Pro, Pol, and Env proteins.

Until recently, MMTV was considered a simple retrovirus, though it encodes two accessory genes, dutpase and sag (superantigen). In 2005, MMTV was found to encode a third accessory protein termed regulator of export/expression of MMTV mRNA, or Rem, which is homologous in function to Rev from HIV and Rex from human T-cell leukemia virus (HTLV) (206,289). Possession of this trans-activating factor ranked

MMTV among complex retroviruses, the first murine retrovirus to be classified as such

(289).

The 70S genome of MMTV consists of twin copies of non-segmented, positive- sense, single-stranded RNA (2,114,121). The two RNA strands are held together non- covalently through sequences near the 5’ end of the genome that comprise the dimer linkage structure (DLS) (29,314,334). The putative DLS region of the MMTV genome lies within the MA domain of the gag coding region (138). Each 35S genomic RNA monomer is 8,600 nt long and like all retroviral genomes, contain a 5’ methylated cap and a 3’ polyadenylated tail (345).

The 5’ end of the MMTV RNA genome contains the 5’ untranslated region or

UTR. This area consists of a 15 nt direct repeat sequence (R) followed by the 120 nt unique region (U5) and the primer binding site (PBS), a short span of nucleotides that participates in the priming of reverse transcription. Next are the three overlapping open reading frames (ORFs) that encode the viral proteins: gag, pro, dutpase, pol (which encodes the reverse transcriptase and integrase enzymes), env, rem, and sag.

Overlapping with the coding sequences of env and sag, the 3’ end of the MMTV genome is composed of the 1,200 nt unique 3’ region (U3) flanked by the second R sequence

(Figure 2.1).

10

2.5 Proteins of MMTV

2.5.1 MMTV mRNA and ribosomal frameshifting

The viral genes gag, pro, and pol are translated from the unspliced 35S full- length RNA that also serves as the genomic RNA, whereas the synthesis of other viral proteins is directed by subgenomic transcripts: env and sag are produced from singly- spliced mRNAs and rem from doubly-spliced RNA. Because splicing is required for the production of proteins integral for the assembly of infectious virus particles, the positive- sense MMTV RNA genome is not infectious.

To produce all three Gag, Pro, and Pol proteins in different overlapping ORFs from one RNA transcript, two ribosomal frameshift events must occur during translation

(208,305). A -1 frameshift at the end of Gag allows for read-through of Pro, and a second -1 frameshift after Pro allows for translation of Pol. Interestingly, the frameshifts appear to be mediated by a heptanucleotide sequence near the boundaries of Gag-Pro and Pro-Pol, in addition to a RNA pseudoknot structure just downstream that appears to stall ribosomes in translation allowing the shift to occur (84,86). Collectively, ribosomal frameshifting yields the non-glycosylated polyprotein precursors of Pr77Gag, Pr110Gag-Pro, and Pr160Gag-Pro-Pol which are produced in a ratio of 30:10:1, respectively

(12,102,113,191,208) (Figure 2.1). These larger polyproteins are subsequently cleaved by the viral protease and possibly cellular proteases (284) to yield the protein components found within mature virus particles.

2.5.2 Group specific antigen (Gag)

The Gag polyprotein serves as the dominant structural protein that drives assembly in the formation of new MMTV virus particles. Within immature virus particles,

Gag molecules align radially to form a spherical structure (285), and are subsequently

11

Figure 2.1 Features of the MMTV genome. (Top) Illustration of the integrated MMTV proviral genome, containing the flanking duplicate long terminal repeats (LTR) consisting of the unique 3 (U3), direct repeat (R), and unique 5 (U5) regions. Directly below, promoters within the MMTV genome are indicated as triangles. The classical MMTV promoter is marked with the filled triangle, and potential promoters used for the sag

mRNAs are designated with open triangles. Within the coding sequence lie 3 open

reading frames (ORFs), designated as ORFs 1-3, containing the MMTV genes gag, pro/dutpase, pol, env/rem and sag. (Middle and below) Diagram of the MMTV RNA

transcripts (black lines with “T” ends) and the protein products (open boxes) that they

yield after translation. The unspliced MMTV mRNA serves as the genomic RNA for

packaging, as well as the template for Gag, Gag-Pro, and Gag-Pro-Pol translation.

Splicing sites are noted with arrows for splice donor (SD) and splice acceptor (SA).

Transition between ORFs during translation of Gag-Pro and Gag-Pro-Pol occurs via -1

frameshifts of the ribosome (indicated by zig-zag arrows). The letter “M” in a small circle

indicates the myristic acid modification of MA at the N-terminus of Gag. Subsequent

cleavage products of Gag, Pro, and Pol are also indicated within the protein product

boxes. Spliced RNA products are shown below, with angled thin lines denoting the

excised intron regions between fused exons; Env is produced from a single splicing

event, Sag is produced from four possible mRNA transcripts that are singly-spliced, and

Rem results from a doubly-spliced mRNA. Subsequent cleavage products of Env are

indicated within the protein product box. Not shown are the glycosylation sites that exist

within the Env and Sag proteins.

12

13 cleaved upon maturation of the virus (discussed in section 2.7.8). From N-terminus to

C-terminus, Gag spans 592 amino acids and consists of the domains: MA (matrix/p10), phosphoprotein 21 (pp21), p3, p8, n, CA (capsid/p27) and NC (nucleocapsid/p14)

(12,113,191,192,274,396). The MA, CA, and NC domains are functionally conserved among all retroviral Gag proteins, though domains from different viruses may share little sequence homology with one another (345).

2.5.2.1 Matrix (MA)

The most N-terminal domain of all retroviral Gag polyproteins is the membrane- associated matrix or MA protein. During assembly, the 10 kD MA is responsible for directing the nascent particle towards the membrane, and within the virion MA is associated with the (81,285,345,357,448,460). This membrane association is attributed to the hydrophobic nature of MA (81,269,274) as well as a post- translational covalent modification of its second amino acid residue (glycine) by myristic acid (192,392). The myristoylation of MMTV Gag as well as the Gag proteins of other retroviruses is necessary for virus assembly and budding (414,479), however not every retroviral Gag protein is myristoylated. For example, the Rous sarcoma virus (RSV) Gag polyprotein is acetylated at its N-terminus and contains a stretch of basic amino acid residues within MA that mediates plasma membrane trafficking (74).

Additionally, MA bears a region of 18 amino acid residues termed the cytoplasmic targeting-retention signal (CTRS). Studies of the homologous sequence in

MPMV shows that the CTRS targets Gag to the pericentriolar region of the cell for immature particle assembly; transport of MPMV Gag to this site has been shown by

Sfakianos et al. to be mediated by the dynein/dynactin motor complex and is microtubule-dependent (89,362,364,397). Additionally, the mutation of a single residue within the MPMV CTRS is capable of converting MPMV Type B assembly to Type C

14 particle production at the plasma membrane. Though some of the MPMV virions produced by this altered pathway were not infectious, the study indicates that the CTRS region of MA is important for particle assembly in the cytoplasm (362).

2.5.2.2 In media res: The unique pp21, p3, p8, and n domains of Gag

Beyond the Gag domains common to all retroviruses, MMTV also encodes four noncanonical peptides, the properties of which are poorly understood. The phosphoprotein 21 (pp21) domain is the second largest domain of Gag after CA, and it is the major phosphorylated component of the virus (322,380). A similar phosphoprotein element exists within MPMV, which has been shown to bear a late domain (L) sequence

(PPYX4PSAP) that is imperative for particle budding and release (170). However, the

sequence of MMTV pp21 (and the remainder of Gag) lacks a similar identified entity.

Even less information is known about the smaller Gag peptides p3, p8, and n.

The 33 amino acid long p3 region is rich in acidic amino acids, whereas the shorter p8

peptide (24 amino acids) is basic residue rich. The small “n” domain of only 17 amino

acids is located between p8 and CA, however, a peptide corresponding to this region of

Gag has yet to be identified within virions (192). Mutational studies that delete p3, p8,

and n of Gag from MMTV proviral constructs found that mutants missing the p8 and n

regions were severely defective in budding and proper virion formation (480). Though it

appears that p8 and n are contributing to Gag assembly, the mechanisms for how this

may be occurring are currently not understood. Additionally, it has been reported that

the p3-p8 intermediate cleavage product of the Gag polyprotein is likely ubiquitinated

(330), however the significance of this finding is not known.

15

2.5.2.3 Capsid (CA)

The p27 CA domain of MMTV Gag is the largest of the Gag domains and is the major structural protein that forms the shell encapsidating the viral genome in mature virions (448). CA contains a hydrophobic subdomain (269) and exists in both phosphorylated and unphosphorylated forms (380). As with most retroviral CA proteins,

MMTV CA is one of the more antigenic domains of Gag (448) and antibodies are easily generated against CA for use in detection assays. Unique to the CA domain is the presence of the major homology region (MHR), a stretch of approximately 20 amino acids that are conserved among all retroviruses except spumaviruses (266,339,460).

2.5.2.4 Nucleocapsid (NC)

The most C-terminal domain of the Gag polyprotein is NC, a highly basic 14 kD protein that functions to moderate the activity of the viral RNA; NC specifically binds to the viral genomic RNA during assembly and in the virion, promotes RNA dimerization, stimulates reverse transcription by RT, and binds to viral DNA after reverse transcription

(414,448). MMTV NC contains dual copies of the zinc finger motif (Cys-X2-Cys-X4-His-

X4-Cys; Cys-His box) which binds zinc and is conserved among all retroviruses (except

for spumaviruses) (34,448). The structure of the MMTV NC has been determined via

nuclear magnetic resonance (NMR) spectroscopy and found to have an N-terminal Cys-

His box similar to that of HIV while the C-terminal Cys-His box possesses a more unique

structure (225). This difference in structure may allow for the two independent Cys-His

domains, associated by a flexible linker region, to participate in separate nucleic acid

binding events (225). The RNA-packaging properties of NC conferred by the Cys-His

boxes will be discussed further in section 2.7.7. Additionally, the NC of MMTV is

diubiquitinated (330), though the importance of this post-translational modification is not

known.

16

2.5.3 Deoxyuridine triphosphatase (dUTPase or DU)

Proteolytic cleavage of the frameshifted MMTV Pr110Gag-Pro and Pr160Gag-Pro-Pol

polyprotein precursors yields the 30 kD dUTPase (p30), whose protein coding region lies

at the junction of Gag-Pro and overlaps with the C-terminal sequences of the Gag NC

domain (32,191,229). The first 95 amino acids of the MMTV dUTPase are derived from

NC with the following 154 amino acids derived from the 5’ ORF of pro (191). The MMTV

dUTPase is enzymatically active as a trimer which may directly associate with the viral

RNA by virtue of the NC domain sequences (32). dUTPases are enzymes that catalyze

the conversion of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate

(dUMP) and pyrophosphate, which is important for nucleotide biosynthesis and

prevention of uracil incorporation into DNA sequences. In addition to retroviruses,

dUTPase enzymes are found within eukaryotic and prokaryotic organisms, as well as

herpesviruses and poxviruses (229,278). Within retroviruses, the dutpase gene exists in

some B/D type viruses and nonprimate lentivirus species and is speculated to increase

the fidelity of reverse transcription by decreasing dUTP pools and providing dUMP for

thymidine biosynthesis (32,420). It is also speculated that dUTPase may serve a

structural role in the virus as an alternate form of NC; dUTPase is an abundant protein

that can bind to genomic RNA but possesses low catalytic activity (32). Though the

deletion of dUTPase in some retroviruses does not affect viral replication in vitro, there

appears to be a requirement for dUTPase in certain cell types. This cell type

dependency on dUTPase may reflect a profusion of intracellular dUTP, which could be

misincorporated into viral DNA and lead to mutations (discussed in (229,345)).

Unlike the conserved gag-pro-pol-env gene order found within all retroviral

genomes, the dutpase gene location can be found either in the ORF encoding pro

(MMTV and other B/D type retroviruses) or in the ORF encoding pol (lentiviruses) (278).

The location of the dutpase gene in the viral genome therefore corresponds with the

17 abundance of the protein, as retroviruses yield more Gag-Pro polyproteins than Gag-

Pro-Pol (32,229). Though the dUTPase activity is conserved between the B/D type retroviruses and the lentiviruses, the gene sequences for this protein are not closely related between the two groups (345).

2.5.4 Protease (Pro or PR)

Depending on the genus of retrovirus examined, the virally-encoded protease

(pro) can appear either in the same ORF as gag (example: RSV), the same ORF as pol

(example: HIV), or in its own ORF, as is the case with MMTV. MMTV Pro is 13 kD in size and is packaged into virions as part of the Gag-Pro and Gag-Pro-Pol precursor polyproteins. Pro is the viral enzyme responsible for the ordered processing of the polyproteins into their respective mature subunits, resulting in the transformation of immature Type A particles into mature infectious Type B particles (286,414). In most retroviruses, Pro functions late in assembly to facilitate cleavage of polyproteins after or concurrent with budding. However, MMTV protease begins processing polyproteins intracellularly in a manner that is not dependent on membrane targeting or budding of immature particles (479). This curious phenomenon correlates with the observation of occasional mature virus particles among immature cores in the cytoplasm of infected cells (33,156,325). Though the significance of early processing by Pro is not understood, it does raise the possibility that Pro may cleave cellular proteins that play a role in viral replication or pathogenesis (414); it has been shown in independent studies that MMTV

Pro is capable of cleaving the translation elongation initiation factor eIF4G (9) and poly(A) binding protein 1 (PABP1) (8) (discussed in section 2.7.6).

The retroviral protease has been classified as an aspartic proteinase, which is characterized by the presence of two aspartate amino acid residues within the conserved Asp-Thr/Ser-Gly motifs. Unlike eukaryotic aspartic proteinases, retroviral

18 proteases contain only one Asp-Thr-Gly motif and therefore require the dimerization of

Pro for its catalytic activity (286,414). Retroviral proteases primarily target hydrophobic residues within a 7-8 amino acid stretch for cleavage, though the specific target sequences are variable (127,414). Although Pro is essential for the proper assembly, budding, and maturation of retroviruses, it is not known what regulates its activation within the immature virus particle as opposed to before or during assembly. It has been suggested that a change in pH within the virion (MMTV Pro is most active at pH 4-6) or the association of Gag-Pro dimers in the immature cores may allow for protease to undergo autocatalytic cleavage (286,414). The activation of MMTV Pro appears to be loosely regulated compared to Pro of MPMV, as MMTV proteolytic cleavage occurs in the cytoplasm of infected cells prior to budding and MPMV requires membrane targeting of immature capsids before processing of Gag can occur (479).

2.5.5 Reverse transcriptase (RT)

The hallmark of any retrovirus is the possession of reverse transcriptase (RT), the RNA- and DNA-dependent DNA polymerase that also has nuclease activity (420).

MMTV RT is a 66 kD transframe protein that consists of 27 amino acids from the Pro- coding region fused to the N-terminal portion of the Pol ORF (133,419). The polymerase activity of RT is situated within its N-terminus, while the RNase H activity that cleaves the RNA strand of RNA-DNA hybrid complexes is within the C-terminus of

RT (420). The MMTV RT has been shown to be enzymatically active as a monomer with an optimum pH of 7.5 and a preference for Mg2+ ions for polymerase activity

(115,419). Like all retroviruses, MMTV uses a host-encoded tRNA primer for initiation of

reverse transcription. It has been shown that MMTV preferentially packages tRNALys

and uses tRNALys3 for priming at its primer binding site (PBS) near the 5’ end of the viral

RNA genome (343,455). RT lacks 3’ and 5’ exonuclease proofreading capabilities,

19 lending to the high mutation rate, significant genetic diversity, and rapid viral evolution and adaptation of retroviruses (420). In comparison to host replication machinery, retroviral RT is much slower and is poorly processive (420).

2.5.6 Integrase (IN)

The second half of the MMTV Pol ORF encodes the viral integrase (IN), which catalyzes the insertion of the reverse-transcribed DNA genome into host cell DNA. IN is included in the virion as part of the Gag-Pro-Pol polyprotein precursor, the cleavage of which yields the active IN. IN recognizes, cuts, and then joins specific viral DNA sequences at the long terminal repeat (LTR) ends to random sites within host DNA in the process of retroviral integration.

The specific characteristics of the MMTV integrase have not been extensively studied, though it is assumed to share properties that are conserved among all retroviruses. Retroviral range in size from 280 to 450 amino acids in length

(MMTV IN is 320 amino acids), and consist of three domains: the N-terminal HHCC domain, the central catalytic core domain, and the C-terminal DNA-binding domain (58).

All retroviral integrases share a highly conserved zinc-finger-like His-Cys motif (H-X(3-7)-

H-X(23-32)-C-X2-C) within the HHCC domain, as well as a triad of essential acidic amino acids known as DD35E within the catalytic core (132,210). The HHCC region stabilizes the catalytic core of integrase and promotes tetramerization of the enzyme, whereas the catalytic core is responsible for specifically integrating the viral RNA into host DNA and the variable C-terminal domain of integrase participates in DNA binding (58). The integrase tetramer complex bound to viral DNA ends has been shown in vitro to be the minimal components necessary for the concerted integration of both viral LTRs that border the genome (139). This process occurs by specific recognition of the viral DNA

20 ends, which are flanked by inverted CA/TG sequences that are essential for integrase substrate targeting (58).

2.5.7 Regulator of export/expression of MMTV mRNA (Rem)

Rem is the most recently discovered component of the mouse mammary tumor virus. It was discovered using MMTV proviral constructs that were disrupted in the env gene region which were found to be defective for Gag production and did not export unspliced RNA from the nucleus (289). Another research group simultaneously identified Rem by comparing MMTV with the human endogenous retrovirus type K

(HERV-K), another betaretrovirus (206). Subsequently, the Rem protein was found to have chromosome region maintenance 1/exportin 1 (CRM1/Xpo1)-dependent RNA export activity similar to that found in HIV (Rev) and HTLV (Rem), which mediates transport of unspliced viral RNA from the nucleus to the cytoplasm for translation

(206,289). The 301 amino acid peptide of Rem is the product of a doubly-spliced RNA transcript, consisting of 98 amino acids from the Env leader peptide, 162 amino acids from the Env surface protein (SU or gp52) and 41 amino acids from the Env transmembrane protein (TM or gp36) (206,289). Interestingly, MMTV Rem sequences are in the same ORF as those of Env (Figure 2.1), as opposed to a -1 or -2 shifted reading frame.

The N-terminus of Rem contains regions that are conserved with other retroviral

RNA exporting proteins, while the very long C-terminus appears to have negative self- regulating activity (289). Rem contains both a nuclear localization signal (NLS) and a nucleolar localization signal (NoLS) that mediate nuclear/nucleolar trafficking, with an overlapping arginine-rich RNA binding domain (206,289). CRM1-mediated nuclear export of Rem is facilitated by a canonical leucine-rich nuclear export signal (NES) (206).

Rem also bears two asparagine-linked glycosylation sites that, when filled with

21 oligosaccharide units, increases the size of Rem by 5 kD (72,206). Other modifications of Rem, including myristoylation, amidation, and phosphorylation have also been predicted (289). Rem functions by binding to a Rem responsive element (RemRE) located at the env/U3 border within viral RNA (288,311). The ~480 nucleotide sequences within the RemRE generate extensive secondary structures that are important for Rem recognition and are similar to RNA response elements from other retroviruses (288).

Though Rem possesses features that are shared among other retroviral transport proteins, the MMTV Rem protein is unique in many ways. Rem, at 33-39 kD, is significantly larger than other retroviral export proteins, which range in size from 12 to 21 kD (206,289). Surprisingly, all of the motifs necessary for RNA export are localized within the first 98 amino acids of Rem, within the envelope signal peptide (289). The large C-terminus of Rem remains unaccounted for in terms of function, but may serve as a negative autoregulator for Rem activity or may be secreted as an immune system regulator in infection (72,289). This arrangement of the MMTV Rem domains is similar to those of Rec from HERV-K, another betaretrovirus with an unusually long Env signal peptide region (289).

Signal peptides traditionally target proteins to the ER for membrane insertion and glycosylation, and are later cleaved off by signal peptidase. Before the identification of

Rev, it was reported that the MMTV Env signal peptide curiously localizes to nucleoli of infected cells and interacts with nucleolar protein B23, which is similar to what has been reported for nucleolar retroviral RNA export proteins such as Rev (24,193,194). After the identification of Rem in 2005, the nucleolar localization of the signal peptide made more sense, as it is sufficient on its own to mediate export of viral unspliced RNA from the nucleus (289). Since then, it has been shown that a fraction of Rem localizes to the

ER where its C-terminus is glycosylated, and the N-terminal signal peptide region (p14

22 or SP) is removed for subsequent trafficking to nucleoli and RNA export functions

(72,122).

2.5.8 Envelope (Env)

The env gene of MMTV encodes two proteins termed surface unit (SU or gp52) and transmembrane (TM or gp36) which form the knobs and stalks, respectively, of the glycoprotein spikes that are so prominent on MMTV virion surfaces (378). The Env polyprotein is translated N’-gp52-gp36-C’ from a 24S, 3.6 kb singly-spliced mRNA

(111,120,262,395,396) (Figure 2.1). The Env mRNA transcript begins at the R of the 5’

UTR and continues 288 nucleotides downstream to the splice donor site (138). This short initiating RNA region is fused with the splice acceptor at the start site of env and continues through the ~2 kb env coding region to terminate in the 3’ LTR, slightly overlapping with the 5’ end of the sag ORF (186,345).

Translation of the subgenomic Env mRNA transcript results in the production of non-glycosylated Pr67Env, which is synthesized on the rough ER as a type I

transmembrane protein (111). Subsequently, the 98 amino acid signal peptide (that is

shared with the 5’ end of Rem) is cleaved at the Thr-Gly*Glu-Ser-Thr sequence to

produce a Pr61Env intermediate (18,111,186). Within the ER, the MMTV Env precursor

trimerizes, and maintains as a trimer while it traffics through the cell to the plasma

membrane (54,357,448). The predecessor Pr61Env protein then undergoes a quintuple glycosylation to produce the gPr73Env, which has three- and two- asparagine N-linked

oligosaccharides in SU and TM, respectively (359). The post-translational glycosylation

step of Env is imperative for proper processing and trafficking of the Env protein (146).

After ER glycosylation, gPr73Env travels through the trans-Golgi for secondary glycosylation and proteolytic processing by cellular proteases into the mature gp52 (SU) and gp36 (TM) proteins prior to transport to the plasma membrane (112). The cleavage

23 of gPr73Env occurs by removal of the dipeptide Lys-Arg at the interface of the gp52-gp36 proteins (186,359). Though the two Env subunits are cleaved from one another, they remain associated via noncovalent interactions (111). The proper proteolytic cleavage of gPr73Env precursor is essential for the membrane fusion event needed for entry of virions into host cells (see section 2.7.1) (448).

The Env SU is responsible for binding to the receptor of host cells to mediate viral entry. In the case of MMTV, the host receptor is mouse transferrin receptor 1

(mTfR1) (167,371). In addition to mediating host receptor binding, the SU of MMTV has been implicated in a variety of other roles, including: Sag presentation to T cells to stimulate T-cell proliferation and interleukin 2 production (163); enhancing mouse mammary epithelium sensitivity to prolactin by increasing the production and membrane accumulation of prolactin receptors (46,48); facilitating viral infection via interactions with surface toll-like receptor 4 (TLR4) (358); and potentially participating in mammary tumorigenesis with an immunoreceptor tyrosine-based activation motif (ITAM) within SU that has been shown to induce transformation in cell cultures and in mice (217,370).

The TM protein of Env is anchored within membranes through a 26 amino acid hydrophobic domain near its C-terminus (448). TM has also been shown to interact with the membrane-associated MA domain of Gag in virus particles (357). Trimerization of

Env in the ER is mediated by TM (414) and allows for the production of the 230 kD envelope spikes on the virion surface (357,378).

2.5.9 Superantigen (Sag)

Of all the proteins that are encoded by the MMTV genome, superantigen (Sag) is by far the most characterized and well studied. Sag is encoded within the U3 region of the genome and is translated on ER-associated ribosomes as an immature 37 kD protein (52,91,118,356). Interestingly, Sag can be translated from a variety of singly-

24 spliced mRNA transcripts that can differ in their promoter region; mRNAs from four different promoters have been described in the literature

(17,88,129,177,294,315,439,457,483) (Figure 2.1), some of which appear to be cell-type specific (474). After translation, Sag is glycosylated with 5 N-asparagine-linked oligomannosyl carbohydrates to produce a 47 kD type II transmembrane glycoprotein with a half-life of 1.5-2 hours (52,226,231,235,462). The Sag protein consists of a non- essential 45 amino acid cytoplasmic domain at its N-terminus, a conserved hydrophobic stretch of 22 amino acids that comprise the transmembrane domain, and a long extracellular domain that contains the five glycosylation sites and a polymorphic C- terminus that varies between Sags of different MMTV strains (35,52,91,226,231,462). At least one of the N-linked oligosaccharides must be present on Sag for it to be effectively presented on the cell surface (279).

Next, the gPr47Sag moves to the Golgi for further processing with the addition of complex-type glycans (to produce precursor gPr82Sag) and proteolytic cleavage by furin

and cathepsin L enzymes (108,296,462). Cleavage occurs at 3 variable positions,

yielding non-glycosylated C-terminal pieces of p16/p18, glycosylated N-terminal

segments of gp50/gp70, and the p10 transmembrane region (462). This proteolytic

processing is critical for Sag presentation at the cell surface (296,336). The mature

complex at the plasma membrane consists of an extracellular 18 kD non-glycosylated

protein (the C-terminus of the Sag precursor) (463) noncovalently tethered to the

transmembrane N-terminal portion of Sag, both of which are required for superantigen

function (238,462).

As its name states, Sag acts as an immune system superantigen upon host

infection, causing non-specific activation and expansion of T-cells with large amounts of

cytokine release (90,149,271,466,467). The Sag-mediated proliferation of T-cells

provides a large pool of immune cells which can be subsequently infected by MMTV

25

(reviewed in (101,184). Stable Sag presentation on the cell surface requires association with major histocompatibility complex (MHC) class II molecules that engage T-cells

(172). The 18 kD extracellular portion of Sag bears an MHC class II peptide binding motif (201) as well as regions that interact with the variable β chain of T-cell receptors

(TCRs). Variation in the TCR-specific region accounts for the ability of Sags from different MMTV strains to delete specific TCR Vβ subsets of T-cells (reviewed in

(101,184). Further discussion of Sag’s role in infection will follow in section 2.6.2.

Following Sag-induced T-cell expansion, there is a subsequent apoptotic depletion of the

Sag-activated cells which results in an altered immune cell repertoire for the host (386).

Consequently, Sag modulates the host’s susceptibility/resistance to infectious and

autoimmune diseases (25,38,39,169,244,254,415,440). Sag expression is absolutely

required for milk-borne transmission of MMTV infection (reviewed in (101); without it, the

virus is not transported to its targeted tissue, the mammary gland, where it is able to

effectively replicate and be secreted into milk. In addition to its role in immune

modulation, Sag has been reported to transactivate MMTV LTR-driven transcription

(437) and may participate in MMTV tumor progression (310,432).

2.5.10 Negative acting factor (Naf)

An additional protein encoded by the MMTV genome within the long terminal repeat is termed negative acting factor (Naf) (375). The existence of Naf is somewhat controversial, as only one research group has reported on its functions and it appears to overlap with Sag coding sequences. Naf was described in 1990 as a factor that is involved in the trans-negative regulation of viral mRNA levels (375), and in later reports it was shown that Naf had separable activities from those of Sag (291,464). Unlike Sag,

Naf activity requires sequences within the gag gene for function (464). Most recently,

Naf was found to reduce levels of cellular proteins, implying a role in modulating cell

26 growth and host gene expression (291). However, to date, a protein corresponding to

Naf activity has not been identified, and it is still not clear whether one single gene product or two encodes the activity of both Naf and Sag (291).

2.6 Spread of MMTV

Through several years of study, the MMTV infection cycle has been elucidated as a complicated pathway of intimate virus-host interactions. The properties of MMTV allow it to circumvent host immune defenses, overcome spatial barriers in terms of trafficking from the initial site of infection (stomach) to the final site of replication and carcinogenesis (mammary glands), and exploit the biological signals of the host’s body during puberty and pregnancy for viral transmission. Due to the multitude of factors involved in infection and carcinogenesis, different strains of mice have varying susceptibilities to and tumor outcomes from MMTV infection (368). MMTV infection can be acquired by mice via one of two mechanisms (Figure 2.2): either exogenously from nursing on milk from an infected mother, or endogenous passage of proviral sequences through the germline. Regardless of the mode of transmission, mice bearing integrated

MMTV genomes in their host DNA express Sag and exhibit corresponding depletions of specific TCR Vβ pools. However, only mice with complete, intact MMTV proviral genomes (typically of exogenous origin) will go on to develop mammary tumors.

2.6.1 Endogenous transmission of MMTV (Mtvs)

MMTV proviruses that have stably integrated into a host cell’s germline DNA can be vertically transmitted to offspring by genetics that follow Mendelian rules of inheritance (31,441). As many as 2-10 endogenous MMTVs or Mtvs can exist in the germlines of common laboratory mice (96,233,372,386), as well as wild mice strains. It is thought that some Mtvs have been evolving within murine genomes for as long as 20

27 million years (20), a testament to the long-standing interplay of virus and host. Most

Mtvs no longer encode a complete MMTV genome and are defective for replication and mammary tumorigenesis (233), though some strains of mice develop tumors later in life due to Mtv activity (31,438). However, these mutated virus remnants are more than silent passengers in the murine genome. The majority of Mtvs still encode functional

Sag genes that activate and subsequently cause deletion of Sag-reactive T-cells, resulting in alteration of the host immune system (25,386). It is speculated that Sag- expressing Mtvs have been maintained within murine genomes for protection against infection by exogenous MMTVs that possess Sags with similar T-cell reactivities

(165,166,185). On the contrary, expressed Mtv proteins may provide a pool of “self” antigens during T-cell selection that subsequently allows for a degree of tolerance for future exogenous MMTV infections (4). Modulation of the immune cell repertoire by Mtv

Sag may not only confer a selective advantage to the host against MMTV infections but against other pathogens as well, and may explain why this particular Mtv gene is more preserved than others in mouse genomes (368). At present, more than 50 Mtvs have been indentified (43,74,204,233,424).

2.6.2 Exogenous transmission of MMTV

The acquisition and subsequent infection of exogenous MMTV is highly dependent on exploitation of the host immune system by the virus (4). The life cycle of exogenous MMTV begins when young mouse pups acquire the virus by digesting milk from an infected mother within the first 2 weeks of life (Figure 2.2) (171,303). Though some milk-borne virus particles are digested in the stomach, most pass through to the small intestine, where virions enter the Peyer’s patches via microfold cells (M cells) in the intestinal epithelium (179,216). Here, MMTV encounters a spatial-time barrier: the virus has to somehow reach the ultimate target cells of the mammary gland, but at this

28 point in the development of the young host the mammary cells are not yet activated and will not begin to divide until puberty (167). MMTV thus infects and activates cells of the immune system, starting with the dendritic cells (DCs) of the Peyer’s patches

(68,272,434). The Env protein of MMTV interacts with and activates DCs by virtue of

TLR4 on the DC surface (68,99,358), which also facilitates migration of DCs to the lymph node. DC activation consequently increases cell surface expression of transferrin-1 (TfR1), the receptor for MMTV entry (discussed in section 2.7.1) (68), thus facilitating DC infection. B-cells within Peyer’s patches are also activated through TLR4 by MMTV (145) and consequently infected (15,36,168). Initial infection is restricted to the Peyer’s patches, where viral DNA is detectable within 4 days of birth (216). Through production of cytokines from activated cells, immune suppression is achieved to allow for maintenance of the infection.

Next, infected DCs and B-cells produce and present MMTV Sag in conjunction with MHC class II molecules on their cell surfaces (4,5,37), and present these molecules to T-cells bearing cognate Vβ TCRs (4,5,16,203,280,352). As already discussed in section 2.5.9, the stimulation of TCRs by Sag results in a massive T-cell expansion, with

5-35% of the total mouse T-cell repertoire becoming activated and undergoing rapid proliferation (4,15,184). Due to the increased population of immune cells available for infection, MMTV generates a reservoir of infected cells that migrate from the Peyer’s patches to the lymph nodes and lymphoid organs of the host, including the mucosa- associated lymphoid tissue (MALT) of the mammary gland (reviewed in (368).

Therefore, lymphocytes serve not only as reservoirs for viral amplification, but also as vehicles to transport MMTV to the mammary gland (144,167), which is particularly

29

Figure 2.2 The infection cycle of MMTV in mice. Left pathway: Exogenous acquisition of MMTV. Young mouse pups ingest milk from an infected mother, and the virus proceeds through the stomach to the small intestine. There, MMTV enters the

Peyer’s patches and infects lymphocytes and dendritic cells. Infected cells express

MMTV Sag on their surface (shown in pink on B-cell), which is presented to receptors on

T-cells (yellow on T-cell) in conjunction with MHC Class II molecules (shown in green on

B-cell). Sag presentation of infected cells results in a massive proliferation of T-cells, which subsequently produce large amounts of cytokines that stimulate B-cells to activate and expand. This cumulative proliferation of lymphocytes allows for MMTV to infect large numbers of cells. Consequently, when lymphocytes return to circulation through the body, they carry the virus to the mammary gland where it establishes infection and replicates with hormone stimulation from pregnancy. New virus particles are released into milk and the cycle begins again with infection of offspring. Infected individuals eventually undergo a deletion of Sag-responsive T-cells that bear specific TCR Vβ receptors, and this altered immune cell repertoire is the hallmark of MMTV infection in mice.

Right pathway: Endogenous acquisition of MMTV. Parent mice vertically transmit MMTV through germline cells that have proviral sequences permanently inserted into the gamete DNA. Baby mice are then born, with copies of MMTV in every cell of their body.

In this pathway, MMTV transmission follows Mendelian rules of inheritance. Typically, these endogenous sequences contain deletions that prevent full virus particles from forming. However, Sag is still produced, resulting in the deletion of Sag-reactive T-cells in the host.

30

31 important because with the exception of virus in the milk, exogenous virus in infected mice is cell-associated and likely requires cell-to-cell contact (101,144,164,316,372).

MMTV establishes infection within the mammary gland when the cells are hormonally stimulated to divide during puberty and pregnancy (372). MMTV viral transcription is driven by a hormone response element (HRE) that lies within the viral

LTR (discussed further in section 2.7.5). Production of new virus particles and mammary tumorigenesis only occurs if virus amplification proceeds in the gland tissue

(165,372). In infected virgin mice, who undergo fewer cell divisions in their mammary epithelia than procreative mice, there are fewer MMTV infected cells and thus a lower incidence and longer latency of tumor induction (372). Therefore, females that complete multiple pregnancies have much higher levels of MMTV infection in the mammary tissues (316), purportedly due to the increased susceptibility of dividing epithelium to lymphocyte-mediated infection as opposed to spread from infected mammary cells

(167). The production of virus is highest during the lactation phase of late pregnancy, thus maximizing the excretion of virus to newborn pups (368). Additionally, male mice can produce mammary tumors when treated with the hormone, estrogen (316).

2.7 The MMTV replication cycle

The MMTV replication cycle entails a series of steps that involves adsorption of virions to host cells followed by entry, reverse transcription of genomic RNA to a DNA copy, trafficking of the preintegration complex (PIC) to the nucleus, integration of the proviral DNA into host chromosomes, transcription and translation of virally-encoded products, assembly of nascent particles, and finally the release of virions that undergo maturation to produce infectious virions.

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2.7.1 Adsorption and entry into host cells

Infection of cells by MMTV is initiated by Env-mediated recognition of a cellular surface receptor and adhesion or adsorption to the cell exterior (Figure 2.3, step 1). One such receptor for MMTV was proposed to be MTVR-1, located on murine chromosome

16 and expressed primarily in mouse cells (188). A second reported receptor of unknown function, MTVR, was mapped to chromosome 19 and found to be expressed ubiquitously among mammalian cell lines (167). MTVR is a newly discovered membrane-associated protein of 18.7 kD and biochemical analysis predicts that it contains a hydrophobic domain, a putative N-linked glycosylation site, 3 O-linked glycosylation sites, and 6 putative myristoylation sites (167). Additionally, two human homologs of the mtvr gene have since been described (408). However, MTVR confers only low levels of infection to virus-resistant cells, even when expressed at high levels

(126,167), indicating that it may serve as a low-affinity receptor or assist in virus-cell adhesion (371).

More recently, studies have focused on MTRV-1, which has since been identified as mouse transferrin receptor 1 (TfR1), as the primary MMTV cell-entry receptor

(371,452). This receptor mediates iron uptake by shuttling iron-loaded transferrin from the membrane to acidified early endosomes, where iron is released prior to transport of transferrin back to the cell surface (349,371). TfR1 is a type II glycoprotein that possesses a single transmembrane domain and a short cytoplasmic tail, both of which facilitate the internalization of substrate-loaded receptor to early acidic endosomes

(97,482). Interestingly, previous studies observed that treatment of MMTV-infected tumor cells with acidic pH induced cell-cell fusion (360), and peptides binding to TfR1 outside of its transferring-binding domain could also trigger its endocytosis and acidification (219). Collectively, this suggests a pathway in which MMTV cell entry is mediated by binding to TfR1 at the cell surface, followed by endocytosis through clathrin-

33 coated pits (Figure 2.3, step 2) and trafficking to acidic endosomes (452). The low pH triggers membrane fusion of the virus with the plasma membrane, depositing the viral nucleocapsid into the cytoplasm (360,371) (Figure 2.3, step 3). Data showing that

MMTV infection can be blocked with treatment of cells with methylamine, a lysosomotropic alkalinizing agent (47), supports this model of viral entry and penetration.

2.7.2 Reverse transcription

Once in the cytoplasm of the host cell, reverse transcription is activated inside the viral nucleoprotein core complex (Figure 2.3, step 4). The protein makeup of this core is not well characterized, however, all of the constituents needed for the reverse transcription reaction (except for the deoxyribonucleotides) are available within its confines. The nucleoprotein, NC, is present within the nucleoprotein complex and enhances RT processivity and template switching (420). The presence of the two RNA genome copies increases the probability of successful reverse transcription and, through recombination, permits the production of a DNA copy bearing sequences from both

RNAs (420). The majority of retroviruses complete reverse transcription in the host cytoplasm, though some viruses such as avian leukosis virus (ALV) complete this process after trafficking to the nucleus (420). The viral genomic RNA is reverse transcribed by RT to produce a blunt-end, double-stranded DNA complex (59,374)

(Figure 2.4). This collinear DNA molecule possesses terminal duplications that make up the long terminal repeats (LTRs) consisting of U3, R, and U5 that flank the gene encoding regions (420). The LTR regions are produced from two template switching or strand transfer events during the reverse transcription process, which initiates with the tRNALys transfer RNA annealed to the primer binding site (PBS) near the 5’ end of the

RNA genome. After production of a short transcript towards the 5’ end of the genome

(called minus-strand strong-stop DNA) (Figure 2.4, step 1), RT’s RNase H activity

34

Figure 2.3 The MMTV replication pathway. The infection and replication of MMTV in host cells is a multi-step process. (1) Virus particles adhere to the cell surface via cellular receptors (mouse transferrin receptor, in green) and enter (2) via receptor- mediated endocytosis into endosomes. The acidic compartment of the early endosome mediates fusion of the cell and viral membranes, facilitating the deposit of the viral nucleocapsid complex into the cytosol (3). In the cell cytoplasm, reverse transcription of the viral RNA occurs within the preintegration complex (PIC) to produce a double- stranded linear DNA copy (4). The PIC is transported to the cell nucleus (5) where the proviral DNA integrates into the host genome (6). RNA polymerase II facilitates the transcription of viral mRNA (7), and both spliced and unspliced viral mRNAs are transported to the cytoplasm for translation on ribosomes (8). Glycosylated protein products of spliced mRNAs, such as Env, Sag, and Rem, are produced and modified in the endoplasmic reticulum (ER) and shuttled to their respective cellular targets. In this diagram, Env (purple) is depicted traveling to the plasma membrane in vesicles (9); Rem and Sag are not structural proteins of the virus and therefore are not packaged in new virions. After translation, Gag, Gag-Pro, and Gag-Pro-Pol polyproteins (multi-colored rods) are directed by the cytoplasmic targeting/retention signal (CTRS) in matrix (MA) to assemble particles in the perinuclear region of the cell (10). The assembled immature

Type A particles are then guided by the myristoylation and membrane-binding signals of

MA to the plasma membrane where budding occurs (12). At some point after assembly or during/after budding, the viral protease enzyme cleaves the polyproteins into their respective components, yielding a mature infectious virus particle (13).

35

36

Figure 2.4 Retroviral reverse transcription. This diagram represents the multiple steps involved in reverse transcription by the MMTV RT enzyme. The enzyme begins with the viral genomic RNA as a template and undergoes several rounds of DNA synthesis and RNase H digestion, and two strand transfers, to produce a double- stranded linear proviral DNA product. A detailed, step-wise description is presented in the text of section 2.7.2. The genomic RNA is represented in faded colors. The proviral

DNA sequences are represented in brighter colors. Boxes mark the LTR regions of U3,

R, and U5; straight lines denote the viral coding sequences. The primer binding site

(PBS) and polypurine tract (PPT) are highlighted in green and orange, respectively.

Dotted lines later in the pathway denote DNA synthesis in the marked 5’ to 3’ direction.

37

38 digests the RNA from the RNA-single-stranded-DNA (ssDNA) hybrid (Figure 2.4, step 2) and the ssDNA complex “jumps” to the 3’ end of the RNA (first strand transfer) (Figure

2.4, step 3). There, the R region from the ssDNA anneals to the 3’ R region of the RNA, priming the continuation of minus-strand DNA synthesis accompanied by RNase H removal of the RNA template (Figure 2.4, steps 4 and 5). The RNase H degradation is not complete, however; it leaves behind a short segment of RNA known as the polypurine tract (PPT), which primes the second or plus-strand DNA synthesis (Figure

2.4, step 6). Plus-strand synthesis continues until it reaches the tRNA primer of the minus-strand template, where it halts to produce plus-strand strong-stop DNA. RNase removes the primer tRNA (Figure 2.4, step 7), exposing regions of the plus-strand DNA

(PBS-site) that are complementary to the 3’ end of the minus-strand DNA; a second strand transfer event occurs (Figure 2.4, step 8), resulting in the assembly of a structure that allows for the DNA to be elongated using both the positive and negative strands as templates. The DNA copy of the viral genome is completed when RT copies the plus and minus strands entirely (420) (Figure 2.4, step 9).

2.7.3 Nuclear trafficking of the preintegration complex (PIC)

MMTV, like most oncogenic retroviruses, relies on actively dividing target cells

(mammary epithelia) for its replication (reviewed in (58). After entry, the viral core (or preintegration complex, PIC) must face the challenge of trafficking to the cell nucleus

(Figure 2.3 step 5) where it can integrate its reverse-transcribed DNA into the host genome. Although the mechanism for MMTV’s migration to the nucleus is not understood, it may use the host cytoplasmic dynein and microtubule network similar to

HIV to localize to the perinuclear region (277). Regardless of how it travels to the nucleus, the large PIC complex is not able to pass through the nuclear pores, so it must gain access to the nucleus when the nuclear membrane is disassociated during mitosis

39

(58). Alternately, lentiviruses such as HIV are able to infect non-dividing cells through use of nuclear localization signals and active, energy-dependent import through the nuclear pores (reviewed in (58). Although it has been found that host factors, such as

LEDGF and BAF, can interact with the PICs of other retroviruses to mediate nuclear translocation and integration, the cellular constituents involved in MMTV trafficking are not known at this time (137).

2.7.4 Integration of the MMTV provirus

Incorporation of a linear DNA copy of viral RNA (provirus) into the host cell genome is a fundamental event in the life cycle of retroviruses. This process is mediated by the virally-encoded integrase through a specific, step-wise process that joins a precise nucleotide site in the viral DNA termini to non-homologous sites in the host DNA (263) (Figure 2.3, step 6). For in vitro integration reactions, the only viral components needed are the IN enzyme and the 10-15 nucleotide ends of the viral DNA

(58), but other viral and cellular proteins may participate in the regulation of the in vivo integration event.

Integration begins with the recognition and endonuclease processing of specific nucleotides at the 3’ ends of the linear DNA molecule by IN (58). This cleavage removes the terminal 2 nucleotide bases from each 3’ end of the DNA (117), producing recessed 3’ hydroxyl groups that serve as the proviral attachment sites to the host DNA.

This step may precede or follow the PIC nuclear entry step, depending on whether the virus needs to finish reverse-transcribing the RNA genome into a DNA copy in the nucleus (58). After binding of the host DNA by the PIC, IN catalyzes a transesterification reaction in which the 3’ hydroxyl ends of the viral DNA attack the phosphodiester bonds on opposite strands of the host target DNA and completes the strand transfer (58). With

MMTV IN, the host DNA target sites are staggered by 6 bases in the 5’ direction

40

(117,263). As no external energy source is needed for the viral DNA integration reaction, it is thought that the energy released from breaking the target DNA bonds is used in the joining reaction (58).

At this point in the integration reaction, the viral DNA is united with the host DNA, but a 6-nucleotide gap remains in the host DNA strands and 2 base pair, 5’ overhangs exist as flaps from the viral DNA sequences. To remedy the gaps in these junctions, viral RT or host repair enzymes, such as polymerase β or δ, extends the host 3’ hydroxyl groups with DNA synthesis (58,131,477). The 5’ viral flaps are also removed, possibly by a host flap endonuclease (55,131,477), and ligation by cellular ligases (55,477) completes the proviral integration process. The provirus, consisting of the retroviral coding sequences flanked by twin 1,300 nt LTRs (117) and a pair of 6-nucleotide host-

DNA repeats (117,263), is subsequently replicated along with the host cell DNA as a permanent fixture of the host genome (58).

In general, the location of retroviral integration in the host genome is essentially random and most host sequences can serve as integration sites, though research has shown a preference of some retroviruses for selected target regions. Integration tends to occur within the major groove of “kinked” DNA that is wrapped around nucleosomes or warped by nearby DNA-binding proteins such as nuclear matrix (reviewed in (58,471).

Steric hinderance of DNA-bound proteins can also preclude regions of host genome for integration (471). The tethering model of integration, proposed by Fred Bushman, suggests that host DNA-binding proteins interact with the viral PIC and influence target site selection (69). Interestingly, large-scale analyses of the integration sites for murine leukemia virus (MLV) and HIV uncovered a strong bias towards transcriptionally active gene regions, although ALV does not share this preference (471). Early reports on

MMTV integration suggested that it did not have a preference for specific host sequences (117). A study in 2007 by Derse et al. predicted that MMTV, based on

41 sequence conservation of IN, would integrate randomly in host DNA with no preference for genomic features, similar to HTLV and ALV (109). A follow-up survey on MMTV integration sites confirmed that prediction, demonstrating that MMTV has the most random dispersion of integration sites among retroviruses studies thus far, without the slightest preference for any particular target location (137). Such random integration of the MMTV proviral genomes can lead to aberrations in host gene regulation and oncogenesis, especially when insertion occurs near a cellular proto-oncogene

(discussed in section 2.8).

2.7.5 Viral transcription

Like all retroviruses, transcription of the MMTV provirus is mediated by the host cell RNA polymerase II (Pol II) (Figure 2.3, step 7). The efficiency of transcription can be influenced by the location of the provirus within the host genome, termed chromosomal position effect (141,355), meaning that some integration sites may promote proviral transcription better than others. As previously mentioned, the MMTV provirus contains 4 different promoters (Section 2.5.9) (Figure 2.1: triangles). Transcription of sag mRNA can occur from all 4 promoters, though all other known MMTV genes and the genomic

RNA are primarily transcribed from the standard LTR promoter at nucleotide 1196 of the genome (Figure 2.1, filled triangle). This principal promoter sits at the U3/R junction and governs tissue-specific transcription in ductal epithelial cells of the lungs, kidneys, mammary, salivary and prostate glands as well as in Leydig and lymphoid cells (92).

Retroviral are essentially treated as cellular RNA, and thus are subjected to the all of the cellular RNA processing events. Cellular machinery caps the 5’ end of all proviral

RNA transcripts with m7G5′ppp5′Gmp and polyadenylates the 3’ end with a 200-300 sequence of noncoding adenylic acid residues (43,345,355). Retroviruses appear to have no control over where their RNA transcripts terminate, and the RNAs often extend

42 past the viral template (355). Processing of the 3’ ends by endonucleases aids in making the transcripts more uniform after transcription and before polyadenylation; cleavage occurs at the R/U5 border and is regulated by the AGUAAA sequence in the

MMTV U3 region (355). Host machinery also moderates the splicing of MMTV proviral

RNA to produce subgenomic mRNAs encoding Env, Sag and Rem (355).

With the standard promoter of MMTV located at the U3/R interface, and the presence of two LTRs containing a U3/R boundary, the inevitable question arises as to why transcription only occurs from the 5’ LTR. This dominance of the 5’ LTR over the 3’

LTR for transcription is referred to as promoter occlusion, and may be mediated by elongating 5’ LTR transcription complexes that disrupt or prevent the assembly of transcription factor complexes in the 3’ LTR (reviewed in (355). However, transcripts originating in the 3’ LTR have been found when the 5’ LTR is deleted or normal transcription termination is altered (reviewed in (355).

The MMTV standard promoter contains a number of DNA-binding sites for transcriptional regulation that control tissue-specific and steroid hormone responsive gene expression. These include: a TATA box sequence (TATAAAA) located -41 to -23 bp upstream of the transcription initiation site (425); a basal promoter element located 2-

12 nucleotides downstream of the initiation site that binds the initiation site binding protein (ISBP) required for efficient LTR transcription (140,346); a hormone responsive element (HRE) located -200 to -80 nt upstream of the transcription start site (reviewed in

(176); and hormone sensitive DNA-binding sites for the octamer transcription factors

Oct-1 and Oct-2 which stimulate basal and hormone driven transcription, nuclear factor 1

(NF1)/CCAAT transcription factor (CTF) which is required for glucocorticoid-induced transcription, and TATA binding protein (TBP)

(13,14,60,61,63,65,98,202,221,222,224,293,365,425,427).

43

Before MMTV was even defined as a virus, it was known that hormones played an important role in the development of mammary tumors in mice afflicted with the

Bittner agent (41). Since those early days, huge strides have been made in the understanding of how hormone regulation plays a role in virus transcription and replication. Steroid hormones (glucocorticoids, androgens, and progesterone) produced during puberty and pregnancy enhance transcription by the standard LTR promoter and increase protein production (reviewed in (176). This response to hormones is mediated by the HRE, leading to a dramatic increase in the amount of MMTV expression during lactation (297). Four glucocorticoid receptor (GR) binding elements (GREs) within the

HRE bind hormone receptors that recruit distinctive groups of coactivators to the MMTV promoter to upregulate transcription (19,147,250,340,382).

Additionally, the MMTV LTR contains 3 contiguous negative regulatory elements

(NREs) that are located between nts -645 and -264 upstream of the transcription start site: the distal (d) NRE, the junctional (j) NRE, and the proximal (p) NRE. The NREs repress transcription and control tissue specific expression of MMTV

(51,181,200,245,295,308,369,486). Two cellular transcriptional repressors regulate

NRE repression. Special AT-rich-sequence binding protein 1 (SATB1), which is most abundant in the thymus, represses transcription in lymphoid cells, and the homeodomain protein CCAAT displacement protein (CDP) prevents transcription in undifferentiated mammary gland cells (253,484-486). CDP is expressed in most undifferentiated tissues, but its transcriptional repression activity is removed once its expression is reduced with cellular differentiation, such as during mammary gland cell division during lactation

(486). Other transcriptional factors, such as transcription factor Ku (159), transcriptional enhancer factor 1 (TEF-1) (261), and transforming growth factor-beta (TGFβ) (83,292) also bind to the NRE regions and repress MMTV transcription.

44

The 5’ promoter region of MMTV also includes a mammary gland-specific enhancer (MGE) that increases basal and hormone-induced transcription from the

MMTV LTR and directs high level expression in the mammary glands of transgenic mice

(297,476). The bipartite MGE consists of the BanII element (located nearly 1 kb upstream of the transcription start site) and the mammary-specific enhancer of MMTV

(MEM) element (-938 to -862 above the transcription start site) (247,476). The BanII element interacts with a mammary gland-specific nuclear factor complex consisting of several host components (247,283), whereas the MEM element binds mammary specific factors such as mammary gland factor (MGF) and others that respond to hormones

(354).

Additional miscellaneous cis-acting transcriptional elements also exist within the

MMTV LTR. One such constituent is the 13 bp tannic acid response element that binds

a DNA helicase domain-containing protein (Sμbp-2) and negatively regulates LTR

transcription (429). Several regions within the 250 nts upstream of the transcription start

site also serve to bind the forkhead transcription family member Forkhead box A

(FoxA1), which modulates chromatin structure alterations that allow for increased basal

transcription while decreasing glucocorticoid receptor-induced transcription (196).

Lastly, a 411 bp region within the MMTV env gene serves as a specific transcriptional

activator for activated T-cells called MMTV env transcriptional activator (META) (294).

2.7.6 Translation of viral proteins

After transcription, unspliced and spliced MMTV mRNAs are exported from the nucleus to the cytoplasm for translation (Figure 2.3, step 8). Unspliced mRNAs, that can serve as the viral genome or the template for Gag-Pro-Pol translation, are exported from the nucleus via the virally-encoded Rem protein and the Rem response element

45

(RemRE) located near the 3’ end of the viral RNA (see section 2.5.7). Rem-dependent

RNA export is sensitive to the drug Leptomycin B (LMB), confirming that Rem uses the

CRM1 nuclear export pathway for unspliced RNA trafficking (206). On the other hand, spliced MMTV RNA export is LMB-insensitive (206), despite the fact that all MMTV

RNAs (spliced and unspliced) possess the 3’ RemRE sequence (287). This LMB insensitivity implies that spliced MMTV transcripts exit the nucleus via an alternate pathway, most likely by a standard cellular export mechanism for spliced mRNA products such as Tap (reviewed in (227,361).

The MMTV viral proteins are translated from both unspliced, full-length mRNA

(Gag, Pro, Pol) as well as singly- and doubly-spliced subgenomic mRNAs (Env, Sag,

Rem) on free and membrane-bound ribosomes in the cytoplasm, respectively (414)

(Figure 2.3, steps 8, 9). The translation of spliced mRNAs which produce Env, Sag, and

Rem is completed on ER-associated ribosomes, where the proteins are processed and modified in the ER prior to transport to their respective cellular locations (Env and Sag to the membrane, Rem to the nucleus).

As discussed previously, two ribosomal frameshift events must occur in order for the precursor proteins to be produced from the unspliced RNA transcript (section 2.5.1).

The ORF of dUTPase/Pro overlaps with 16 nts of the Gag C-terminus, whereas the Pol

ORF begins 13 nt upstream of the Pro termination codon (208). A 7 nt sequence termed the shift site, as well as host tRNAs and nearby G-C rich RNA pseudoknots, create a

“pause” in ribosome read-through, allowing the frameshift to occur (84,208).

Approximately 23% of translating ribosomes complete the first frameshift at the Gag-Pro junction, with 8% of those ribosomes going on to complete the second frameshift at the

Pro-Pol site (208,305). The result of ribosomal frameshifting is the production of precursor polyproteins in ratios that are biologically relevant for the structure and enzymatic needs of new virus particles (see section 2.5.1).

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Due to the presence of the 5’ cap structure and 3’ poly(A) tail on all retroviral

RNAs, it is generally thought that translation occurs by standard cap-mediated translation initiation followed by downstream ribosomal scanning to the start codon

(reviewed in (407). However, this seems to be quite a cumbersome task for the translation initiation complex, considering the extensive secondary structure of the 5’ viral RNA (packaging sequences, pseudoknots, dimerization interface, primer binding site, etc) (reviewed in (23). Additionally, the 5’ untranslated regions of many retroviral

RNAs include AUG codons that are not the anticipated start site for translation and would have to be somehow bypassed by the scanning ribosome (414). In 1988, internal ribosome entry sites (IRESs) were discovered in picornaviruses and found to mediate direct ribosome binding near the AUG codon without 5’ cap-mediated translation initiation (reviewed in (23). Since then, several retroviruses, including MLV, ALV, HIV-2, and simian immunodeficiency virus (SIV), have been found to possess IRESs (23).

Interestingly, it had been reported that the protease of MMTV is able to cleave translation initiation factor eIF4G (9) and poly(A) binding protein (PABP) (8); eIF4G interactions with PABP mediate translation initiation, and degradation of eIF4G leads to the abrogation of 5’ cap-mediated protein synthesis but not IRES-mediated translation.

Cleavage of both eIF4G and PABP is common among other retroviruses (23). It was therefore not surprising that recent work has elucidated the existence of an IRES in the

5’ UTR of MMTV that mediates viral protein translation (436).

2.7.7 MMTV virion assembly

After translation, the structural Gag, Gag-Pro, and Gag-Pro-Pol polyproteins come together to form the immature nucleocapsid core or Type A particle that contains the viral genomic RNA (Figure 2.3, steps 10, 11). These spherical immature particles

(Figure 2.5), which are stabilized by disulfide bonds (405), are approximately 80 nm in

47 diameter (see section 2.3.2) and consist of the Gag molecules radially aligned like a wagon wheel. MA is located near the periphery and NC/Pro/Pol near the interior where the two viral RNA molecules are located (285,341). These immature structures form in the pericentriolar region of the cytosol, due to the CTRS located within MA of Gag (see section 2.5.2.1). Under electron microscopy (EM), the Type A particles appear as a double ring structure and are distributed throughout the cytoplasm, near the nucleus and associated with the cytoplasmic periphery of vacuoles/vesicles (33,156,285,325,406). It is currently not known whether these immature particles form in association with cytoplasmic structures of the cell. In general, little is known about either the viral or cellular requirements for MMTV assembly (368).

The MMTV Gag proteins form the core structure via protein-protein interactions that take place between the CA-NC domains of adjacent molecules; weak interactions also take place between Gag domains throughout the entire length of Gag to promote the Gag-Gag oligomerization (481) (Figure 2.5). It is estimated that 1,500 molecules of

Gag exist within each retroviral virion (23,53,87,105,449), though the numbers will vary with the range of particle sizes.

Packaging of the genomic RNA (gRNA) is mediated by the NC domain of Gag, which also facilitates the dimerization of the two RNA molecules (reviewed in (335).

Though it is not known where MMTV Gag and RNA first unite in the cell, the gRNA must somehow traffic to the site of Gag oligomerization where it is used as a scaffold for building nascent particles (reviewed in (335). An RNA switch mechanism has been proposed for packaging, wherein the NC-binding sites in psi are sequestered by base- pairing in the monomeric RNA until they become exposed following dimerization

(reviewed in (335). As the full-length viral RNA serves dual purposes for packaging and translation, it is not yet understood how the virus designates RNA for these two

48

Figure 2.5 Organization of the immature and mature MMTV virus particles.

Top: Schematic diagram of an immature retroviral particle. On the outer-most surface of the virus particle, the envelope glycoproteins (purple/white projections) are embedded in the host-derived lipid bilayer (white circle). Under the viral membrane, the Gag polyprotein is arranged radially with the N-terminal matrix domain (MA, orange) interacting with the envelope transmembrane region (not shown). MA is followed by the pp21, p3, p8, and n regions of Gag (blue), the capsid (CA, purple), and nucleocapsid

(NC, tan) domains. For the Gag-Pro and Gag-Pro-Pol polyproteins that are also part of the immature structure, Pro is drawn as a small red circle and Pol is a green pentagon.

Two copies of the viral RNA are packaged into each virion (black X in center).

Bottom: Schematic diagram of a mature retroviral particle. For the virus to be in its

mature, infectious form, the Gag polyproteins must be cleaved by the viral protease (red

circle) into their respective counterparts. This leads to a dramatic morphological change

in the particle structure. The NC domain condenses in the center of the virion onto the

viral RNA (tan ovals associated with black X), the CA domain forms a core around the

NC-RNA complexes (purple ovals), and MA remains associated with the viral membrane

(orange). Pro (red circle) is thought to remain free in the particle, along with some capsid

protein (not shown). The locations of the pp21, p3, p8, and n domains of Gag are not

known in the mature virion. The majority of integrase (green triangles) and reverse

transcriptase (green rectangles) remains near the center of the virion with the RNA.

49

50 activities. Partitioning of RNA for these two functions is surely regulated to allow for enough RNA to fulfill both activities (414). One idea is that two pools of RNA are maintained, with the RNA for packaging sequestered away from the translation machinery, though this has yet to be established for MMTV (70).

MMTV NC, like that of other retroviruses, identifies the viral RNA from all of the other cellular RNA by binding to psi (ψ) sequences located at the 5’ end of the viral genome (376). To date, no studies have elucidated the exact boundaries of the psi signal for MMTV, though the 5’ UTR through the first 400 bp of Gag is known to be sufficient for packaging (6,366). Interestingly, it has been shown that the NC of MMTV, when used to replace the NC of an HIV provirus, is capable of selectively packaging HIV

RNA over MMTV RNA into virus particles, lending evidence that the NC region is important for genomic packaging but is not the sole contributor to selectivity (350).

2.7.8 Envelopment, budding, and release

The Type A particles obtain their viral envelope concurrently with budding through the plasma membrane or endosomal membranes/ER that have been modified with the inclusion of viral Env proteins (Figure 2.3, step 12) (156,401). The MA domain of Gag molecules in the immature particle directs the structure to the membrane where it interacts with the TM region of Env (81,274,285,342,345,357,448,460). The entire retroviral assembly process is speculated to be driven by concentration of Gag-Gag interactions and the presence of envelope/MA and NC/RNA interactions, though the specifics of how MMTV moderates this process are not understood (30). Also packaged within the viral particles are cellular components such as ribosomes, tRNA, cellular mRNAs and some ribosomal RNAs (rRNAs) (156,343,414). Studies have also shown that the mouse protein APOBEC3 can be packaged into virions and, like its APOBEC3 counterpart in humans that restricts HIV infection, inhibits MMTV replication (327).

51

Though it is not certain how APOBEC3 interferes with MMTV replication, it was shown to interact with the MMTV NC protein and may alter the viral RNA through deamination

(327). This form of host restriction is not completely effective, obviously, as MMTV is still a very prevalent disease among mouse populations.

Several studies have reported on the use of Gag late (L) domains to mediate retroviral budding through the employment of cellular factors (ESCRT machinery) involved with the multivesicular body (MVB) sorting pathway and the ubiquitin- proteasome degradation system (reviewed in (273,307). Though MMTV Gag does not bear a more traditional PPPY or PTAP sequence that signifies an L domain, it does possess potential L domain sequences in CA (a PSAP sequence) and 2 YXXL motifs in

MA and pp21 (330). Additionally, studies using proteasome inhibition demonstrated that

MMTV does not require ubiquitin for budding like other retroviruses, in spite of the fact that sequences within Gag are ubiquitinated (330). Collectively, these data demonstrate a unique and undetermined mechanism behind MMTV’s ability to use host cell machinery for the budding process.

The completely assembled MMTV Type A particles preferentially exit from host cells at sites of cell-to-cell contact (155,156,316). MMTV appears to be primarily cell- associated during infection with the exception of free exogenous virions in secreted milk

(368). Transfer of virus from one cell to another may be assisted by the formation of virological synapses that develop when Env proteins on the infected cell surface crosslink to receptors on an adjacent cell surface (reviewed in (307). MMTV particles crossing these intracellular bridges have been observed as elongated structures that undergo simultaneous budding from one cell and endocytosis into the next (155,307).

Use of such a discrete mechanism for virus release may allow the virus to evade immune detection (307).

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During or shortly after budding, the viral protease cleaves the polyproteins within the immature particle to form the mature structural proteins (110,156,405) (Figure 2.5).

A recent study supports that a large portion of this cleavage actually occurs within the cell cytoplasm, as evidenced by the appearance of viral cleavage products in whole cell lysates, which can be reduced with a protease mutation (479). Examination of these cleavage products revealed that Gag polyprotein cleavage occurs in a N’ to C-terminal direction, with MA cleaved from Gag first and the CA-NC intermediate cleaved last

(110,113). Protease cleavage produces the individual proteins of the Gag polyprotein, as well as the virion-associated enzymes; the maturation event of retroviral particles is absolutely necessary for the production of fully infectious virus (414). This final step of virus production results in the gross structural rearrangement of proteins within the virion, resulting in the formation of an electron-dense core observable by electron microscopy (414).

2.8 Mechanisms of MMTV tumorigenesis

The widespread use of MMTV as a murine model system to study human breast cancer is founded on its ability to cause tumors specifically within the mammary tissue.

Transforming retroviruses such as MMTV cause cancer by the action of proviral integration into the host genome as part of the retroviral life cycle, which can disrupt the normal organization of cellular genes and lead to oncogenesis. This process is termed insertional mutagenesis and has been used for years to discern what normal cellular genes can contribute to cancer as a proto-oncogene when abrogated in terms of its cellular expression.

As is typical of most retroviral infections, MMTV oncogenesis occurs after a long

latency period (6-12 months) (5,53,87,373). Many mammary tumors are clonal in

nature, and appear to be derived from several proviral integration events that occur

53 within a single progenitor mammary stem cell (230). Typically, 10 or more proviruses are integrated into different regions of the host genome (78), resulting in the production of proteins not normally expressed in the mammary gland (323). It is not certain whether MMTV-induced mammary tumors are the result of a single initiating integration or are an amalgamation of multiple integrations (368). It does appear, though, that pregnancy-dependent tumors are polyclonal in nature and pregnancy-independent tumors are generally monoclonal (66,74). Tumor formation is not necessary for the replication of MMTV virus but appears to be a by-product of the normal infection cycle

(372).

MMTV proviral integration introduces strong promoter and enhancer elements into the host genome which can perturb normal gene structure in a variety of ways.

First, the provirus could insert upstream of the gene in the same transcriptional orientation. The promoter and enhancer elements in the MMTV LTRs may then promote oncogene expression to levels higher than normal or to levels that are developmentally inappropriate in an event known as promoter insertion (367). Second, the enhancer regions of the proviral LTR can strongly influence gene expression when the provirus is inserted upstream of the target gene in the opposite transcriptional orientation or downstream in either orientation. This is called enhancer insertion (367). The BanII and

MEM enhancer elements within the MMTV MGE have been reported to mediate transcriptional activation of proto-oncogenes via enhancer insertion (75,174). A third mode of provirus-proximal oncogene activation occurs when read-through transcripts initiating in the proviral LTR continue into cellular sequences. The hybrid RNA transcripts are then translated to yield altered proteins with modified or defunct functions

(75,367). Lastly, proviral integration may simply destroy gene regulatory control elements such as silencers, pause sites, polyadenylation signals or methylation patterns, resulting in the loss of regulation (367). Additionally, proviral insertion can inactivate

54 genes that serve as tumor suppressors and contribute to cancer with the loss of cellular factors intended to prevent oncogenesis (367).

By determining the location of genes where proviruses are integrated and cause tumorigenesis, scientists are able to identify cellular factors and pathways linked to human cancers. In this way, several groups of cellular proto-oncogenes have been identified and further examined in transgenic mouse models. Though the MMTV provirus inserts in a fairly stochastic manner in the host genome (see section 2.7.4), integrations frequently occur in oncogenes belonging to families such as the wnt

(wingless-related MMTV integration site), fgf (fibroblast growth factor), notch, and R-

Spondin gene families (74,75,321). Many of these common insertion sites (CISs) afflict oncogenes that regulate and control cell growth, proliferation, differentiation, and tissue homeostasis (75). Regardless of the many oncogenes that can be altered by proviral insertional mutagenesis, tumor induction by MMTV is further complicated by oncogene cooperativity (197,367), the genetic background of the host (197,268), and the presence of hormones (93,344).

The full carcinogenic potential of MMTV has only been recently appreciated with the discovery that virally-encoded proteins can also contribute to tumorigenesis. MMTV

Sag displays oncogenic activity when expressed in certain mammary epithelial cell lines

(310). The MMTV Env protein possesses transforming abilities in cell culture and in mice that are dependent on the ITAM domain located in SU (217,370). Furthermore, the gag gene has been attributed with oncogenic properties. In 2000, Hook et al. compared the sequences and tumor burden of 3 different MMTV strains: the highly tumorigenic

C3H strain; a tumor-attenuated and genetically engineered hybrid provirus, HP; and a newly divergent, tumor-attenuated strain of MMTV called HeJ that is the product of a recombination event between endogenous Mtv1 and exogenous C3H (197). Mapping

data indicated that variable sequences within Gag were responsible for the difference in

55 tumor outcomes within one strain of susceptible mice (197). A follow-up study further defined the transforming regions of Gag by creating viral chimeras containing sequences from tumorigenic and non-tumorigenic MMTV strains (413). Sequences within the CA and NC regions of Gag conferred high tumorigenic potential when derived from the C3H strain, but cancer incidence was reduced when the CA-NC region from C3H was replaced with that from strain HP (413). Differences in tumorigenicity between different strains of mice infected by the same strain of virus implied that a host factor may be playing a role in Gag-mediated oncogenesis (413). Additionally, it was observed in another study that tumors appeared more abundantly and more quickly in mice infected with MMTV as compared to transgenic mice with genetic mutations comparable to those created by insertional mutagenesis (428). These studies provide convincing evidence that insertional mutagenesis alone is not solely responsible for the tumor burden observed in mice infected by MMTV, and that virally-encoded products, such as Gag, may accelerate proto-oncogene-based mammary tumor development by interaction with host factors (413). A putative Gag-interacting host factor, mts, has been proposed to coordinate variations in tumorigenicity between different tumor-susceptible strains of mice (413), however, this protein has not been characterized nor has the work been continued. Currently, the cellular factors that interact with Gag and the mechanisms for how Gag may promote tumorigenesis are unknown.

2.9 The barriers to MMTV study

From early on, scientists realized that working with MMTV in the research laboratory would not be an easy task. After the discovery that an infectious agent was passed through the milk to offspring, the first difficult task was in obtaining enough mouse milk to purify the virions for study (62,171). One laboratory even established a

“large scale mouse dairy” (171). A series of mouse-milking devices were constructed

56 and used for the tedious and delicate milking operation from nursing female mice

(142,171). Though it was easier to obtain mammary tumor tissue for study, the complex nature of tissue components in early studies made examination and purification of

MMTV difficult; milk, on the other hand, was a more simple medium to work with though the caveat was obtaining enough volume (171).

From those initial ground-breaking studies in mice, research on MMTV attempted to move to an in vitro system for ease of manipulation and isolation of MMTV away from other viruses found within in vivo systems. However, that proved to be more difficult than anticipated, and the progress of MMTV research was hampered by lack of a suitable study system. Though infected mouse cell lines from mammary tumors could be maintained in culture, it was quickly found that normal mouse cells could not be infected in vitro, and some other cell types that lack endogenous Mtvs (such as feline and mink) could only be infected with extremely high multiplicities of infection (MOIs)

(79,82,198,199,240-242,435). Through the progressive development of cell culture techniques such as use of the steroid dexamethasone to stimulate virus production and serial passage of virus through feline cells, an in vitro system for MMTV study in a variety of cell types slowly emerged (199,303,442). Poor infectivity still appeared to be a problem (263) in cell culture, but this is most likely due to the cell-to-cell spreading nature of MMTV (see section 2.7.8) as well as the virus tropism for alveolar cells of the mouse mammary gland (305,419).

Another hurdle to MMTV study appeared during attempts to molecularly clone exogenous proviral DNA sequences into plasmid vectors for amplification in Escherichia coli. A region within the gag gene was found to be unclonable as it propagated poorly, if at all, in standard laboratory bacteria (64,116,117,120,138,143,175,262,263,430). This region of gag was designated the “poison” sequence, and impeded the progression of

MMTV genome characterization and development of an infectious proviral clone

57 because it was located in an indispensable part of the genome. Further examination of the poison region mapped its position to the pp21-p3 region of gag, comprising over 150 bps (56). Stable DNA from this gene region of gag was able to be propagated in bacteria only when the poison sequences were disrupted by deletions, mutations, or incorporation of bacterial insertion sequences (56). Though the molecular basis for this poison region’s toxicity remains unknown, its identification has led to the use of alternative techniques for cloning the MMTV proviral genome: Shackleford and Varmus created an infectious hybrid provirus (HP) bearing the 5’ sequences of the endogenous provirus Mtv1 (which is uniquely unaffected by poison sequences) and 3’ sequences from the exogenous C3H strain (398); Morris et al. cloned a full-length pathogenic

exogenous provirus using a lambda bacteriophage DNA vector (309); and Xu et al.

cloned an infectious proviral DNA using a phagemid vector system (473). Though it has

taken longer to overcome the obstacles to study MMTV than other retroviruses, many

more tools are now available to study the mechanisms behind how this virus infects its

host and causes tumor progression.

2.10 Is there a human mammary tumor virus?

The idea of a retrovirus related to MMTV that may be involved in human breast

cancer is one of the longest running controversies in human retrovirology (reviewed in

(450). Since the discovery of MMTV by Bittner in 1936, breast cancer researchers have

sought to answer the question: does a human virus, similar to MMTV, exist and cause

cancer in people? After 7 decades of research, a definite answer to this question is still

not known, though several lines of interesting information have come to light in search of

a viral etiology to human breast cancer.

Early studies reported findings of MMTV-like proteins in human breast cancer

biopsies and the presence of antibodies to MMTV in breast cancer patients

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(50,106,195,290,326). However, a more recent research study reported in 2006 examined the sera of breast cancer patients and was unable to detect any MMTV- specific antibodies via Western blot analysis (161). Additionally, MMTV-specific gene sequences have been reportedly amplified from human breast tumors in several studies

(reviewed in (160,416) and reports have also been published showing evidence of virus particles in tumor samples by EM (reviewed in (282,302). However, for as many publications as there are that show evidence of MMTV in human breast samples, there are just as many that are unable to replicate these results (reviewed in

(160,337,372,410,416). Also, mice typically have traces of virus in some of their normal tissues, but analysis of normal human tissue samples showed no evidence of MMTV infection (134). Variations in sample preparation and the use of differing tools and techniques by researchers appear to be one barrier in finding a clear answer as to whether human breast tumors bear MMTV components.

Other blatant facts complicate the possibility of a MMTV-like human retrovirus causing breast cancer. First, there is no correlation between breast-feeding and cancer incidence among humans, which differs from the mode of exogenous MMTV transmission in mice (423). It has yet to be addressed how HMTV is transmitted in humans. Moreover, rates of breast cancer have been shown to increase in countries where the practice of breast-feeding has declined (150,259). Additionally, breast tumors in mice are known to be instigated with pregnancy whereas pregnancy in humans has a cancer-protective effect (183,215) providing further argument against a milk transmissible agent that increases human breast cancer risk.

A second case against a role of MMTV in human breast cancer is the nature of human breast cancer tumors themselves. The disease is very heterogeneous, with over

60 breast cancer-related genes identified as being up-regulated or altered in spontaneous cancer patients and each patient presenting a unique case that varies in

59 biological, histopathological, epidemiological, metastatic, and genetic characteristics

(267). Such a high degree of diversity indicates multiple etiologies at play (267).

Although an inherited form of breast cancer does occur in humans similar to the endogenous proviruses inherited by mice, the human cancer is attributed to mutations within the BRCA-1/BRCA-2 genes (183,328), not proviral sequences. Furthermore, the histopathology of murine MMTV tumors (hyperplasias) is dissimilar from the various invasive carcinomas of human breast neoplasms (267).

New data in support of MMTV’s role in human cancer has surfaced and enlivened the debate. A correlation between high breast cancer incidence and the geographical range of a house mouse suggested that living in close proximity to MMTV- bearing mice may provide a route of transmission to humans (409). Reports succeeding this finding showed that MMTV could infect and rapidly spread in cultured human cells

(205,207), despite a later finding that human TfR1 (homologue to the mouse MMTV receptor (371)) was not able to support MMTV infection (452).

As it stands, it is estimated that 15-25% of all human cancers are virally linked

(57), and studies of human mammary tumor virus (HMTV) estimate that as many as 38-

40% of human breast tumors bear MMTV-like sequences (134,267,453). With the acceptance that human papillomavirus (HPV) causes cervical cancer in humans, scientists are on the look-out for other viral etiologies to the numerous cancers that exist.

HMTV is certainly one of the strongest candidates for causation of human breast cancer, however, the evidence to support its existence is still tenuous.

2.11 Ribosomal protein L9 (RPL9)

*This section provides background information on the host protein RPL9 that is discussed in Chapter 3 of this dissertation.

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The eukaryotic ribosome serves as the catalytic and regulatory center of protein synthesis in cells as well as a key player in many aspects of cell and structural biology.

This large 80S structure consists of two subunits, the 60S large subunit and the 40S small subunit, whose noncovalent interaction facilitates the process of converting mRNA messages into polypeptide products. The small subunit comprises 32 ribosomal proteins and the 18S ribosomal RNA (rRNA), and is responsible for the binding and decoding of mRNAs (85). The larger ribosomal subunit bears the enzymatic peptidyl transferase site that catalyzes peptide bond formation during protein synthesis. It contains the 25S, 5.8S, and 5S rRNAs in addition to more than 45 proteins (85). In total, the eukaryotic ribosome structure contains approximately 80 proteins that are assembled around highly modified rRNAs that collaborate to decode the cellular mRNA and meet the demands of cellular protein synthesis (reviewed in (119). Despite the number of proteins involved, rRNAs form the bulk of the ribosome structure (two-thirds) and possess the enzymatic activities required for protein synthesis as ribozymes

(reviewed in (119,304).

One of the protein components of the large 60S subunit is ribosomal protein L9 (RPL9). RPL9 is 192 amino acids long (411) and exists as a single copy within the ribosome structure (468). The prokaryotic equivalent to eukaryotic RPL9 is

RPL6, and the properties of RPL9 have been deduced from RPL6 comparison studies.

In E. coli and Bacillus stearothermophilus, RPL6 binds directly to the 23S rRNA of the

large prokaryotic subunit and is located in the aminoacyl-tRNA binding site of the

peptidyl transferase center (162,433,470). The crystal structure of the B.

stearothermophilus RPL6 protein has been resolved, demonstrating that the RPL6

protein contains two domains with nearly identical folds that may have transpired from

an ancient gene duplication event (162). The structure also suggested that the RPL9 N-

terminus participates in protein-protein interactions at the large ribosomal subunit

61 interface while the C-terminal hydrophobic residues bind the 28S rRNA towards the interior of the ribosome (162). A recent study elucidating the structure of the canine ribosome depicts a small portion of RPL9 visible on the large subunit surface (85), which is presumably the N-terminus at the subunit boundary. Use of trypsin digestion to remove ribosomal proteins located on the surface of the large ribosome subunit failed to remove RPL9, indicating that it is positioned more internally on the 60S subunit than other ribosomal proteins (270). Interestingly, RPL9 is one of two ribosomal proteins contacting the 28S rRNA that interacts with the A chain of ricin, a poison derived from castor beans, to mediate its toxic ribosome-inactivating effects (443).

In humans, the RPL9 gene is 5.5 kb in length, contains 8 exons, and is located on chromosome 4 (276). There may be as many as 8 pseudogenes for RPL9 in the human genome with multiples also present in other mammalian genomes (23 RPL9 pseudogenes in rat), but only one gene copy is functional for RPL9 expression

(276,411,468). Ribosomal pseudogenes lack introns and functional promoters, and because of this, only rarely show up in the host transcriptome/proteome.

RPL9 is expressed in all human tissues (as all cells contain ribosomes) but there are variations in RPL9 protein levels among different tissue sites (276). The highest levels of RPL9 were detected in the adult liver and neural retina, two sites known to be extremely active for protein synthesis (276). RPL9 expression levels appear to change through human development, as RPL9 levels are much higher in the fetal brain than in the adult (276). This is not surprising, as young tissues have a greater demand for protein synthesis than adult cells which have completed growth and differentiation.

RPL9 is known to play an important role in proper ribosome formation and normal growth and development. Drosophila melanogaster flies carrying one defective RPL9 allele are characterized by stunted growth, reduced viability, and diminished fertility, and homozygosity for mutant RPL9 results in lethality (390). Homozygous mutations of

62

RPL9 are also embryonic lethal in zebrafish (258), emphasizing the necessity of RPL9 for viability and development.

Human RPL9 has a molecular weight of 21.8 kD and consists of 70 hydrophobic residues, 36 basic residues, and 20 acidic residues (411). By comparison to other ribosomal proteins that have been characterized, the basic regions of mammalian RPL9 most likely contain sequences that target RPL9 to the nucleus/nucleolus for ribosome biogenesis (468), however these signals are currently not mapped in RPL9. The cluster of basic residues within RPL9 is also reminiscent of a bZIP motif and is speculated to mediate protein-protein dimerization and DNA binding (468). RPL9 is fairly conserved in terms of amino acid sequence across archaebacteria, prokaryotes, and eukaryotes

(468), and a number of sequence characterizations have been reported for various species, including human (276), rat (411), fruit fly (390), yeast (which has two isoforms,

A and B) (211,445), and pea plant (306).

2.12 The dynamic nucleolus

*This section provides background information that corresponds to data Chapter

3 of this thesis.

The nucleolus is the most prominent substructure of the eukaryotic cell nucleus and is large enough to be seen through a light microscope. Its central role in the cell is rRNA transcription, pre-RNA processing, and ribosome subunit assembly (reviewed in

(45,403). Like all of the other intranuclear structures, nucleoli are not membrane-bound but rather a dense amalgamation of cellular proteins and nucleic acid (237). The host factors associated with the ribosome assembly process are located within the three subregions of the nucleolus, termed the fibrillar center (FC), the dense fibrillar component (DFC) and the granular component (GC). The innermost region of the nucleolus is the FC, which is surrounded by the DFC, and those two subregions are

63 encompassed by the outermost GC. Transcription of the ribosomal DNA by RNA polymerase I occurs at the FC/DFC margin to produce pre-rRNA transcripts that are modified and processed in the DFC. Also within the DFC are ribosomal proteins that were translated in the cell cytoplasm and transported to the nucleolus for ribosome biogenesis. These protein subunits bind rRNAs in the DFC to assemble early ribosomal subunits. Maturation of the 60S and 40S ribonucleoprotein complexes occurs in the GC region prior to export to the cytoplasm (reviewed in (45,403).

Nucleoli fluctuate in response to signaling events that reflect changes in cell growth, metabolic activity, and stress. Furthermore, nucleoli disappear completely during cell mitosis (reviewed in (45). At any given time, a cell nucleus may possess 2-5 nucleoli ranging in size from 0.5-5 μm in diameter (255). Tumor cells, on the other hand, typically have many more nucleoli that are much larger in size. Early on in cancer research, doctors noted that a patient’s prognosis correlated with the size and number of nucleoli: the larger and more abundant the nucleoli, the worse the prognosis (299).

There are now several lines of evidence that indicate a role of nucleoli in the regulation of tumor suppression and oncogene activities (reviewed in (299,329).

Due to the dense nature of the nucleolus, the structure can be isolated from cells and used for proteome analysis. Currently, over 700 proteins have been identified within the human nucleolus (248) . Though a large number of proteins were associated with ribosome biogenesis as anticipated, a significant portion (nearly 30%) of the nucleolar- associated proteins were of unknown function (11,248). The large scale analysis of nucleolar components also revealed a number of proteins involved in chaperone activity, phosphorylation, DNA repair, DNA replication, RNA modification, RNA helicase activity, splicing, ubiquitination, p53 regulation, and several others (11). The diversity of proteins found within the nucleolus attests to the large number of cellular functions that it is involved in aside from ribosome biogenesis.

64

Considering the large number of proteins trafficking into and out of the nucleolus, there needs to exist a way for proteins to be targeted to this subnuclear structure.

Analysis of several proteins that localize to nucleoli has revealed the existence of nucleolar localization signals (NoLSs) that often work in conjunction with NLSs to mediate nucleolar transport. There does not appear to be a pattern for NoLSs

(130,182), and it is not possible to identify a signal merely by looking at an amino acid sequence. The one unifying characteristic of all NoLSs is a cluster of basic amino acid residues (130,182). Often times, the basic amino acid sequences of the NLS and NoLS overlap, and cannot be separated from one another (130). Rather than serving as a direct targeting sequence, NoLSs appear to serve as targets for other nucleolar proteins or RNAs that help mediate import into the nucleolus (130). For example, a NoLS may facilitate nucleolar targeting by binding to a ribosomal protein, ribosomal DNA, rRNA, or other resident of the nucleolus that then imparts localization. One such resident of the nucleolus that appears to have a major role in protein shuttling is nucleolin, which has been reported to interact with a variety of cellular (49,130) and viral proteins

(76,190,431) to mediate nucleolar localization as well as cell regulatory effects. The fact that several viral proteins contain strong NoLSs alludes that viruses have evolved specific nucleolar functions, though many of these are not yet understood (reviewed in

(190).

2.13 P-bodies (PBs) and stress granules (SGs)

*This section provides background information on host ribonucleoprotein structures that are discussed in Chapter 4 of this dissertation.

The control of mRNA translation and degradation is important in the regulation of eukaryotic gene expression, and the relationship between these two functions has been illuminated by understanding the cellular pathways of degradation. Two major mRNA

65 decay pathways are initiated with deadenylation of the mRNA poly(A) tail and decapping of the 5’ end, subsequently causing the formation of a mRNA associated with a multitude of regulatory proteins that is unable to be translated (reviewed in (338). These translationally repressed ribonucleoprotein complexes accrue in cytoplasmic foci referred to P-bodies (PBs) (218,338)). PBs contain components of the 5’ to 3’ decay machinery, nonsense-mediated decay pathway, and RNA-induced silencing machinery

(reviewed in (218). Under steady-state conditions, PBs exist within the cell cytoplasm as numerous small discrete structures that primarily degrade cellular mRNAs through exonuclease activity (27).

Related to PBs are the ribonucleoprotein complexes known as stress granules

(SGs), which are transiently present in the cytoplasm of eukaryotic cells during periods of environmental stress such as heat, viral infection, oxidative conditions, UV light, or hypoxia (reviewed in (218). SGs consist largely of stalled translation complexes including the small ribosomal subunit and early translation initiation factors (reviewed in

(135,218). During a stressful cellular event, homeostatic protein translation is halted until steady-state conditions are reestablished. SGs serve as storage depots for mRNAs that can resume translation once the stress has passed. PBs and SGs share some mRNA and protein components but also bear a number of unique markers specific for each structure (reviewed in (136,218). In some cases, PBs and SGs appear to communicate with another, as evidenced with immunofluorescent staining showing docking or overlap of the two structures in cells (27,218). This may represent an exchange of ribonucleoprotein complexes from the SG to the PB for degradation, or from the PB to the SG for storage. These extremely dynamic structures have come to the forefront of research just recently, as more and more evidence builds on the multiple roles of proteins and RNAs that are associated with PBs and SGs.

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Chapter 3: Interaction of Mouse Mammary Tumor Virus Gag Protein with

Ribosomal Protein L9 in the Nucleolus

3.1 Abstract

Mouse mammary tumor virus (MMTV) Gag has been shown to confer tumorigenic properties independent of proviral insertional mutagenesis, though the host factors involved in this process are unknown. Through yeast two hybrid analysis we identified ribosomal protein L9 (RPL9), an essential component of the large ribosomal subunit, as an interactor of Gag. Overexpression of RPL9 in mouse cells caused a significant and specific relocalization of MMTV Gag from the cytoplasm to nucleoli.

Fluorescence energy transfer (FRET) analysis was used to detect a direct interaction between Gag and RPL9. Mapping studies using truncations of Gag fused to GFP identified the CA domain of Gag as the interaction site of RPL9. Alternately, mapping of

RPL9 determined that the nucleolar localization signal exists within the N-terminal half, with the Gag interaction region in the C-terminal portion. Lastly, we were able to demonstrate the RPL9-Gag interaction through coimmunoprecipitation of RPL9 with Gag from mammary tumor extracts from MMTV infected mice. With the knowledge that

RibL9 acts as a tumor suppressor, we hypothesize that Gag may sequester RPL9 in nucleoli to enhance tumor formation. Nucleolar trafficking of MMTV proteins, including

Gag, may allow for interactions with host or viral factors that are important for pathogenesis.

3.2 Introduction

Upon its discovery as a milk-transmitted agent in the 1930’s (40), mouse mammary tumor virus (MMTV) has served as an important model in breast cancer research and immunology. This oncogenic retrovirus induces mammary tumors and

67 adenocarcinoma in infected mice through a complex series of cellular signaling pathways (reviewed in (324). Evidence suggests that a MMTV-like virus is associated with human cancer patient samples, however, an infectious agent and mode of transmission has yet to be firmly identified (reviewed in (372).

The 9-kb MMTV RNA genome consists of the common retroviral elements gag, pro, pol and env, as well as a dutpase, sag (superantigen) and rem. MMTV was recently characterized as a complex retrovirus as it encodes Rem, an HIV-1 Rev-like protein that regulates export of unspliced viral RNA from the nucleus (207). After infection of host cells, the viral RNA genome is reverse transcribed into a DNA copy that is integrated into the host genome, where it is transcribed into mRNA by cellular transcription machinery.

Unspliced viral RNA is exported to the cytoplasm where it is either translated to produce the Gag, Gag-Pro, and Gag-Pro-Pol polyproteins or packaged within assembling immature capsids as the viral genome. Unlike most other retroviruses, MMTV assembles immature capsids (Type B particles) in the cytoplasm at a perinuclear location prior to trafficking to the plasma membrane for release and maturation. This assembly pathway characterizes MMTV as a betaretrovirus, along with the well studied

Mason Pfizer monkey virus (MPMV).

Unlike some other oncogenic viruses, MMTV does not possess a virally-encoded oncogene. Rather, the mechanism underlying the development of tumors in infected mice results from a complex interplay of virus infection, immune cell transmission, hormonal stimulation of infected tissues, and host genetics. Integration of the viral DNA genome into the host chromosome disrupts normal cellular genes that regulate growth, differentiation and cell cycle control; alteration of these pathways by viral insertional mutagenesis results in uncontrolled cell growth and tumor formation. Recently, MMTV virally-encoded proteins have been implicated directly in tumorigenesis as oncogenes.

The Env protein is capable of transforming cells in culture, but not in vivo, through

68 expression of an immunoreceptor tyrosine-based activation motif (370). Analysis of chimeric viruses derived from a highly tumorigenic MMTV (C3H) strain and a nontumorigenic endogenous MMTV (Mtv1) strain indicate that sequences within MMTV gag are linked to carcinogenesis (197,217,413,413), however, it is not completely understood how these sequences contribute to tumor formation. The possibility remains that a cellular host factor capable of interacting with Gag may mediate the downstream effects leading to malignancy.

Characterization of several retroviruses indicates that the Gag protein possesses a collection of signals that direct not only viral RNA binding and Gag-Gag interactions required for virus particle assembly, but intracellular trafficking and host factor interactions as well. Rous sarcoma virus (RSV) Gag bears nuclear localization and export signals that allow for Gag to transiently reside in nuclei (385). Several reports also indicate that murine leukemia virus (317), human immunodeficiency virus (HIV-

1)(124) and foamy virus (387) Gag proteins also have the ability to traffic to nuclei.

However, little is known about signals required for trafficking of MMTV Gag within infected cells. The related betaretrovirus, MPMV has been well characterized and possesses a cytoplasmic targeting/retention signal (CTRS)(362) and a myristoylation signal (363) that directs sequential cytoplasmic assembly of nascent virions prior to trafficking to the plasma membrane for budding. Additionally, studies of MPMV trafficking show that small amounts of Gag appear within nuclei and associated with nuclear pores (44), indicating that betaretroviral Gag proteins may also participate in a nuclear trafficking step during the late assembly phase of the virus lifecycle.

MMTV Gag has sequences that resemble the MPMV CTRS as well as putative nuclear localization signals (197) and a myristoylation signal, but the molecular mechanisms and targeting of this protein have not been explored. To better characterize the subcellular trafficking of Gag and how it may contribute to

69 tumorigenesis, we sought to identify cellular factors that interact with MMTV Gag protein.

Initial yeast two hybrid screens identified host ribosomal protein L9 (RPL9), a member of the large ribosomal subunit and putative tumor suppressor, as an interacting partner of

MMTV Gag. Interestingly, overexpression of RPL9 relocalizes a subset of Gag to the nucleolar compartment where Gag is not seen under steady-state conditions. Our observations indicate that MMTV Gag and RPL9 proteins directly interact with one another in nucleoli, a subcellular compartment with a history of linkage to cancer and possibly a partner in the contribution of Gag to tumor formation.

3.3 Results

Yeast two hybrid identification of RPL9-Gag interaction

As mentioned previously, sequences within Gag were found to contribute to oncogenesis in hosts upon infection with MMTV. However, the mechanism behind how this Gag-related tumor induction occurs is unknown. One possibility is that Gag interacts with and disrupts the normal functions of a cellular factor that plays a role in cell cycle regulation or tumor suppression. In order to identify host proteins capable of interacting with MMTV Gag, initial experiments using MMTV Gag as bait in a yeast two hybrid analysis were employed. Using a cDNA library from the mammary glands of a C3H/HeN mouse as prey, members of the Tatyana Golovkina laboratory identified ribosomal protein L9 (RPL9) as a putative interactor of Gag (Figure 3.1). RPL9 is a ~21 kD protein component of the large (60S) ribosomal subunit that is essential for protein synthesis

(390). Interestingly, RPL9 expression is upregulated in a variety of human tumors

(249,251,451,478) and other studies implicate RPL9 as a tumor suppressor (26,298).

The yeast two hybrid also revealed that RPL9 may interact preferentially with

Gag proteins from viruses that cause higher tumor incidence in mice (C3H). The previously reported Gag hybrid proteins (413) that blend sequences from the

70 tumorigenic C3H MMTV Gag and the less tumorigenic HP/Mtv1 Gag were used to assay

RPL9 interactions (Figure 3.1A). Interestingly, only 14 amino acids differ between the

C3H and HP/Mtv1 Gag sequences, implying that subtle amino acid alterations in multiple regions of Gag are important in conferring increased tumorigenesis in MMTV C3H (197).

Figure 3.1C and 3.1D show the greatest beta-galactosidase activity between RPL9 and

HPvirA Gag, which contains all of the amino acid residues that differ from Mtv1 Gag swapped with C3H. RPL9 interactions with Mtv1 Gag were negligible in comparison to

HPvirA-RPL9 interactions. Two other Gag hybrid proteins were also examined, that contain either the pp21-p8 amino acid sequences of C3H (HPvirB) or just the CA-NC region of C3H in context of Mtv1 sequences (HPvirC). Surprisingly, neither the N- terminal nor the C-terminal portion of C3H is efficient in interacting with RPL9 more than

Mtv1 (Figure 3.1E), implying that all of the amino acid residue differences in C3H are important for efficient RPL9 interactions. Due to the possibility of a difference in RPL9 interaction with C3H Gag and Mtv1 Gag, we initially pursued experiments to further characterize the RPL9-Gag interaction using both strains of Gag protein.

Gag localization under steady-state conditions

We next moved to an overexpression system in cell culture to validate the Gag-

RPL9 interaction in normal mouse mammary epithelial (NMuMG) cells, which are commonly used for MMTV studies as a biologically relevant cell line (260,332,333). To first establish how Gag traffics in cells, we fused both C3H and Mtv1 MMTV Gag to green fluorescent protein (GFP) for visualization using confocal microscopy (Figure

3.2A). Under steady-state conditions, Gag from both MMTV strains localized to discrete foci within the cytoplasm of transfected cells (Figure 3.2B,C, left panels). This localization is consistent with the appearance of authentic MMTV capsids using immunofluorescence with anti-CA antibodies in infected cells (Figure 3.B,C, middle

71 panels). The bright cytosolic foci seen within Gag-GFP expressing cells appear to be capable of forming capsid-like structures visible by electron microscopy (EM), although fewer capsids were seen in cells expressing Gag-GFP compared to infected cells

(Figure 3.2D). As a Type B morphogenetic virus, MMTV assembles immature capsids in the host cytoplasm prior to trafficking to and release from the plasma membrane.

Unlike other retroviral Gag proteins, expression of C3H- or HeJ Gag-GFP did not result in release of VLPs from the cells (to be discussed in Chapter 4). The block in the release of VLPs appears to occur after cytoplasmic assembly but prior to membrane targeting of capsids. We therefore used MMTV-infected cells for many experiments to demonstrate biological relevance of our results with Gag-GFP. As shown in Figures

3.2B and 3.2C, the cytoplasmic spots within transfected NMuMG cells were indistinguishable from those of C3H- and HP-infected cells, indicating that the C3H and

Mtv1 Gag-GFP proteins traffic to intracellular sites similar to that of viral Gag. There was no discernable difference in the localization pattern of Mtv1 Gag from C3H Gag.

Examination of RPL9 and Gag localization using confocal microscopy

Considering that MMTV Gag assembles capsids in the cytoplasm and the RPL9 protein is a subunit of the ribosome, we hypothesized that the putative RPL9-Gag interactions would take place in the cytoplasm. To examine the localization of mouse

RPL9, we fused the RPL9 protein to either a FLAG tag for detection by immunofluorescence or the mCherry fluorophore for visualization using confocal microscopy. With overexpression in NMuMG cells, RPL9 alone localized to discrete subnuclear structures that we identified as nucleoli (Figure 3.3A). Regardless of the

FLAG or mCherry fusion, RPL9 colocalized with known nucleolar proteins, fibrillarin and nucleolin (Figure 3.3A). Little to no RPL9 is detectable in the cytoplasm of transfected cells under standard imaging conditions, but a cytoplasmic signal is detectable when the

72 image gain is increased and nucleolar fluorescence is oversaturated (not shown).

Expression of ribosomal proteins fused to protein tags have been shown previously to localize to nucleoli and to the cytoplasm, and result in functional proteins that can be incorporated into ribosomes (234). Immunofluorescence staining of endogenous ribosomal proteins have also shown localization to nucleoli (234), which serve as the site for ribosome assembly of protein subunits. Attempts were made to visualize endogenous RPL9 protein by immunofluorescence without overexpression in this study, however, a suitable antibody was not available.

Interestingly, when RPL9 was overexpressed in context of Gag-GFP transfection or MMTV infected cells, we saw a dramatic relocalization of Gag to nucleoli (Figure 3.3B,

C). There were no observable differences in the relocalization of Mtv1 Gag compared to

C3H Gag, and both were relocalized to nucleoli with RPL9 overexpression. In both the transfected and infected cells, Gag protein remained localized to discrete foci in the cytoplasm, but a subset of Gag also accumulated in the nucleolar compartment where it had not been observed previously. As shown in Figure 3.3B and 3.3C, RPL9-FLAG and

RPL9-mCherry were comparable in their ability to cause Gag to relocalize in Gag-GFP transfected and MMTV-infected cells, indicating that the tags were not responsible for the observed relocalization nor did the tags impede the ability of RPL9 to relocalize Gag.

Additionally, the relocalization of Gag was still apparent when the fluorophore tags were swapped; RPL9-GFP relocalized Gag-mCherry as well as viral Gag (Figure 3.3D).

To verify that the nucleolar relocalization of Gag was specific in response to

RPL9 overexpression, and not an artifact of expressing high amounts of a nucleolar protein, we overexpressed CFP-fused nucleolar markers fibrillarin, nucleolin, and B23 in

C3H infected cells. Upon examination using confocal microscopy, no alteration of C3H

Gag’s localization from steady-state conditions was observed (Figure 3.4A), indicating that general overexpression of nucleolar proteins did not evoke Gag relocalization. We

73 also tested overexpression of GFP-fused ribosomal proteins RPL4 (of the large ribosomal subunit) and RPS6 (small subunit) to identify if Gag relocalization was a ribosome-related phenomenon. Figure 3.4B shows that C3H Gag within infected cells did not colocalize with either ribosomal protein when overexpressed, indicating that the

RPL9-Gag colocalization event was specific. Due to the similarities in localization, Mtv1

Gag was not tested with the nucleolar markers or other ribosomal proteins at this time.

The data up to this point suggest that overexpression of RPL9 specifically “traps” Gag in

nucleoli, as Gag localization to nucleoli is not visible by confocal microscopy at

endogenous levels of RPL9 (without overexpression).

We were also interested in whether mouse RPL9 could relocalize the Gag

proteins of other retroviruses, or if this relocalization is very specific to the MMTV Gag

protein. To this end, we expressed GFP-tagged Gag from RSV and HIV-1 in quail and

human cells, respectively, with mouse RPL9-mCherry overexpression. RSV and HIV

Gag proteins fused to GFP have been shown to be localized to the cytoplasm under

steady-state conditions (124,383). As seen in Figure 3.4C, neither Gag protein was

altered in its localization from the cytoplasm. It is unlikely that RSV and HIV Gag were

unresponsive due to overexpression of mouse RPL9 as opposed to that from quail or

human; as shown in Table 1, the RPL9 protein is very conserved among higher

eukaryotes, with more than 98% homology between mouse and human or chicken.

Although we did not examine the Gag protein of another Type B retrovirus (such as

MPMV) in context of RPL9, it is possible that the ability of RPL9 to relocalize Gag to

nucleoli is specific to MMTV and is not universal among all retroviral Gag proteins.

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Validation of RPL9 interaction with Gag using fluorescence energy transfer (FRET) and coimmunoprecipitation.

Acceptor photobleaching FRET was used to verify the yeast two hybrid data that indicated a direct protein-protein interaction between RPL9 and C3H MMTV Gag. Unlike colocalization, which only indicates that two proteins occupy the same area, FRET can measure the exchange of energy between fluorophore pairs that can only occur when two proteins are in very close proximity to one another. We chose to use a GFP (FITC) donor/mCherry acceptor FRET pair, which has been noted as a stable FRET fluorophore couple (7,426). Upon bleaching RPL9-mCherry in nuclei of MMTV C3H infected cells, we were surprised to observe an obvious visual increase in the intensity of the donor fluorophore between the pre- and post-bleach images (Figure 3.5A), indicative of a highly positive FRET. Such a change was not observed in the raw FRET data of the negative control pair, Rem-GFP and RPL9-mCherry, both of which localize to nucleoli

(Figure 3.5B). The graph in Figure 3.5C shows the percent FRET values obtained between RPL9 and Gag of C3H infected cells, in comparison to several control pairs that represent no protein-protein interactions. The very strong positive FRET signal between

RPL9 and C3H Gag confirms that the two proteins interact directly with one another within the nucleoli of NMuMG cells. This strong Gag-RPL9 interaction dominates over the % FRET values of the other FRET protein pairs that were not expected to interact with one another (mCherry + Gag of C3H infected cells, Rem + RPL9, and mCherry +

GFP). The low FRET values for these control protein pairs signify the absence of interactions as expected. Preliminary FRET data of RPL9 with Gag from HP-infected cells (not shown) indicates that Mtv1 Gag also interacts with RPL9 via direct protein-

protein interactions (77.5% positive FRET), though this data has yet to be repeated.

Due to the inability to distinguish C3H Gag interactions with RPL9 from those of Mtv1

75

Gag, we continued the characterization of Gag-RPL9 interactions using only the C3H strain of Gag.

To further validate that we could detect Gag-RPL9 interactions in our cell culture system, we performed coimmunoprecipitation assays from C3H chronically infected cells with endogenous levels of RPL9 (Figure 3.6A) and in uninfected NMuMG cells overexpressing Gag-GFP and RPL9-FLAG (Figure 3.6B). In either situation, we were able to detect Gag-RPL9 interactions; viral Gag and free CA protein were immunoprecipitated with RPL9 antibodies from infected cell lysates (Figure 3.6A) and

RPL9 was immunoprecipitated with antibodies to GFP in context of Gag-GFP overexpression (Figure 3.6B). The RPL9-Gag interaction may be weak or transient, as it was difficult to observe the interactions via coimmunoprecipitation, with only modest amounts of pulled down protein detectable on the Western blots. Despite several replicates of the experiment, the RPL9-Gag coimmunoprecipitation data was not readily repeated. Attempts were also made to fractionate and purify nucleoli for coimmunoprecipitation, however these experiments were fraught with problems and negative results. Though the coimmunoprecipitation data alone do not consistently support Gag-RPL9 interactions, taken in combination with the FRET data we are confident that RPL9 and Gag interact with one another directly in MMTV infected cells.

Mapping regions of Gag involved in RPL9 interaction

Based on the confocal microscopy data that showed a dramatic relocalization of full-length Gag-GFP to nucleoli with RPL9-mCherry expression, and the FRET data indicating that RPL9 and Gag are directly interacting with one another, we used a series of C-terminal truncations of Gag fused to GFP to determine if a particular domain(s) of

Gag contributed to the RPL9 interaction (Figure 3.7A). When each truncation was expressed alone in NMuMG cells (Figure 3.7B), the GFP-fused proteins were distributed

76 primarily in the cytoplasm with a small amount in the nuclei of cells and complete exclusion of the nucleoli. As anticipated, the GagΔNC-GFP and subsequent C-terminal deletions were diffuse and not within cytoplasmic foci as seen with full-length Gag; the

NC region of Gag proteins is important for Gag-Gag interactions that facilitate the formation of capsids and the bright foci seen in the cytoplasm of transfected cells (see section 2.5.2.4).

RPL9-mCherry was then coexpressed with each Gag-GFP truncation to look for relocalization of Gag domains to nucleoli (Figure 3.7C). We observed the most dramatic effect with GagΔNC-GFP, which had little visible protein in the cytoplasm as nearly all of it relocalized to nucleoli with RPL9. Removal of the CA domain, and subsequent Gag domains, revealed a consistent distribution of Gag in the cytoplasm with a subset of protein still localizing to nucleoli. Even the smallest Gag truncation, MA.pp21-GFP, had a small amount of nucleolar relocalization, indicating that regions throughout Gag may interact with RPL9. A GFP-only control was included to show the diffuse distribution of the GFP in cells that appears unaffected by RPL9 overexpression. The MA domain alone was not examined as its molecular weight with GFP is below the threshold for nuclear import (~50 kD) and therefore freely diffuses throughout the cell like GFP (see

Chapter 4). Importantly, the loss of the CA domain (GagΔCA.NC-GFP) from GagΔNC yielded the most striking phenotype change, alluding to CA as the dominant region of

RPL9 interaction.

To investigate this possibility further, we examined the contributions of the CA and NC domains to relocalization with RPL9. We also included a p3.p8.n.CA construct that removes the N-terminal domains (MA.pp21) which were sufficient for nucleolar relocalization with RPL9 in Figure 3.7C, and the NC domain which yielded the dramatic, entirely nucleolar-localized phenotype. Interestingly, NC-GFP alone localized to nuclei and nucleoli (Figure 3.8A), indicating that NC possesses the inherent ability to traffic to

77 nuclei and is retained in nucleoli through nuclear localization and nucleolar retention signals within its amino acid sequence. However, as shown in Figure 3.7C, Gag lacking the NC domain is still able to relocalize to nucleoli with RPL9 overexpression, demonstrating that Gag traffics to nucleoli independently of its NoLS in NC and may do so by traveling with RPL9. In contrast, the p3.p8.n.CA-GFP and CA-GFP constructs localized to the cyto- and nucleoplasm of transfected NMuMG cells and excluded nucleoli (Figure 3.8A) when expressed alone. In context of RPL9-mCherry overexpression (Figure 3.8B), NC-GFP appears unaffected in its distribution within cells, while p3.p8.n.CA-GFP and CA-GFP are completely relocalized to nucleoli. The relocalized phenotype of the CA domain reiterates that it possesses sequences important for interaction with RPL9.

Identification of RPL9-Gag interaction regions

To begin mapping regions of RPL9 that participate in interactions with MMTV

Gag, we first looked at homologues of RPL9 in other species (Table 3.1). RPL9 is a conserved protein among higher eukaryotes (95-99% homology), however, yeast RPL9 isoforms A and B showed the least amount of homology (69%) and identity (50%) to mouse among the species examined. Considering the sequence conservation between mouse, yeast and E. coli (most divergent), we expressed RPL9 from these different species in context of MMTV Gag to look for conservation of the nucleolar relocalization phenotype. We rationalized that conservation of RPL9-Gag interactions may correlate with conserved amino acid sequences, allowing for mapping of the Gag interaction region within RPL9.

Although both yeast isoforms of RPL9 localized to not only the nucleoli but also to the nucleoplasm (Figure 3.9A, middle panel), in either case MMTV Gag relocalized to the nuclear/nucleolar compartments in response to yeast L9 overexpression (Figure

78

3.9A, left panel). These observations indicate that conserved amino acid regions of mouse and yeast RPL9 may contain the Gag interaction domain(s). Interestingly, E. coli

RPL6 accumulated in nucleoli (Figure 3.9B) despite the fact that bacteria lack nuclei.

When overexpressed in C3H infected cells, though, the nucleolar E. coli RPL6 did not relocalize the Gag protein, indicating sequence differences that may alter the ability for interaction with Gag. Similar results were obtained when yeast RPL9 isoforms and E. coli RPL6 were coexpressed with C3H Gag-GFP (not shown).

Upon looking at the amino acid sequence comparison between mouse RPL9, yeast RPL9, and E. coli RPL6 proteins (Figure 3.9C), we noticed many regions of homology throughout RPL9 between all three species. As the bacterial RPL6 did not relocalize MMTV Gag, we focused on the areas of conservation between yeast and mouse for the Gag interaction domain. One area that drew our attention was a stretch of 16 nearly identical residues between mouse and yeast near the C-terminus of RPL9

(Figure 3.9C, horizontal red line). As little is known in terms of the putative domains of eukaryotic RPL9, we chose to divide mouse RPL9 in two portions for further mapping based on structural data from the prokaryotic equivalent, RPL6 (162) (Figure 3.9C, vertical red line; 3.10A). We examined the localization of the N-terminal (NTD) and C- terminal (CTD) halves of RPL9 fused to mCherry and found that the two halves vary greatly in their cellular distribution (Figure 3.10B, middle panel). RPL9 NTD-mCherry localized primarily to nucleoli whereas RPL9 CTD-mCherry excluded the nucleus and existed within aggregated cytoplasmic foci. Interestingly, when overexpressed with Gag-

GFP (Figure 3.10B, left panels) or within infected cells (Figure 3.10C), the RPL9 NTD did not relocalize Gag to nucleoli, but some Gag foci did colocalize with cytoplasmic aggregates of RPL9 CTD. This observation led us to hypothesize that RPL9 interacts with Gag through its CTD and uses a NLS/NoLS within its N-terminus to traffic to

79 nucleoli. Both the NoLS and the Gag interaction region of RPL9 are necessary to see relocalization of Gag to nucleoli.

To test our hypothesis, we fused the known NoLS of HIV Rev (94) (Figure 3.10D) to the RPL9 CTD to restore nucleolar trafficking and observe whether Gag could move to nucleoli via interaction with the Rev NoLS-containing RPL9 CTD. As shown in Figure

3.10E, Gag-GFP localization was not affected by the Rev NoLS control fused to mCherry, but addition of the Rev sequence to RPL9 CTD was able to restore the ability of Gag to be relocalized to nucleoli. This data confirms that the CTD of RPL9 is sufficient for interactions with MMTV Gag.

We further examined the region of homology at the most C-terminal end of RPL9 that was nearly identical between mouse and yeast RPL9 sequences (Figure 3.9C, red horizontal line) to determine if it was responsible for the Gag-RPL9 interaction. The expression of a mouse RPL9 construct lacking the last 26 amino acids of its CTD

(RPL9ΔCTD26-mCherry), however, was still able to relocalize Gag-GFP (Figure 3.11A), indicating that amino acids 86-172 of the mouse RPL9 (Figure 3.9C) possess the Gag interaction region. In addition, we found two NLS/NoLS sequence motifs within the

RPL9 NTD using the cNLS mapper prediction program (232) and based on Timmers et al. 1999 (Figure 3.11B) (422). Confocal microscopy revealed that either NLS/NoLS region of RPL9 is sufficient for nucleolar localization with some signal in the nucleoplasm

(Figure 3.11C, top left panels), but greater nucleolar localization (with little-to-no mCherry signal in the nucleoplasm) is achieved when the two signals work together

(Figure 3.11C, top right panel). These data suggest that the two NLS/NoLS motifs act as a bipartite signal to enhance nucleolar targeting of RPL9.

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3.4 Discussion

MMTV serves as a well-studied model for understanding the mechanisms of tumor progression in breast cancer. However, little is known about the intricate host-cell interactions that occur upon infection with MMTV. Here we report the novel findings that

MMTV Gag interacts with a ribosomal protein, L9, and is capable of localizing to nucleoli in context of RPL9 overexpression. Recent studies have elucidated dynamic properties of nucleoli that extend beyond their traditional role in ribosomal biogenesis, including roles in stress detection and cancer. Viruses have been privy to this information for some time, as scientists discover that many viruses co-opt nucleolar machinery as part of their replication strategies (reviewed in (189,190). Additionally, ribosomal proteins are known to have functions independent of protein synthesis that contribute to regulation of transcription, DNA replication, cell growth and tumor suppression (10,236,469). This compilation of information points to the involvement of RPL9 in MMTV Gag-linked tumorigenesis. We initially investigated the possibility that RPL9 differentially interacted with the Gag of the more tumorigenic strain of C3H over that of the less tumorigenic

Mtv1. Beyond the yeast two hybrid studies, there were no observable differences in

C3H and Mtv1 Gag relocalization or Gag-RPL9 interactions as analyzed by FRET, and from our data it appears that RPL9 is able to interact with Mtv1 Gag. This does not exclude the possibility of RPL9 interacting with Gags of different strains with different binding affinities or kinetics that ultimately results in variable tumor progression, which would need further study to validate.

We have not completely ruled out the participation of RPL9 in virus assembly, though it does appear that the RPL9-Gag interaction plays only a transient role within the infected cell; we are unable to detect RPL9 in virus particles collected from MMTV infected cells (Figure 3.12), indicating that RPL9 does not continue with Gag through the assembly process. Additionally, the expression levels of RPL9 do not appear to be

81 altered in infected cells in comparison to uninfected cells (Figure 3.12B). We attempted to use siRNA and shRNA to knockdown RPL9 in MMTV infected cells, to see if there was an effect on virus particle production when RPL9 expression was decreased or absent. Unfortunately, the outcomes of these experiments were uninterpretable due to inconsistent or unobservable decreases in RPL9 expression and unhealthy-looking transfected cells. According to the siRNA/shRNA manufacturers, knockdown of RPL9 has not been attempted previously with their products (personal communication with

Dharmacon and SA Biosciences representatives); RPL9 knockdown may not be possible, knowing that RPL9 deletions in fish and fruitfly systems are embryonic lethal

(258,390). However, other ribosomal proteins have been reported to be knocked down in the literature using siRNA methods (103,104,148,417), and further problem-shooting with the siRNA/shRNA transfection may yield an answer to whether or not RPL9 contributes to assembly. Alternately, we attempted to overexpress RPL9 in infected cells to look at the effect on virus output. Transfection of infected NMuMG cells with

DNA constructs using Lipofectamine 2000 (Invitrogen) yields the highest transfection efficiency (~20%) compared to other reagents tested, however those transfection levels are not sufficient for testing a population effect on virus production.

Interestingly, we noted the presence of both Gag and the Gag cleavage product,

CA, in coimmunoprecipitations with the RPL9 antibody from infected cells (Figure 3.6).

This is not too surprising, considering that our data points to the CA domain as the main interaction region of Gag with RPL9. It has been shown by Zabransky et al. that the Gag polyprotein undergoes early processing by the viral protease prior to release of virus particles from the (479). This intracellular CA would then be available to interact with RPL9 along with uncleaved Gag during the lysis of cells for coimmunoprecipitation. While it is clear that CA can interact with RPL9 from our studies, we are not certain whether the RPL9-CA interaction that we detect is the biologically

82 relevant interaction over the RPL9-Gag interaction. Though most of our studies have focused on the full-length Gag protein, we cannot rule out the possibility that CA interacts with RPL9 during entry of the virus as part of the preintegration complex (PIC) nuclear trafficking step.

Our data suggests that during infection, Gag/CA is carried to the nucleolus via interactions with the C-terminus of RPL9 and the nucleolar targeting sequences located in the N-terminus of RPL9. This nucleolar trafficking occurs transiently, as only small amounts of Gag are detectable in the nuclei of cells under steady-state conditions

(Figure 3.2B and C), and modest amounts of Gag-RPL9 were detected with coimmunoprecipitation (Figure 3.6). Gag possesses a NoLS of its own within the NC domain (Figure 3.7), which has basic amino acid residues scattered throughout its sequence that could contribute to a NoLS. The nucleolar targeting of NC may also play a role in virus entry with nuclear targeting of the viral PIC. Despite its NoLS, our data shows that RPL9 can relocalize Gag to nucleoli in the absence of NC (Figures 3.7, 3.8).

We also have preliminary data indicating that RPL9 can be coimmunoprecipitated with

GagΔNC (data not shown). Gag primarily interacts with RPL9 through its CA domain

(Figures 3.6, 3.7, 3.8), however, Gag-RPL9 may be stabilized with weaker interactions

throughout the length of Gag, as truncations of Gag as small as MA.pp21 still show

nucleolar relocalization with RPL9 overexpression (Figure 3.7).

Strikingly, the two domains of Gag that we found to localize to nucleoli either

independently (NC) or in association with RPL9 (CA) are the same two domains that

were reported to contribute to mouse tumorigenesis in virus hybrid studies (413).

Considering the collective data of Gag interacting with RPL9 to mediate nucleolar

trafficking, the connection of Gag to increased tumor propensity in mouse studies, and

the large body of data linking ribosomal proteins and nucleoli to cancer (reviewed in

(299), we have deduced a model for Gag-RPL9 mediated tumor progression (Figure

83

3.13). Once in the nucleolus of infected cells, we speculate that Gag sequesters RPL9 from extraribosomal activities that suppress tumor progression, either directly or through downstream molecules that interact with factors such as p53. By altering the anti-tumor activities of RPL9, Gag may enhance cancer progression in cells that have undergone insertional mutagenesis upon infection.

Though the extraribosomal functions of RPL9 are currently unknown, protein interaction studies predict RPL9 as a binding partner for several proteins involved in cell cycle control and tumor suppression (Table 3.2). NEDD8, a ubiquitin-like protein, is predicted to modify RPL9, and has been shown to have an important role in modifying proteins that regulate p53, including many ribosomal proteins, cullins, and UBC (472).

Additionally, several ribosomal proteins that interact with RPL9 are known to play a significant role in p53 regulation. For example, interactions of RPL5, RPL11, RPL23,

RPL26, RPS3 and RPS7 with MDM2, a negative regulator of p53, lead to alteration of p53 levels in response to cellular stress (reviewed in (454,475). This alludes that through protein-protein interactions or through its own, currently unknown abilities, RPL9 may participate in the regulation of the key tumor suppressor protein, p53. Though we may not yet understand the mechanisms for how Gag and RPL9 play a role in tumorigenesis, there is a well established link between cancer, nucleoli, and ribosomal proteins which may provide the foundation for future understanding.

Although this is the first account of MMTV Gag trafficking to the nucleus/nucleolus, it is not the first MMTV viral protein reported to do so. Rem (289) and p14, a derivative of the Env protein (24), have been shown to localize independently to nucleoli of MMTV infected cells. Several other retroviral proteins, including Rev and Tat of HIV-1, are known to traffic to nucleoli. Though their nucleolar activities have been studied and their NoLSs described the purpose of targeting viral proteins specifically to the nucleolus remains largely unknown. Recent studies have revealed nucleoli as

84 cellular control hubs for not only ribosome biogenesis, but for RNA processing, cell cycle regulation, senescence, stress management, and gene silencing. Considering the importance of the nucleolus in basic cell homeostasis, it is not hard to envision that viruses may usurp these subnuclear structures to promote viral replication strategies.

3.5 Materials and Methods

Plasmids

pNucleolin-GFP, pFibrillarin-GFP and pB23-GFP (123), pRPL4-GFP and pRPS6-

GFP (234), pHIV Gag-GFP (187), pRSV Gag-GFP (74) and pGFP-Rem (289) were previously described. pNucleolin-GFP and pFibrillarin-GFP were modified using PCR cloning to exchange CFP for GFP (made by E.Ryan/T.Lochmann). pRPL9-FLAG was cloned by insertion of amplified mouse RPL9 (NCBI: NM_011292) into the BglII site of the pCMV-FLAG-MAT-2 vector (Sigma). pRPL9-mCherry was created by inserting the

RPL9-FLAG sequence into pmCherry.N2, which was made by replacing GFP in pEGFP.N2 (Clontech) with mCherry from pRSet8.mCherry (399). pRPL9-GFP was

made by amplification of RPL9-FLAG and insertion into the HindIII/SalI sites of

pEGFP.N2. pGag-GFP was created by PCR amplification of MMTV Gag from MMTV

strains C3H and Mtv1 (template DNA from T. Golovkina) and insertion into pEGFP.N2

using BamHI-HindIII; 21 nts of the upstream untranslated region of Gag were included

in the pGag-GFP constructs. Similarly, pGag-mCherry was made by amplification of

C3H Gag and insertion into mCherry.N2 using the HindIII/ApaI sites. GFP-fused

truncations of MMTV C3H Gag ΔNC (1-1485), ΔCA.NC (1-804), Δn.CA.NC (1-753),

MA.pp21.p3 (1-681), MA.pp21 (1-582), p3.p8.n.CA (583-1485), and CA (805-1485) were

PCR amplified at the noted nucleotides of gag (primers available upon request) and

ligated into BamHI-HindIII of pEGFP.N2. The NC domain of Gag (1486-1767) was PCR

amplified and cloned into BamHI/BglII of pEGFP.N2 with Klenow. pYRPL9A-mCherry

85 and pYRPL9B-mCherry were made by amplification of RPL9 isoform A or B sequences

(yeastgenome.org: YGL147C and YNL067W) from yeast genomic cDNA (gift from A.

Hopper) and insertion into pmCherry.N2 using HindIII-SalI. pEcoliRPL6-GFP was made by amplification of RPL6 (NCBI: AP009048) from Escherichia coli DH5α genomic DNA and ligation into pEGFP.N2 via HindIII-SalI. pRPL9 NTD-mCherry (1-255), pRPL9 CTD- mCherry (256-576), pRPL9 NLS#1-mCherry (61-108), pRPL9 NLS #2-mCherry (142-

183), pRPL9 NLS #1-#2-mCherry (61-183), and pRPL9ΔCTD26-mCherry (1-499) were made by PCR amplification of the indicated RPL9 nucleotide bases and ligated into pmCherry using HindIII-SalI.

The NoLS of HIV-1 Rev (94)(NCBI K03455) was amplified from pRev-YFP and cloned into pmCherry or pRPL9 CTD-mCherry via SalI-ApaI. pRev-YFP was made by amplifying Rev from pCMV-Rev (264,265) and cloning into SalI-ApaI of pEYFP.N2. All created plasmids were sequenced and shown to be faithful copies of the corresponding genes. The NLS/NoLS sequences of RPL9 were predicted using the free online cNLS mapper program (232) accessible at http://nls-mapper.iab.keio.ac.jp/.

Yeast Two Hybrid

This experiment was conducted by Ingrid Swanson, Leonid Yurkovetskiy, and

Tatyana Golovkina of the University of Chicago in the laboratory of Stephen Goff at

Columbia University. MMTV Gag proteins were cloned into the multiple cloning site of the pNLexA Bait vector to create a Gag-LexA fusion protein. The HP/Mtv1, HPvirA,

HPvirB, and HPvirC Gag proteins were previously described (413). A C3H/HeN mouse mammary gland cDNA library was used to clone prey into the pGADNOT vector multiple cloning site, which produced GAL4-cDNA fusions. Over 300,000 GAL4-cDNA plasmid clones were made, representing each mouse genome gene with 10x redundancy. MMTV Gag sequences were also cloned into the pGADNOT vector to

86 serve as Gag-Gag homodimerization positive controls. Positive protein interactions between bait and prey were measured with beta-galactosidase activity on Xgal plates.

Cell Culture and Transfection

NMuMG (normal mammary gland; ATCC CRL-1636) cells, MMTV strain C3H

(125,213) chronically infected NMuMG cells, and proviral HP and HPvirA stably- transfected infected NMuMG cells (413) were cultured in DMEM supplemented with 10%

fetal bovine serum, penicillin, streptomycin and Amphotericin B (HyClone). HeLa cells

(ATCC CCL-2) were maintained in Dulbecco’s media supplemented with 5% FBS,

glutamine, penicillin, streptomycin, sodium bicarbonate and Amphotericin B. HeLa and

NMuMG cells were transfected using Lipofectamine 2000 (Invitrogen) according to

manufacturer’s directions. Quail fibroblast (QT6) cells were cultured as previously

described (100) and transfected via the calcium phosphate method.

Microscopy and Immunofluorescence

Cells were seeded in 35-mm dishes containing #1.5 glass coverslips, fixed in 4%

paraformaldehyde, washed in PBS, permeabilized with 0.25% Triton X-100/PBS and

blocked with 3% BSA/PBS. Primary antibodies used were mouse anti-MMTV CA (353)

or mouse anti-FLAG (Sigma). After washing in PBS, coverslips were stained with sheep

anti-mouse IgG-Cy3 (Sigma), goat anti-mouse IgG-FITC (Sigma), or goat anti-mouse

IgG Alexa Fluor 514 (Invitrogen). Cell nuclei were stained with DAPI, and SlowFade

reagent (Molecular Probes) was used for mounting coverslips. Fixed cells were

examined using a Leica AOBS SP2 confocal microscope with excitation of GFP at 488

nm, CFP at 458 nm, Cy3 and mCherry at 543 nm, and Alexa 514 at 514 nm. Cell

images were false-colored for overlay comparison using ImageJ and image intensities

were increased using CorelDRAW X3.

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Acceptor Photobleaching FRET Analysis

Using fixed and immunostained cells, prebleach images were obtained for GFP

(excitation at 488 nm, emission at 492-533, 25% laser power) and mCherry (excitation at

543 nm, emission at 558-599, 50% laser power) or Cy3 (excitation at 543 nm, emission at 558-599, 50% laser) channels using sequential scanning. For photobleaching, nuclei were selected as the region of interest, and mCherry or Cy3 fluorophores were bleached using the 543-nm laser at 100% power until the fluorescence intensity was reduced to

20% of prebleach levels or for 5 minutes. Percentage FRET efficiency was calculated as previously described (220). FRET analysis was performed using a minimum of 10 different cells on two different days, and the mean and the standard error of the mean were calculated for graph construction (GraphPad Prism).

Coimmunoprecipitation

Coimmunoprecipitations were performed using either the Pierce Crosslink

Immunoprecipitation Kit (Thermo Scientific) or a protocol based on the previously described Lischka et al. (252). For the commercial kit, the manufacturer’s directions were followed, with use of an alternate lysis buffer (150 mM NaCl, 20 mM Tris-HCl, 1%

NP-40, 1 Roche protease inhibitor tablet); cells were lysed on ice for 1 hr with vortexing every 10 min, and insoluble debris was removed from lysates with centrifugation for 25 min at full speed in a microfuge. A total of 2-4 mg of protein was used per column. Anti-

GFP (Roche) antibodies were used for crosslinking. Briefly, the Lischka method lysed cells in lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 5 mM EDTA with 1% NP40 and protease inhibitors) for 20 min on ice and debris was pelleted. Supernatants were cleared with protein A agarose beads prior to incubating overnight on a rotator at 4 C with anti-RPL9 antibody (Genway Biotech). Beads were added for 1 hour prior to

88 pelleting and 6 successive washes. All immunoprecipitated protein samples were then analyzed by SDS PAGE and Western blotting (see Supplemental Methods).

Virus Collection and Western blot

Media samples were collected from 2 confluent 100 cm2 dishes of MMTV C3H chronically infected NMuMG cells and spun through a 25% sucrose pellet for 1 hour at

90,000 x g (Beckman SW41) at 4 C. Cell lysates were prepared by lysing cells in boiling 2x SDS-loading dye, or lysing cells in RIPA lysis buffer followed by a Bradford assay. Protein from lysates and pellets were separated on 12% SDS PAGE and transferred to PVDF membrane. After blocking with 5% nonfat dry milk in TBS-T, blots were probed with primary antibody to detect RPL9 (GenWay Biotech, Santa Cruz) or

CA/Gag (353) with HRP-linked secondary antibodies (Sigma) and detected with enhanced chemiluminescence (Thermo Scientific, GE Healthcare).

Online databases for interacting partners of RPL9

Internet protein databases were searched for potential binding partners of RPL9 to provide insight into the possible downstream roles of RPL9 outside of the ribosome.

Lists of interacting proteins were found for human RPL9 at: Entrez Gene

(http://www.ncbi.nlm.nih.gov/gene), Gene ID 6133; UniProt (http://www.uniprot.org/), ID

P32969; and GeneCards (http://www.genecards.org), ID GC04M039131 (accessed

December 6th, 2010).

3.6 Acknowledgements

We would like to thank the following scientists for their contributions of reagents and materials used in this study: R. Tsien, University of California at San Diego; D.

Goldgaber, The State University of New York, Stonybrook; M. Olson, University of

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Mississippi Medical Center; T. Krüger, University of Würzburg, Germany; M. Resh,

Memorial Sloan-Kettering Cancer Center; J. Dudley, University of Texas at Austin; and

A. Hopper, Ohio State University and B. Cullen, Duke University. We would also like to acknowledge the Penn State College of Medicine Imaging Core Facility for confocal and electron microscopy imaging, and members of the Parent Laboratory (E. Ryan and T.

Lochmann) for DNA cloning.

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Figure 3.1 Yeast two hybrid data implicating RPL9 as a binding partner of MMTV

Gag. A) Diagram of the Gag proteins that were used in the yeast two hybrid screen for

Gag-binding host factors. HP/Mtv1 (white box) represents the hybrid provirus which produces lower tumor incidence in mice while the C3H (black box) produces high tumor incidence in susceptible mice. Between the two Gag proteins, there are approximately

12 amino acid residues that differ in the HP and C3H strains (conserved residues are shown in black and white, while nonconserved residues are shown in color). The

HPvirA, HPvirB, and HPvirC Gags are hybrid Gags containing both HP/Mtv1 and C3H sequences as indicated. This figure is adapted from (413). B) Positive control yeast two hybrid plate of yeast transformed with Gag bait constructs and Gag prey constructs. The blue color (produced by the beta-galactosidase cleavage of the Xgal substrate) indicates a positive interaction between the protein pairs. C) Experimental yeast two hybrid plate showing the cellular protein RPL9 interacting with the different Gag constructs. The initial screen of Gag with the mouse cDNA library (not shown) yielded RPL9 as a possible interacting factor with Gag. D,E) Quantitation of the beta-galactosidase activity of the Gag-RPL9 interactions on the plate in panel B. D) Comparison of the less tumorigenic Mtv1 Gag with the more tumorigenic HPvirA Gag in terms of RPL9 interaction. E) Comparison of the Mtv1, HPvirB, and HPvirC Gag proteins in terms of

RPL9 interaction. Note that the Y-axis of the graphs in panels C and D differ in range and intervals. The experiments represented by this figure were conducted by my collaborator, Tatyana Golovkina, and members of her laboratory at the University of

Chicago.

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Figure 3.2 Localization of MMTV Gag in transfected and infected cells. A) Diagram of MMTV Gag with C-terminal fusion to GFP. B) Confocal images of NMuMG cells transfected with C3H Gag-GFP alone (left), infected with MMTV C3H and immunofluorescently stained to detect viral Gag protein (middle), and C3H-infected cells transfected with C3H Gag-GFP (green) and stained for Gag (red) to detect overlap

(yellow, right). C) Confocal images of NMuMG cells transfected with Mtv1 Gag-GFP alone (left) and cells stably transfected with provirus HP and immunofluorescently stained to detect viral Gag protein (right). D) Transmission electron micrographs of virus-like particles in the cytoplasm of NMuMG cells transfected with C3H Gag-GFP (left) and virus particles within C3H chronically infected NMuMG cells (right).

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Figure 3.3 Localization of mouse RPL9 in context of nucleolar markers and MMTV

Gag in NMuMG cells. A) Confocal microscopy of NMuMG cells expressing FLAG-fused or mCherry-fused RPL9 and CFP-fused nucleolar markers, fibrillarin and nucleolin.

Images are false-colored. B) Upper panel, overexpression of RPL9-FLAG with C3H

Gag-GFP in transfected NMuMG cells. Lower panel, overexpression of RPL9-FLAG with Mtv1 Gag-GFP in transfected NMuMG cells. C) Upper panel, overexpression of

RPL9-mCherry within C3H chronically infected NMuMG cells. Lower panel, overexpression of RPL9-mCherry within stably NMuMG cells stably transfected/infected with provirus HP. RPL9-FLAG was detected with immunofluorescence staining for

FLAG with Cy3-conjugated secondary antibody, and viral Gag protein was detected with staining for CA and FITC-labeled secondary antibody. D) Confocal microscopy of

NMuMG cells transfected with C3H Gag-mCherry and RPL9-GFP (top) and C3H infected cells transfected with RPL9-GFP (bottom). Viral Gag is visualized by immunofluorescence staining for CA with Cy3-conjugated secondary antibody.

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Figure 3.4 Examination of nucleolar cellular proteins with MMTV Gag and viral

Gag proteins with mouse RPL9 in NMuMG cells. A) C3H infected NMuMG cells were transfected with either fibrillarin-CFP, nucleolin-CFP, or B23-CFP and imaged by confocal microscopy. Gag proteins were immunofluorescently stained with anti-CA antibodies followed by Alexa-514 conjugated secondary antibodies. All images are false-colored. B) GFP-fused ribosomal proteins RPL4 and RPS6 were overexpressed in

C3H infected NMuMG cells. Viral Gag was detected with immunofluorescence staining for Gag with Cy3-conjugated secondary antibody. All images are false-colored. C)

Upper panel: HeLa cells were co-transfected with GFP-fused HIV Gag and mouse

RPL9-mCherry. Lower panel: QT6 quail fibroblasts were co-transfected with GFP-fused

RSV Gag and mouse RPL9-mCherry. Quail cells are shown at a higher magnification than the HeLa cells.

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Table 3.1 Amino acid comparisons of different species to mouse RPL9 (NCBI:

NM_011292)

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Species Homology Identity Accession No. Human 99 98 NM_001024921 Rat 99 98 NM_001007598 Chicken 98 94 XM_423225 Zebrafish 95 89 NM_001003861 Fruit fly 80 64 NM_057813 Yeast A 69 49 NP_011368 Yeast B 69 50 NP_014332 E. coli RPL6 43 21 BAE77986

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Figure 3.5 RPL9-MMTV Gag interactions as shown by FRET analysis. A,B)

Confocal images representing raw FRET image data. Pre-bleach images were acquired prior to beginning the FRET analysis. Post-bleach images were obtained after nucleoli containing RPL9-mCherry were specifically bleached with a 543-nm laser. A) RPL9- mCherry overexpression in immunofluorescently stained MMTV infected NMuMG cells.

B) Rem-GFP and RPL9-mCherry expression in NMuMG cells. C) Comparison of %

FRET values obtained from the following protein pairs in NMuMG cells: RPL9-mCherry and viral Gag (FITC-stained); empty mCherry vector and viral Gag (FITC-stained);

RPL9-mCherry and MMTV Rem-GFP; empty mCherry and GFP vectors.

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Figure 3.6 RPL9 immunoprecipitates with Gag from infected and transfected

NMuMG cells. A) Coimmunoprecipitation was performed with anti-RPL9 antibody or beads only from chronically infected NMuMG cells (Lischka method). Western blotting with anti-CA (left) was performed to detect Gag (77 kD) and the viral cleavage product

CA (27 kD); the same blot was stripped and reprobed (right) to detect RPL9 (21 kD).

The heavy smear in the RPL9 IP lane is from antibody heavy chain; the identity of the higher molecular weight bands is unknown in the RPL9 Westerns. Whole cell lysate lanes are shown to indicate the presence of Gag/CA and RPL9 in lysates at endogenous levels. B) NMuMG cells were transfected with RPL9-FLAG and Gag-GFP or RPL9-

FLAG alone and used for coimmunoprecipitation with anti-GFP antibody (Pierce cross- linking kit). Blots were probed to detect Gag with anti-CA antibody (top) or with RPL9 antibody (bottom).

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Figure 3.7 C-terminal truncations of C3H Gag in context of RPL9-mCherry overexpression. A) Diagram of the C-terminal truncations of Gag made by sequentially deleting the C-terminal domains of Gag and fusing GFP. B) Confocal microscopy images of Gag protein truncations fused to GFP expressed alone in fixed NMuMG cells.

C) The Gag C-terminal truncations fused to GFP coexpressed with RPL9-mCherry in fixed NMuMG cells.

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Figure 3.8 Localization of C3H Gag domains in context of RPL9 overexpression.

A) Confocal microscopy images showing the localization of NC-GFP, p3.p8.n.CA-GFP and CA-GFP in fixed NMuMG cells. B) Expression of the Gag domains fused to GFP in context of RPL9-mCherry overexpression.

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Figure 3.9 Examining the conservation of RPL9-Gag interactions across species with confocal microscopy. A) Yeast RPL9 isoforms A and B were fused to mCherry and overexpressed in MMTV infected cells with immunofluorescence to detect Gag with

FITC conjugated secondary antibody. B) E. coli RPL6 was fused to GFP and overexpressed in infected cells stained to detect Gag with Cy3 conjugated secondary antibody. Images are false-colored. C) Amino acid comparison of RPL9 from yeast

(NCBI, NP_011368, NP_014332) and mouse (NM_011292), and RPL6 from E. coli

(BAE77986). Black boxes highlight sequence similarity among all four sequences, gray boxes outline conserved residues among three species. The vertical red line signifies the division between the N-terminus and the C-terminus of RPL9. The horizontal red line marks the region of the C-terminus with high sequence homology between mouse and yeast sequences. Generated with Geneious Pro 5.1.3 software; www.geneious.com.

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Figure 3.10 Mapping the Gag interaction domain within RPL9. A) Schematic of the

N-terminal and C-terminal domains of RPL9 fused to mCherry. The RPL9 NTD consists of amino acids 1-85, and the RPL9 CTD consists of amino acids 86-192. B) Confocal microscopy of NMuMG cells co-transfected with C3H Gag-GFP and mCherry-fused

RPL9 domains. C) Confocal microscopy of C3H infected NMuMG cells transfected with the mCherry-fused RPL9 domains. Gag is detected with immunofluorescent staining with antibodies to detect CA and FITC-conjugated secondary antibody. D) Graphic of the HIV Rev nucleolar localization signal fused to the N-terminus of mCherry and inserted into the RPL9 CTD-mCherry construct between the RPL9 and mCherry sequences. E) Confocal images of Gag-GFP coexpressed with Rev NoLS-containing constructs.

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Figure 3.11 Further detailed mapping of RPL9 sequences. A) A RPL9 construct missing the last 26 amino acids at the C-terminus was fused to mCherry and transfected into NMuMG cells with Gag-GFP. Cells were fixed and examined via confocal microscopy. Gag-GFP is shown in green, RPL9ΔCTD26 is shown in red, and the overlay of the two channels is shown on the far right. B) Regions of RPL9 bearing putative NLS/NoLS amino acid residues were fused to mCherry singly or in tandem

(NLS1&2). Plasmids were then transfected into NMuMG cells (C) which were fixed and viewed under confocal microscopy. Full-length RPL9-mCherry and empty mCherry vector were included for comparison of localization.

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Figure 3.12 RPL9 is not detected in MMTV virions and equally expressed in infected and uninfected cells. A) Western blot of MMTV strain C3H infected cell lysates and media probed for the presence of RPL9 (left) and then stripped and re- probed to detect Gag (right). The identity of the higher molecular weight bands above

RPL9 was not investigated. Gag is 77 kD, CA is 27 kD, and RPL9 is 21 kD in size. B)

Western blots of whole cell lysates from uninfected and MMTV infected NMuMG cells lysed in RIPA buffer. Protein concentration was determined by Bradford assay and equal amounts of protein were loaded (10 μg or 25 μg) in each lane of the gel. The blot was probed with anti-RPL9 antibody to examine the amount of endogenous RPL9 expression in each cell line.

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Figure 3.13 Model for downstream pathways of RPL9 that may be affected by interactions with MMTV Gag and lead to deregulation of tumor suppressive activities. This model is based off of Table 3.2 that shows cellular proteins predicted to interact with RPL9. NEDD8, a ubiquitin-like protein, is predicted to modify RPL9, and has been shown to have an important role in modifying proteins that regulate p53, including many ribosomal proteins, cullins, and UBC, which are also predicted to interact with RPL9. Several ribosomal proteins that interact with RPL9 (RPL5, RPL11, RPL23,

RPL26, RPS3 and RPS7) are known to play a significant role in p53 regulation by interacting with the p53 negative regulator, MDM2. Many of these RPL9-protein interactions appear to be connected to the p53 regulation pathway, alluding that RPL9 may also participate in tumor suppression through a p53-related mechanism.

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Table 3.2 Potential RPL9 interacting proteins derived from internet protein databases Entrez Gene (http://www.ncbi.nlm.nih.gov/gene), Gene ID 6133; UniProt

(http://www.uniprot.org/), ID P32969; and GeneCards (http://www.genecards.org), ID

GC04M039131 (accessed December 6th, 2010)

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ARFRP2/ARL15 EMG1 PCNA RPL27 RPS15A SNORD55

ARHGEF4 ERCC4 PIAS1 RPL29 RPS16 SNORD68

CUL1 FAM100A PIAS2 RPL30 RPS2 SORBS2/ARGBP2

CUL2 GCN1L1 PRPF3 RPL31 RPS20 SSRP1

CUL3 H2AFX RPL10A RPL35A RPS23 SUMO3

CUL4A HARS RPL11 RPL4 RPS26 TARS

CUL4B HIST2H2BE RPL12 RPL5 RPS27A UBA52

CUL5 KARS RPL13 RPL6 RPS4X UBB

DDB1 MAP3K14 RPL18 RPL7 RPS6 UBC

DHPS MCM4 RPL21 RPL7A RPS7 UBE2MP1

EEF1A1P9 MCM5 RPL23 RPL8 RPS8 YWHAG

EEF1A2 MOCS2 RPL23P8 RPS11 SF3B3 YWHAH

EEF2 NAE1 RPL24 RPS13 SNORD36B

EIF2A NEDD8 RPL26 RPS14 SNORD38B

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Chapter 4: Trafficking and Budding Requirements of the Anomalous Mouse

Mammary Tumor Virus Gag Protein

4.1 Abstract

Mouse mammary tumor virus (MMTV) is an oncogenic betaretrovirus that causes mammary carcinoma in infected mice. Betaretroviruses, such as MMTV and Mason

Pfizer monkey virus (MPMV), differ from other retroviruses in that they assemble particles in the cytoplasm prior to transport to the plasma membrane for egress, as opposed to particle assembly at the plasma membrane concurrent with particle release.

Although there is an abundance of information available about MPMV assembly as the prototypical betaretrovirus, little information is known about the characteristics of MMTV particle formation and release.

MMTV Gag contains putative nuclear import and export signals within the protein sequence that are similar to those previously reported for Rous sarcoma virus (RSV)

Gag, and their role in subcellular targeting were examined. Mutational analysis of putative nuclear trafficking sequences and treatment with Leptomycin B showed no effect on MMTV Gag localization and suggests a CRM1-independent mode of nuclear export. Fractionation assays and expression of Gag truncations indicate that Gag is able to traffic through the nucleus, and most likely uses a nuclear localization signal found within the p8 domain.

Trafficking of GFP-tagged MMTV Gag within the cell was observed via confocal microscopy and was found to be concentrated in discrete cytoplasmic foci, excluding the nucleus. A subset of Gag was found to colocalize with intracellular ribonucleoprotein complexes called P-bodies and stress granules, which serve as key players in mRNA translational control, silencing, storage and degradation. Our colocalization data

121 suggest that these RNA granules may be involved during the formation of new virus particles and represent a novel step in the retroviral assembly pathway.

Additionally, overexpression of MMTV Gag was found to be insufficient for the production of virus-like particle (VLP) formation and release, despite the dogma of Gag serving as the minimal component necessary for particle formation. The roles of Env and RNA targeting signals were also examined with Gag expression but were unable to rescue VLP formation. Thus, further investigation will be needed to determine the minimal components necessary for VLP formation with MMTV Gag. In total, the characterization of MMTV Gag suggests that while it possesses some signals and features common to other retroviruses, the peculiarity of its properties leads to the conclusion that MMTV is unique even among betaretroviruses and offers the prospect of novel discoveries relating to retroviral assembly.

4.2 Introduction

The retroviral Gag polyprotein is considered necessary and sufficient for the assembly of virus particles and serves as the major structural protein of nascent virions

(reviewed in (460). Within its amino acid sequence, Gag possesses multiple signals that mediate packaging of viral RNA into new particles, allow multimerization with other

Gag molecules, and direct Gag to participate in interactions with host factors to traffic throughout the cell and to the plasma membrane. Also, signals within the Gag protein determine if it will assemble particles concurrently with budding at the membrane as a

Type C retrovirus, or if Gag will assemble immature capsids within the cytoplasm prior to membrane targeting via the Type B/D morphogenetic pathway (364). Ultimately, the translation and cellular trafficking of Gag lead to the final release of virions from the host plasma membrane.

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For many retroviruses, what happens to Gag after its translation on cellular ribosomes and before the release of virions at the host membrane is only vaguely known. Historically, it was thought that the progression of Gag from the ribosome to the membrane was a direct path, and somewhere in between the psi packaging element- containing viral RNA was specifically selected and used as an assembly platform

(77,312,402,448). Complex retroviruses rely on virally-encoded proteins to transport the unspliced viral RNA needed for translation and packaging from the nucleus to the cytoplasm, whereas simple retroviruses must exploit alternate nuclear RNA export pathways. However, it is not understood if retroviruses maintain separate pools of translatable and packageable RNA, or if the RNA serves a dual purpose. Somehow,

Gag selects the proper psi-containing viral RNA and carries it to the virion assembly site for packaging.

Recent evidence suggests that a more complicated route for Gag trafficking after translation is possible. For the avian retrovirus Rous sarcoma virus (RSV), which follows a Type C morphogenetic pathway, it was discovered that the Gag protein participates in a nuclear trafficking step prior to arriving at the plasma membrane for assembly. RSV Gag uses nuclear localization signals (NLSs) within its MA and NC domains to exploit the nuclear import pathway of cellular karyopherins, and exits the nucleus via a CRM1-dependent nuclear export signal (NES) located in its p10 domain

(71,383,385). Bypass of the nuclear trafficking step through addition of strong membrane binding signals to RSV Gag leads to the reduction of packaged RNA, which implies the necessity of nuclear trafficking for efficient RNA packaging (154). At least for

RSV, nuclear trafficking of Gag provides a way for the virus to seclude and select its unspliced genomic RNA for packaging.

Although RSV is the only well characterized example of Gag nuclear trafficking in retroviruses, the ability of other retroviral Gag proteins to traffic to the nucleus has been

123 reported. Approximately 18% of murine leukemia virus (MLV) Gag protein is present in the nucleus under steady-state conditions, with additional Gag nuclear accumulation when the N-terminal myristic acid moiety is removed (317). The Gag protein of human immunodeficiency virus (HIV) bears a CRM1-dependent NES within its MA domain that allows for nuclear trafficking (124), while deletion mutants of the HIV NC domain partially localizes Gag to the nucleus (173). Additionally, it has been reported that Type

B/D retroviruses Mason Pfizer monkey virus (MPMV) and foamy virus (FV), and the yeast retrotransposon Ty3, which all form intracytoplasmic particles, have the ability to transiently traffic through the cell nucleus (44,239,387). Despite evidence that these other retroviruses can participate in nuclear trafficking, there have not been continued studies to characterize if mechanisms similar to RSV Gag nuclear import/export are employed.

In conjunction with other studies in our laboratory, the opportunity to study the subcellular trafficking and localization of mouse mammary tumor virus (MMTV) Gag arose. MMTV is a complex Type B retrovirus that causes tumor formation in the mammary glands of infected mice. Although it has served as an important model for understanding the role of oncogenes in human breast cancer, the molecular mechanisms of MMTV infection and virus assembly are poorly understood. Much of what is known about the MMTV Gag protein has been inferred from studies on MPMV, which is a betaretrovirus similar to MMTV. A cytoplasmic targeting/retention signal

(CTRS) has been mapped to the MA domain of MMTV Gag based on similarity to MPMV

(89,362) that targets Gag to the pericentriolar region for cytoplasmic assembly and the

N-terminus of Gag is myristoylated for membrane targeting of assembled core complexes (363) (Figure 4.1). Additionally, MMTV possesses a KR box that is conserved among betaretroviruses and was shown in MPMV to be important for RNA packaging and virus replication (44).

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Beyond these known similarities with other betaretroviruses, ambiguity surrounds

MMTV Gag. It does not have a single specific Gag-Gag interaction domain like that found in MPMV’s p12 domain (481) nor does it possess readily identifiable late domains for budding (330). Upon examination of the MMTV Gag amino acid sequence, we were surprised to find three sequence motifs that are homologous to the NES mapped for

RSV. Moreover, two putative NLSs also exist within MMTV, as reported by Hook et al.

2000 (197). Using our knowledge and skills from previously characterizing the nuclear trafficking of RSV Gag, and driven by the interest to look at a retrovirus following the

Type B morphogenetic pathway, we set out to explore whether MMTV Gag can also participate in nuclear trafficking.

In the development of a GFP-tagged Gag system for live-cell imaging in our nuclear trafficking studies, we came upon two interesting discoveries that prompted further investigation: despite the dogma that retroviral Gag protein is sufficient for virus- like particle (VLP) formation, the expression of MMTV Gag alone is not sufficient for the production of VLPs; and expression of MMTV Gag in cells results in the formation of discrete cytoplasmic foci, presumably sites of Gag assembly, that are reminiscent of cellular ribonucleoprotein structures. Here we report our observations thus far on the intracellular trafficking, localization, and budding requirements for the MMTV Gag protein.

4.3 Results

Examination of MMTV Gag localization under steady-state conditions.

To examine the localization of MMTV Gag protein in transfected mouse mammary epithelial cells, we developed a GFP-fusion at the C-terminus of Gag for live cell microscopy. Upon examination of the MMTV Gag amino acid sequences of two strains, C3H, a highly tumorigenic exogenous virus, and HeJ, a less tumorigenic

125 exogenous virus that is a recombinant of C3H and the endogenous Mtv1 virus (197), we noted that a few of the 14 amino acid differences between the 2 strains lie within the putative NLSs (Figure 4.1). As such changes could alter the localization of the different

Gag proteins, we chose to study both C3H Gag-GFP and HeJ Gag-GFP as representatives of MMTV Gag protein trafficking.

Under steady-state conditions in transfected NMuMG cells, the expression of

Gag-GFP from both C3H and HeJ results in the production of Gag that is distributed throughout the cytoplasm with only small amounts of Gag detected in the nucleus of some cells (Figure 4.2A, top). The foci vary somewhat in size, and the number and intensity of the punctate spots appears to vary with the expression level of Gag protein.

In comparing the expression of HeJ Gag-GFP to that of C3H Gag-GFP, there are no observable differences.

We also examined the GFP-tagged Gag proteins in the presence of other viral proteins to determine if there is an effect on Gag distribution. Gag overexpressed in the stable Env expression NMuMG cell line, Q4 (discussed below) and in C3H chronically infected NMuMG cells, is not altered in its cytoplasmic distribution nor is it distinguishable from Gag-GFP expression alone in normal NMuMG cells (Figure 4.2A, middle and bottom panels). Overexpression of Gag in infected cells (Figure 4.2A, bottom right panel) appears to be somewhat cytotoxic, as very bright green cells have many Gag foci that accumulate in large aggregates near the perinuclear region and the cells do not appear normal in terms of morphology. Though we cannot discern different locations of the Gag foci without suitable markers for cellular compartments, we address what the cytoplasmic Gag foci may be in another section of this report.

Additionally, we examined the location of Gag protein from infected NMuMG cells through immunofluorescent staining with an anti-CA antibody. As it is known that Gag undergoes early proteolytic cleavage within infected cells prior to budding (479), the anti-

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CA antibody would detect Gag as well as free CA that exists within immature particles in the cytoplasm. As shown in Figure 4.2B, Gag and CA within C3H infected cells as well as HP infected cells (which is a low tumorigenicity hybrid MMTV that possesses Mtv1

Gag similar to HeJ) form intracytoplasmic foci that are distributed throughout the cell cytoplasm similar to those seen in Gag-GFP transfected cells. Interestingly, we did not observe accumulations of foci at the plasma membrane indicative of membrane trafficking and budding, as seen previously with RSV (383). Though MMTV forms its immature particles in the cytoplasm as opposed to at the membrane like RSV, MMTV still traffics its particles to the membrane for budding as evidenced by the production of virions that can be collected from infected cell media (Figure 4.9B). However, the kinetics of MMTV assembly and budding is currently not understood, and the lack of particles visualized at the plasma membrane may reflect a very slow trafficking process; the addition of the hormone dexamethasone, which is known to stimulate MMTV production, may increase Gag production and allow for quicker assembly and release of particles.

As mentioned in the introduction, one of our early observations in this study was that the Gag-GFP proteins did not produce VLPs in the media of transfected cells

(Figure 4.8). We therefore questioned whether the GFP tag was negatively affecting its localization within transfected cells. We attempted to produce Gag tagged with smaller peptides, such as HA or FLAG, however those constructs did not yield Gag expression.

Coexpression of Gag-GFP with another fluorophore-tagged Gag, Gag-mCherry, showed similar localization to Gag-GFP, as expected with the similarly-sized fluorescent tag

(Figure 4.2C, top). As we were unable to express Gag with small peptide tags to properly address whether Gag localizes to biologically relevant sites, we next asked whether the Gag-GFP foci were the same as those seen in chronically infected cells.

Understanding that anti-CA antibody will stain both viral Gag as well as Gag-GFP

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(Figure 4.2C, middle panel), we stained Gag-GFP transfected, chronically infected

NMuMG cells. Every cytoplasmic foci containing Gag-GFP will appear both red and green (yellow in overlay) but foci containing viral Gag only would appear red. As shown in Figure 4.2C (bottom panel), nearly every cytoplasmic focus has both red and green associated with it, indicating that Gag-GFP is localizing to the biologically relevant Gag foci of infected cells. We presumed that these Gag foci are sites of Type B morphogenetic Gag assembly, which we examine later in this report.

Lastly, we also examined the localization of Gag-GFP in other cell lines available in our laboratory to determine if cell-type differences affected the localization of Gag under steady-state conditions. Expression of both C3H and HeJ Gag-GFP resulted in foci formation within human embryonic kidney epithelials (293 cells) and mouse 3T3 fibroblasts, which are similar to foci formed from YFP-tagged Gag expressed in QT6 quail fibroblasts (Figure 4.2D). It appears that regardless of the cell type or fluorophore tag used, Gag bears the inherent information to traffic to distinct cytoplasmic sites, and the cellular factors involved in this process are conserved.

Evaluating the nuclear trafficking capabilities of MMTV Gag.

As already mentioned, two putative NLSs exist within the pp21 and p8 domains of MMTV Gag (Figure 4.1). Based on the mapped CRM1-dependent NES of RSV, which contains 4 key hydrophobic residues over a stretch of 11 amino acids, we identified 3 putative NESs within the MA and pp21 domains of MMTV Gag (Figure 4.3A).

NES1, located in MA, has the most striking similarity to the NES of RSV with 7 homologous amino acid residues within the NES motif region and the identical positioning of 4 hydrophobic residues. If MMTV uses the cellular factor CRM1 for nuclear export, the trafficking of Gag will be sensitive to the drug Leptomycin B (LMB) which interferes with the interaction of CRM1 and NES-bearing cargo. Using RSV Gag-

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GFP as a positive control, we added LMB to both C3H Gag-GFP and HeJ Gag-GFP transfected NMuMG cells. As shown in Figure 4.3B, LMB had only modest effects on

MMTV Gag-GFP trafficking (center panels) that were not nearly as dramatic as the complete nuclear entrapment of RSV Gag-GFP (left-most panels). GFP alone was not affected by LMB indicating no contribution of the GFP tag, and no differences were noted for the effects of LMB between the C3H and HeJ Gags.

Considering the abundance of cytoplasmic foci, we questioned whether we would see accumulation of nuclear Gag if the Gag-GFP is stably associated with the cytoplasmic foci. To abrogate the ability of Gag to multimerize, we created a Gag C- terminal truncation that lacks the NC domain. Under steady-state conditions, this alteration of Gag exists in the cytoplasm in a diffuse state, owing to the loss of RNA- binding by NC, which also mediates Gag-Gag interactions (77,312,330) (Figure 4.3B, lower panels). Even in the absence of cytoplasmic Gag aggregates, though, LMB appears to have only minor effects on MMTV Gag localization, with a small amount of nuclear-accumulated Gag visible. Additionally, no major effect of LMB was observed on the Gag of C3H infected MMTV cells that were exposed to varying concentrations of

LMB, including 6x the amount of LMB effective for RSV Gag nuclear accumulation (not shown). Concurrently, we also mutated the first hydrophobic residue of the MMTV

NES1 sequence within C3H Gag-GFP and looked for nuclear accumulation similar to that of the L219A NES mutant of RSV Gag (385) (Figure 4.3A, mutated residues noted within box). Figure 4.3C shows, though, that the L54A mutation of Gag did not abrogate the cytoplasmic localization of Gag. The insensitivity of MMTV Gag to both LMB and putative NES mutation suggests that if Gag traffics through nuclei, it uses a CRM1- indepenent mechanism for export.

Up to this point, the only compelling evidence that MMTV Gag can go to the nucleus is the small amount of Gag visualized in the nucleus of transfected and infected

129 cells, and when the RPL9 protein is overexpressed in the presence of Gag (Chapter 3).

To determine whether Gag can be found in the cell nucleus under steady state conditions without RPL9 overexpression, we fractionated nuclei from transfected cells and assayed for the presence of Gag by Western blotting. Replicable data shown in

Figure 4.4A (upper) indicates that both HeJ and C3H Gag are present in both the cytoplasmic and soluble nuclear fractions of transfected NMuMG cells. These fractions are distinct by detection of calnexin in the cytoplasmic but not the nuclear fractions

(Figure 4.4A, lower). Additionally, the insoluble nuclear pellet obtained in the fractionation was found to contain the cellular protein fibrillarin (Figure 4.4B, lower), and most likely represents isolated nucleoli, substructures of the nucleus. Interestingly, C3H

Gag was detected in more abundance in the insoluble fraction than was HeJ Gag, implying a potential difference in the ability of Gag to localize/accumulate in nucleoli

(Chapter 3). However, this represents a preliminary result, and collection of the insoluble nuclear fraction for this experiment must be repeated for verification.

Additionally, consideration must be taken that the fractionation process may pellet cytoplasmic Gag aggregates with the nuclear pellet, yielding a false-positive detection of

Gag in the nuclear fraction. Use of additional cellular cytoplasmic markers to distinguish the cytoplasmic fraction, a truncation of Gag (GagΔNC) that prevents Gag aggregate formation in the cytoplasm, and confocal microscopy examination of isolated nuclei for the presence of Gag foci can be used to validate the presence of Gag in the nucleus.

Ideally, C3H and HP infected cells would be used for fractionation, to avoid the issue of transfection efficiency, and all cells would have similar levels of Gag expression from the infection. At the time that the fractionation was performed, our laboratory did not yet have the infected cell lines available.

As the fractionation data did not provide convincing evidence of Gag nuclear trafficking, we pursued confocal microscopy experiments to examine whether alterations

130 of the Gag protein lead to nuclear accumulation. A series of C-terminal truncations of both C3H and HeJ Gag fused to GFP were created (Figure 4.5A) and examined for cellular localization. The rationalization for the Gag truncations is that removal of a particular domain may expose a signal that increases or decreases Gag nuclear localization. Two noticeable changes are apparent with the localization of the Gag truncations. As already mentioned the removal of NC disrupts the formation of the Gag cytoplasmic foci, and it also appears to increase the amount of green protein visbile in the nucleus (Figure 4.5C and D). The removal of NC may expose a NLS signal that is normally masked, possibly serving as a way for NC to regulate appropriate nuclear trafficking activities in conjunction with RNA and Gag binding. Secondly, the green protein nuclear localization of transfected cells becomes reduced after the removal of p8 from C3H and HeJ Gag. The p8 domain is a short basic region that possesses one of the two putative NLSs, and the data presented in Figure 4.5C and D supports its role as a functional signal for Gag nuclear localization. The role of the other putative NLS in pp21 could not be examined in the context of this experiment, as the MA,pp21-GFP and

MA-GFP proteins approached the sizes permissive for passive diffusion into the nucleus

(< 50-60 kD). Unfortunately, many of the truncated Gag proteins expressed a high level of free GFP, which complicates interpretation of the green protein localization in the confocal microscopy images (Figure 4.5B). Therefore, further study of these putative

NLSs in isolation from the rest of Gag will determine how functional they are in terms of directing cargo to the nucleus.

Cytoplasmic localization of Gag to ribonucleoprotein complexes.

With the observation that Gag in transfected and infected cells form intracytoplasmic foci, we questioned whether these potential Gag assembly sites localized with constituents of the cellular cytoplasm. The numerous, compact Gag

131 puncta distributed throughout the cytoplasm look similar to ribonucleoprotein complexes called P-bodies (PBs) and stress granules (SGs) that have been observed in yeast and mammalian cells. PBs and SGs are cytoplasmic aggregates of translationally repressed cellular mRNAs and their associated proteins (see section 2.13). Within P-bodies, mRNA can be degraded, stored or recycled for translation, while stress granules serve as an mRNA sorting center, containing ribosomal subunits and stalled preinitiation complexes. Interestingly, studies have shown that Gag proteins and genomic RNA of the yeast retrotransposon Ty3 accumulate in PBs (28), suggesting that retroviral assembly could involve these cellular RNA granules.

To examine if MMTV Gag localizes to PBs and SGs, we obtained a variety of

Myc- and fluorophore-tagged cellular proteins that are known to be associated with PBs,

SGs, or both (listed in Figure 4.6). PB proteins Mov10 and Dcp1a, and SG proteins YB-

1, PABPC1, HuR, and TIA-1 were transfected into C3H infected cells and examined by confocal microscopy for colocalization with MMTV Gag. We observed striking overlap between MMTV capsids and SG markers TIA-1, YB-1, HuR, and PABPC1 (Figure 4.7, left panels). Additionally, the PB marker Mov10 colocalized with capsids while Dcp1a displayed a “docking” phenotype with capsids adjacent to Dcp1a granules (Figure 4.7, white arrow in overlay and inset). Furthermore, overexpression of HuR, TIA-1, YB1, and

Mov10 altered the distribution of the MMTV capsids, inducing larger cytoplasmic aggregates (Figure 4.7, white arrows in green channel images). The PB/SG proteins were also coexpressed in NMuMG cells with C3H and HeJ Gag-GFP to determine if Gag is sufficient for localization to the RNA granules (Figure 4.7, right panels). As seen with the infected cells, Gag-GFP colocalizes with TIA-1, YB-1, HuR, PABPC1, and Mov10, and localizes adjacent to Dcp1a protein accumulations. These observations suggest that MMTV capsids interact with PB/SG machinery during assembly in the cytoplasm,

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and may interact with host factors or viral RNA within PBs/SGs as part of the virus

assembly pathway.

Investigating the requirements for MMTV Gag-GFP VLP production.

Upon developing the MMTV Gag-GFP construct for use in our cell culture system, one of the first questions we asked was whether MMTV Gag could produce

VLPs that are detectable in the cell media. It is considered dogma that the retroviral

Gag protein is necessary and sufficient for the production of VLPs in the absence of all other retroviral proteins (151,460); some prominent examples are simian immunodeficiency virus (SIV) (107), HIV-1 (157,404), and RSV (456). We therefore anticipated that MMTV would produce VLPs with overexpression.

In early experiments with QT6 cells and Gag-GFP expression, RSV- and MMTV

Gag-GFP is readily detectable in cell lysates but only RSV Gag-GFP is detected in VLP pellets collected from cell media (Figure 4.8A). As avian QT6 cells are not the natural host for MMTV infection, we considered the lack of MMTV VLP formation a result of incompatibility with host factors that facilitate assembly.

To investigate whether MMTV Gag can bud from a biologically relevant cell line,

Gag-GFP was overexpressed in mouse mammary epithelial cells (NMuMG cells).

However, no VLPs are detected in the media of mouse cells with Gag overexpression

(Figure 4.8B). Reports in the literature suggest that some retroviruses, such as MPMV , human FV, and bovine FV, need coexpression with Env to enhance particle production

(21,228,397). As all 3 of these viruses form virus particles within the cell cytoplasm similar to MMTV, we proposed that MMTV Gag may also need Env expression to assist in trafficking of immature particles from the perinuclear region to the membrane for budding. However, expression of C3H and HeJ Gag-GFP in stable Env-expressing cells

(NMuMG.Q4) did not produce detectable virus particles in the media (Figure 4.8B). An

133 attempt was made to express MMTV Gag in another mammalian cell line (HeLa cells) in the event that non-mouse cells express or lack a host factor that permits MMTV Gag budding. Even when MMTV Gag expression was greater than that of HIV Gag-GFP and

RSV Gag-GFP in HeLa cell lysates (Figure 4.8C), MMTV VLPs were not produced. HIV and RSV VLPs were easily detectable in VLP pellets collected from transfected HeLa cell media, indicating that the MMTV Gag protein is incapable of either producing and/or releasing VLPs from transfected cells.

In Figure 4.2, it appears that Gag-GFP localizes to the same cytoplasmic foci that viral Gag does, yet Gag-GFP does not yield VLPs. We next consider the possibility that

Gag-GFP protein is not able to assemble into virions or virus-like particles in the cytoplasm. Additionally, GFP-tagged Gag is not detected in virions collected from Gag-

C3H infected NMuMG cells transfected with Gag-GFP, indicating that the tagged Gag is not incorporated into virus (not shown). An issue we have had throughout our MMTV studies is that the transfection efficiency of NMuMG cells is not ideal (~20%), resulting in a pool of cells that do not homogenously express Gag and possibly hindering detection of small amounts of VLPs. To evaluate this further, we created a selected Gag-GFP expressing cell line by transfecting NMuMG cells and sorting for Gag-GFP-positive cells by flow cytometry. The sorted cells were then grown in cell culture with G418 selection to maintain the GFP plasmid. As shown in Figure 4.9A, the sorting/selection process yielded a population of cells in which the majority were green and had the appearance of cytoplasmic foci as expected for Gag-GFP expression.

With a population of Gag-GFP expressing cells, we asked again whether VLPs are present in the cell media. One immediate observation was that despite loading similar cell-equivalents of lysate in the gel for Western analysis, MMTV infected cells produce larger amounts of viral protein than the selected Gag-GFP cells (Figure 4.9B).

Furthermore, the media of 6-confluent 100 mm plates was collected and pooled from the

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Gag-GFP cells, yet no VLPs are detectable. Virus collected from a single 100 mm plate of confluent C3H infected cells is more than enough to be detected on the Western blot with anti-CA antibody.

To determine whether Gag-GFP is assembly competent, we examined the selected Gag-GFP cells under electron microscopy to look for the presence of immature capsids in the cytoplasm. Figure 4.10 (top row) shows that VLPs are indeed formed in the cytoplasm of Gag-GFP expressing cells and are associated with intracellular vesicles and membranes. However, these VLPs are difficult to find in the Gag-GFP cells, and exist only as sporadic single particles. In comparison, C3H infected cells were also imaged and found to contain an abundance of immature capsids in large groups near intracellular membranes (Figure 4.10, bottom row). These data along with the confocal microscopy suggest that Gag-GFP localizes to the right intracellular site for assembly and may form immature capsids similar to infected cells, but something is inherently wrong with Gag-GFP in that the capsids are not stable or able to be assembled properly.

At this point, we assume that unlike HIV and RSV, MMTV Gag could not be fused to

GFP and result in VLP production. Expression of an “untagged” Gag was attempted by removal of GFP from the Gag-GFP DNA construct, however the resulting CMV-driven

Gag protein was not expressed in transfected cells, as determined by confocal microscopy and Western analysis (not shown). Therefore, another strategy was pursued.

Production of MMTV Gag from DNA constructs bearing RNA transport signals.

In addition to creating a tagless form of MMTV Gag for expression and testing

VLP production, we created several constructs of MMTV Gag bearing a variety of RNA export signals. Our rationale is based on the findings of Swanson et al. that found retroviral assembly to be linked to the pathway that viral RNA uses for export from the

135 nucleus (412). Though it is obvious that Gag-GFP mRNA is exported and translated in transfected cells (protein is detected by Western blotting and confocal microscopy), it is possible that the RNA is not directed to the proper location for Gag translation and priming of the assembly process. Based on the constructs used by Swanson et al., we cloned MMTV Gag constructs that possess 4 copies of the MPMV constitutive transport element (CTE), a copy of the HIV Rev response element (RevRE), or no specific RNA signal (delta) (Figure 4.11). In addition, we created Gag-GFP constructs bearing the endogenous MMTV RNA transport sequences. As a complex retrovirus, MMTV encodes the regulator of export/expression of MMTV mRNA (Rem), a Rev-like protein which mediates nuclear export of the unspliced viral genomic RNA bearing the Rem response element (RemRE) (206,289). In addition to cloning the RemRE into our constructs, we also cloned the MMTV 5’ untranslated region (UTR), which carries the psi-packaging sequence of MMTV genomic RNA that allows specific selection of the viral RNA for packaging into virus particles (Figure 4.11).

Expression of the MMTV Gag proteins from the RNA transport signal-bearing plasmids was first examined with transfection and confocal microscopy of NMuMG cells.

All of the constructs yielded Gag protein that localized in cytoplasmic foci indistinguishable from Gag-GFP and viral Gag (Figure 4.11, right panels).

Subsequently, expression was examined by Western blotting for the presence of Gag protein in cell lysates and VLPs in the media. As shown in the preliminary data presented in Figure 4.12 (upper) (needs to be replicated), every DNA construct produced detectable levels of Gag protein, though in varying amounts. In the absence of specific RNA targeting signals, Gag-Δ has the lowest level of expression. The addition of the RevRE alone to Gag seemed to increase Gag production, while pairing

RevRE with transfection of Rev increased Gag production perceptively. A higher level of

Gag production was also achieved with Gag from RNA bearing the MPMV 4xCTE.

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Interestingly, the Gag-GFP constructs with or without endogenous viral RNA transport signals had the best expression levels, though they are driven by a CMV promoter similar to the untagged Gag constructs. Regardless of the added RNA transport sequences, no Gag proteins indicative of VLP production were detected in the media samples (Figure 4.12 bottom). As mentioned, this data is preliminary in that the experiment needs to be replicated. However, the information collected thus far appears fairly convincing that the addition of RNA transport elements does not contribute to Gag budding. For the time being, the search continues for the sufficient components of

MMTV needed for the production and release of virus-like particles.

4.4 Discussion

After searching the literature on MMTV, we have found this study to be the first to examine the localization of MMTV Gag protein in transfected or infected cells. While the results of this study are a step towards understanding the MMTV Gag protein, we are still left with more questions than answers, and the investigation of Gag trafficking and budding is far from over. Our current data suggest that MTMV Gag possesses the capability to traffic into and out of the cell nucleus, but what purpose would this serve to the virus? One possibility, based on studies with RSV, is that Gag traffics to the nucleus to capture its unspliced RNA for packaging into newly formed virions. The field of virus assembly concerning the location and timing of retroviral genomic RNA packaging is still in its infancy. It is not certain whether retroviral RNA serves the dual role of translation template and genome, or if separate pools of RNA are maintained for these functions.

MMTV is a complex retrovirus that encodes the Rem protein for export of its unspliced

RNA to the cytoplasm for translation, but that does not exclude the possibility that MMTV

Gag may transverse the nucleus for capture of the genome for packaging. Once we are able to abrogate the nuclear trafficking of MMTV Gag, we will be able to further delineate

137 if RNA plays a role. It is also possible that Gag uses nuclear trafficking signals to target cellular proteins in the nucleus that play a role in assembly or pathogenesis. Use of

LMB and mutation of a hydrophobic residue within a putative MMTV NES suggest that unlike RSV, MMTV does not use a CRM1-dependent mechanism for nuclear export.

One possibility for MMTV export is the use of β-catenin, a non-conventional nuclear translocating protein that does not rely on the traditional importin α/β pathway for nuclear entry and uses a CRM1- and -independent mechanism for nuclear export (128,459).

Interestingly, β-catenin is activated by the cellular Wnt signaling pathway (reviewed in

(348), one that is commonly altered by MMTV insertional mutagenesis (74,75,321).

With the small amount of Gag accumulation observed in the nucleus of transfected and infected cells, and data from the fractionation assays, it is probable that

MMTV Gag undergoes a nuclear step at some point in the assembly of virions. Further examination of the two NLSs of MMTV Gag, perhaps fused to an unrelated cargo protein, will demonstrate the ability of these signals to relay nuclear trafficking capabilities and allow for mutagenesis to be performed within these signals of Gag.

Additionally, alteration of signals such as the CTRS may create an accumulation of Gag in the nucleus that is no longer targeted to its cytoplasmic assembly site. Once a method to alter the nuclear translocation of Gag is obtained, we can begin to understand how this alternate trafficking step of MMTV Gag plays a role in the virus life cycle and assembly. It is also possible that the nuclear localization signals may serve to target the preintegration complex (PIC) to the nucleus during the early steps of viral infection, and the localization of full-length Gag during virus assembly is a vestigial phenomenon.

This report is also the first to indicate a role of P-bodies and stress granules in the assembly pathway of MMTV Gag. In both transfected and infected cells, MMTV Gag colocalizes with a subset of PB and SG proteins, though direct protein-protein

138 interactions have yet to be examined. The dynamic nature of the PB/SG ribonucleoprotein complexes provides a tempting notion that Gag may use these structures to rendezvous with its genomic RNA, away from translation machinery, and begin the assembly process with other Gag molecules- an idea already proposed by leaders in the PB/SG field (27). Again, the location of the viral RNA would need to be examined for its ability to traffic to the cytoplasmic RNA granules, and a fluorophore based RNA tracking system for following the course of viral RNA is currently being developed in our laboratory.

MMTV is not the first retrovirus to be implicated with PBs and SGs; as already mentioned, early studies showed that the yeast retrotransposon Ty3 Gag and RNA localize to PBs for assembly (28). Since then, subsequent communications in the literature have tied the life cycles of retroviruses and other viruses to the proteins of cytoplasmic RNA granules. More recently, the host defense protein APOBEC3G that is packaged in retroviral virions was found to be localized to PBs (153,458), and HIV replication is inhibited by PB factor Mov10, which is also packaged into virions (67).

Furthermore, factors involved with RNA-induced silencing associate with PBs and SGs and are linked to endosomal-sorting-complex-required-for-transport (ESCRT) proteins that participate in virus budding (158,246). Considering the current surge of interest in the PB and SG field and the many cellular pathways involved, the early data presented here provides the impetus to continue work with MMTV Gag and RNA granules. This project has been inherited by another graduate student in our laboratory who will use drugs to ablate or induce PBs and SGs, perform coimmunoprecipitations to look for protein interactions between Gag and PB/SG proteins, examine the trafficking of MMTV

RNA with a fluorophore tagging system, and attempt siRNA knockdowns of PB and SG components to look for an effect on MMTV assembly.

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Lastly, the question lingers as to what components of the virus along with Gag

are needed for the formation of VLPs, and several avenues of possibilities have yet to be

explored. First, we have yet to examine the expression of Gag without GFP in the

presence of Env, which may assist in the stable production of particles once the GFP tag

is removed. An early study in 1993 alluded that interactions between Env and Gag in

the cell cytosol were important for membrane trafficking and release of viral proteins

(214); the data from this study imply that Env may be necessary to mediate proper VLP

budding but this has been impeded in our studies by the GFP tag on Gag. We are also

very curious to examine cells that overexpress the Gag constructs without GFP by

electron microscopy to see if VLPs can form in the cytoplasm. If particles are not seen,

that would indicate that something is intrinsically wrong with the Gag molecule itself and

that the addition of the GFP tag is not to blame. Conversely, if VLPs are observed in

abundance, an additional factor may be needed to spur migration to the membrane for

budding and release.

One thing that we have considered is whether MMTV Gag is myristoylated at its

N-terminus with expression of our Gag DNA constructs. The mammalian cells that we

perform our studies in are equipped to handle the co-translational modification, and it is

difficult to fathom that every Gag construct we have tested is defunct for myristoylation.

During the characterization studies of Gag, a Gag-GFP construct bearing a Gly to Ala

substitution at the second residue of Gag was created to produce a Myr- protein. The construct expressed poorly in cells, but those cells with Myr- Gag-GFP looked identical to wildtype Gag-GFP expression, complete with cytoplasmic foci (not shown). The lack of alteration in the localization of Myr- MMTV Gag-GFP is not terribly surprising, as the

removal of myristoylation from MPMV Gag also had no effect on Gag localization in the

cytoplasm, although it did affect the ability of MPMV Gag to bud (363). As we are able

140 to detect some capsid assembly by EM in Gag-GFP expressing cells, the issue may be that they are not able to be directed to the membrane efficiently for budding to take place. While expression of Env may assist this membrane trafficking as shown with

MPMV and FV (21,228,397), we are currently creating a Gag construct bearing the strong membrane binding sequences of the Src gene. The addition of Src sequences has been used previously to target the RSV Gag protein to mammalian cell membranes to promote budding (461). We will be interested to see if forcing Gag to the membrane by Src signals will rescue Gag budding so that we may further address the sufficiency of

Gag in VLP egress.

An additional factor to consider with the inability of MMTV Gag to yield VLPs is that the budding process may be inhibited by a cellular factor. A prominent example is the host defense mechanism of tetherin, which “tethers” virus particles to the host cell surface and prevents their release (318). It has been shown that tetherin exhibits broad- spectrum effects on the VLP formation of Gag proteins from a number of retroviruses, including the betaretrovirus MPMV (212). HIV-1 circumvents tetherin by expression of the virally-encoded Vpu protein (318), and other retroviruses use alternate viral protein proteins such as Env and Nef to mediate Vpu-like anti-tetherin effects (reviewed in (381).

Though no VLPs were noticed in EM images as being “stuck” on the membrane of Gag-

GFP expressing cells, and no Vpu-like sequences are found within the MMTV genome

(personal survey), the possibility of tetherin-mediated inhibition of MMTV VLP release cannot be dismissed. We have acquired a Vpu expression vector and intend to coexpress it with our Gag constructs in NMuMG cells to determine if particle release is induced. Should Vpu restore Gag budding, it will be exciting to map the MMTV sequences responsible for anti-tetherin activity.

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One last consideration for budding of MMTV Gag is that we simply do not have

enough Gag expression to meet threshold levels needed for assembly and release. As

shown in the EM images of Figure 4.10, few immature capsid structures were noticed in

Gag-GFP expressing cells. Additionally, Western blots included in this chapter show

that transfection of Gag constructs into cells do not amount to the levels of expression

seen in lysates of infected cells, even in the selected Gag-GFP cells (Figures 4.9 and

4.12). In future experiments we may need to address this with use of higher DNA

concentrations in transfections, better methods that increase efficiency of transfection, or

with the development of a Gag construct using the native viral promoter that is sensitive

to dexamethasone stimulation of expression. With continued exploration of MMTV Gag

in terms of its budding and localization, we hope to unveil the molecular mysteries

surrounding this unique retrovirus that has evaded study for so long.

4.5 Materials and Methods

Plasmids

pRSV Gag-GFP (73), pRSV Gag L219A-GFP (384), and pMyc-Mov10, pMyc-

PABPC1, pMyc-HuR, pMyc-YB-1, pmRFP-DCP1a and pYFP-TIA-1 (153) were made as

previously described. pGag-GFP was created by PCR amplification of Gag from MMTV

strains C3H and HeJ (template DNA from T. Golovkina) and insertion into pEGFP.N2

(Clontech) using BamHI-HindIII; 21 nts of the upstream region of Gag were included in the pGag-GFP constructs, flanked by an introduced upstream EcoRV screen site. pmCherry.N2, was made by replacing GFP in pEGFP.N2 (Clontech) with mCherry from pRSet8.mCherry (399). pMMTV C3H Gag-mCherry was made by amplification of C3H

Gag and insertion into the pmCherry.N2 vector via the HindIII/ApaI sites. pGag-YFP

was created by PCR amplification of Gag from MMTV strains C3H and HeJ and insertion

into the HindIII/BamHI sites of YFP.N2, which was made amplifying YFP from YFP.C1

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(Clontech) and swapping with GFP from pEGPF.N2 at the BamHI/BsrGI sites. GFP- fused truncations of MMTV C3H and HeJ Gag ΔNC (1-1485), ΔCA.NC (1-804),

Δn.CA.NC (1-753), MA.pp21.p3 (1-681), MA.pp21 (1-582), and MA (1-294) were PCR

amplified at the noted nucleotides of gag (primers available upon request) and ligated

into BamHI-HindIII of pEGFP.N2. QuickChange site-directed mutagenesis (Stratagene)

was used to create pMMTV C3H Gag L54A-GFP, which has an alanine at position 54 of

Gag-GFP in place of lysine. pMMTV C3H Gag Myr--GFP was made using a forward

primer to amplify Gag containing mismatched nucleotides to introduce a Gly to Ala

mutation at position 2 of Gag within pC3H Gag-GFP. pMMTV C3H 5’ UTR Gag-GFP

was created by amplifying the 5’ R and U5 regions of MMTV C3H and insertion into the

BglII/EcoRV sites of pGag-GFP, and using QuickChange mutagenesis (Stratagene) to

remove the aforementioned EcoRV screen site. pMMTV C3H 5’ UTR Gag-GFP RemRE

was made by amplification of the MMTV Rem response element from C3H infected

NMuMG genomic DNA (QIAGEN DNeasy tissue kit) and insertion into the NotI site downstream of GFP in pMMTV C3H 5’ UTR Gag-GFP; clones were screened for directionality with BamHI. pRem-mCherry was made by amplification of Rem from pGFP-Rem (289) and cloned into the HindIII/SalI sites of pmCherry.N2. pMMTV C3H

GagΔ, Gag-RevRE, and Gag-4xCTE were created by PCR amplification of C3H Gag

and insertion into the pHIV GPV-Δ (412), GPV-RevRE (412), and GPV-4xCTE (465) constructs (gifts from M. Malim), respectively in place of the HIV sequences using the

HindIII/EcoRI restriction sites. pRev-YFP was made by amplifying Rev from pCMV-Rev

(264,265) and cloning into SalI-ApaI of pEYFP.N2. All created plasmids were sequenced and shown to be faithful copies of the corresponding genes.

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Cell culture

NMuMG (normal mammary gland; ATCC CRL-1636) cells, MMTV strain C3H

(125,213) chronically infected NMuMG cells (gift from T. Golovkina), proviral HP stably-

transfected infected NMuMG cells (gift from T. Golovkina) (413), NMuMG stably

expressing MMTV Env (NMuMG.Q4, gift from S. Ross) (217), and mouse 3T3 cells

(ATCC CRL-1658) were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin, streptomycin and Amphotericin B (HyClone). HeLa cells (ATCC CCL-2) and

293 cells (ATCC CRL-1573) were maintained in Dulbecco’s media supplemented with

5% fetal bovine serum, glutamine, penicillin, streptomycin, sodium bicarbonate and

Amphotericin B. NMuMG, HeLa, 293, and 3T3 cells were transfected using

Lipofectamine 2000 (Invitrogen) according to manufacturer’s directions. Quail fibroblast

(QT6) cells were cultured as previously described (100) and transfected via the calcium phosphate method. Leptomycin B (LMB) drug (Sigma) was added to culture dish media for a final concentration of 8.14 nM and incubated with the cells for 1.5 hr prior to imaging.

Production of the selected C3H Gag-GFP NMuMG cell line

After transfection with pC3H Gag-GFP, NMuMG cells were grown and passaged for 1 week with G418 selection (1 mg/mL). Cells were then washed in phosphate buffered saline (PBS) and resuspended in clear DMEM prior to cell sorting on a MoFlo high performance 6-color cell sorter (Cytomation). Nearly 5 x 107 cells were sorted using gated parameters to select live, single, green cells. Approximately 3 x 105 cells were

selected and collected in sterile tubes. Three-quarters of this sample was pelleted and

used immediately for EM sample preparation, while the other quarter was cultured in

NMuMG primary growth media with G418 selection. After culturing cells for several

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weeks, the selected Gag-GFP cells were grown on 60 mm Permanox dishes (EM

Sciences) and used for additional EM sample preparation.

Sample preparation for electron microscopy

Cell pellets or cell monolayers grown on a Permanox dish were washed in 0.1 M

sodium cacodylate (pH 7.4) and fixed in 4% paraformaldehyde-0.5% glutaraldehyde for 1 h at 4°C. Cells were postfixed in 1% osmium tetroxide-1.5% potassium ferrocyanide

overnight at 4°C, washed in 0.1 M sodium cacodylate, and serially dehydrated in ethanol.

Cell pellets or monolayers were embedded in Epon 812, thin sectioned, stained with

uranyl acetate and lead citrate, and viewed with a JEOL JEM1400 Digital Capture

Transmission Electron Microscope (TEM). Micrographs were captured with an Orius

SC1000 side mounted CCD camera and DigitalMicrograph software.

Confocal microscopy and Immunofluorescence

Cells were seeded in 35-mm dishes containing #1.5 glass coverslips, fixed in

3.7% or 4% paraformaldehyde, washed in PBS, permeabilized with 0.1% Triton X-

100/PBS and blocked with 3% bovine serum albumin or nonfat dry milk in PBS. Primary antibodies used were mouse anti-MMTV CA (353) and rabbit anti-Myc (Abcam). After washing in PBS, coverslips were stained with sheep anti-mouse IgG-Cy3 (Sigma), goat anti-mouse IgG-FITC (Sigma), or sheep anti-rabbit IgG-Cy3 (Sigma). Cell nuclei were stained with DAPI, and SlowFade reagent (Molecular Probes) was used for mounting coverslips. Live cell imaging was performed by growing cells on glass-coverslip bottom culture dishes (MatTek Corp) and imaging with colorless DMEM or tris buffered saline

(TBS). Microscopy samples were examined using a Leica AOBS SP2 confocal microscope with excitation of GFP at 488 nm, mRFP and Cy3 at 543 nm, and YFP at

514 nm. ImageJ was used to false-color cell images for overlay comparison.

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Virus Collection and Western blot

Virus or virus-like particle samples were collected from the media of confluent

cells and spun through a 25% sucrose pellet for 1 hour at 27,000 rpm in a Beckman

SW41 rotor at 4 C or for 45 min at 55,000 rpm in a Beckman TLA100.4 rotor at 4 C.

Virus pellets were resuspended in 2x SDS-loading dye. Cell lysates were prepared by

directly lysing cells in boiling 2x SDS-loading dye, or lysing cells in RIPA lysis buffer

followed by a Bradford assay and addition of 2x SDS-loading dye. Protein from lysates

and pellets were separated on 12% SDS PAGE and transferred to PVDF membrane.

After blocking with 5% nonfat dry milk in TBS-T, blots were probed with primary antibody

to detect CA/Gag (353), MMTV Env protein gp52 (gift from T. Golovkina), fibrillarin

(Abcam), calnexin (Stressgen) or GFP (Abcam, Roche), and secondarily probed with

HRP-linked mouse or rabbit IgG antibodies (Sigma) before detection with enhanced

chemiluminescence (Thermo Scientific, GE Healthcare).

Cell fractionation

NMuMG cells were transfected, washed, scraped from the plates with PBS, and

pelleted with a low speed spin. Cells were resuspended in a low salt lysis buffer (10 mM

Tris pH 7.4, 3 mM CaCl2, 2 mM MgCl2, 0.5% NP-40 and Sigma protease cocktail) and passed 12 times through a 25 gauge needle. Nuclei were harvested at 14,000x g for

20s at 4 C. The supernatant was collected as the cytoplasmic fraction, and the pelleted nuclei were lysed in high salt lysis buffer (PBS, 0.5 M NaCl, 0.5% NP-40 and Sigma protease cocktail) with end-over-end rotation at 4 C for 40 min. The insoluble nuclear pellet was removed with a 30 min spin in a microfuge at full speed at 4 C, and the supernatant was collected as the soluble nuclear fraction.

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4.6 Acknowledgements

We would like to thank the following scientists for their contribution of reagents to this project: M. Malim, King’s College London; T. Golovkina, University of Chicago; J.

Dudley, University of Texas at Austin; S. Ross, University of Pennsylvania; R. Tsien,

University of California at San Diego; and B. Cullen, Duke University. We would also like to thank John Wills for discussion of retroviral budding and Vpu, Roland Myers for

EM sample preparation, Nate Scheaffer for flow cytometry assistance, and the Penn

State College of Medicine Microscopy, DNA sequencing, and Flow Cytometry core research facilities.

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Figure 4.1 The signals and putative motifs of MMTV Gag. MMTV Gag consists of the MA, CA, and NC domains that all retroviruses possess, as well as the pp21, p3, p8, and n domains which are not well characterized. These are arranged in the 5’ to 3’ order of MA, pp21, p3, p8, n, CA, and NC, as shown in the top figure. MMTV Gag is myristoylated at its N-terminus, which assists in targeting of assembled immature cytoplasmic particles to the membrane (blue zig-zag on MA domain). The sequences of both the C3H (highly tumorigenic) and the HeJ (less tumorigenic) strains of MMTV Gag are shown below, illustrating the 14 amino acid differences (highlighted in red) between the Gags that confer a variation in MMTV tumorigenicity (197). The putative late domain sequences, which are involved in host factor recruitment for virus particle budding, are highlighted in orange (330), the cytoplasmic targeting/retention signal (CTRS) is denoted in purple (89,362), and the putative nuclear localization signals (NLSs) are underlined in red (197). Underlined in blue are the putative nuclear export signals (NESs) that we delineated based on sequence similarity to the mapped RSV NES (385). Also marked in the CA domain are the major homology region (MHR, pink) that is conserved among all retroviruses and the two zinc finger binding domains (Cys-His boxes, blue) responsible for selecting the psi-containing viral RNA for packaging into virions.

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Figure 4.2 Visualization of MMTV Gag in cells under steady-state conditions by confocal microscopy. A) Transfection and expression of C3H Gag-GFP and HeJ Gag-

GFP in live normal mouse mammary epithelial cells (NMuMG cells), in live NMuMG cells that stably express the MMTV envelope protein (NMuMG.Q4), and in fixed C3H infected

NMuMG cells. B) Localization of viral Gag in fixed chronically infected C3H NMuMG cells (left) and in the fixed stably transfected-infected HP proviral NMuMG cell line. The

HP provirus contains the Gag protein from the endogenous Mtv1 virus, similar to that of the HeJ strain of virus. C) Colocalization of Gag proteins in live NMuMG cells transfected with both Gag-GFP and Gag-mCherry (top row), in fixed NMuMG cells expressing Gag-GFP and immunostained for the detection of CA (middle row), and in fixed C3H infected cells transfected with Gag-GFP. Colocalization is noted in the overlay channel as yellow coloring. D) Expression of YFP fluorophore tagged MMTV

C3H- and HeJ Gag in live quail fibroblasts (QT6), and GFP tagged MMTV Gags in live human embryonic kidney cells (293) and in live mouse 3T3 fibroblasts.

Immunofluorescence was used to visual Gag in fixed cells by staining with anti-CA antibody followed by FITC or Cy3 conjugated mouse secondary antibody. Blue coloring in the images represents cell nuclei as stained with DAPI.

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Figure 4.3 Examination of the CRM1 nuclear export capabilities of MMTV Gag. A)

Amino acid residues comprising the mapped nuclear export signal (NES) of RSV Gag compared to the 3 putative NESs that we found within the MMTV Gag sequence. NES1 and NES2 lie within the MMTV Gag MA domain, and NES3 is within pp21. Underlined

MMTV residues are conserved with residues found within the RSV NES region. The red amino acids within the RSV sequence are hydrophobic residues known to be important for CRM1 dependent nuclear export activity. Corresponding hydrophobic amino acids are denoted in red within the MMTV sequences. The black box outlines the two leucine residues that were mutated to alanine to create the RSV NES mutant,

L219A, and the MMTV L54A construct that was tested for nuclear export alteration. B)

Leptomycin B (LMB) treatment of live NMuMG cells expressing RSV Gag-GFP, MMTV

C3H Gag-GFP, MMTV HeJ Gag-GFP, and GFP.N2, as well as two truncations of MMTV

Gag that remove the C-terminal NC domain (C3H GagΔNC-GFP and HeJ GagΔNC-

GFP). The rows marked as LMB- did not receive any treatment, while the LMB+ rows had the drug added to the cell media for 1.5 hours prior to imaging on the confocal microscope. C) Comparison of the nuclear restricted RSV mutant Gag L219A-GFP in live QT6 cells with MMTV Gag L54A-GFP in live NMuMG cells.

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Figure 4.4 Nuclear fractionation of NMuMG cells transfected with Gag-GFP constructs. Following the separation of the cytoplasmic-, the soluble nuclear-, and the insoluble nuclear cell fractions, proteins were analyzed by SDS PAGE and Western blotting. A) Detection of C3H and HeJ Gag-GFP in the cytoplasmic and nuclear fractions of transfected cells with anti-GFP antibody. To distinguish the nuclear versus the cytoplasmic fractions, the blot was stripped and probed for the presence of the cytoplasmic protein calnexin, which should not appear in nuclei (lower blot). Equal concentrations of nuclear proteins and cytoplasmic proteins were not loaded and should not be cross-compared for protein band intensity, though equal volumes of similar samples (i.e. C3H Gag nuclear and HeJ Gag nuclear) were loaded. B) Detection of C3H and HeJ Gag-GFP in the insoluble nuclear fractions of transfected cells by Western blotting with anti-CA antibody. The same blot was stripped and reprobed with antibody to fibrillarin, a protein found within nucleoli, to show similar protein concentrations of loaded samples.

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Figure 4.5 Expression and localization of MMTV Gag truncations. A) Diagram illustrating the C-terminal truncations of Gag that were made and fused with GFP. B)

Western blot of cell lysates from transfected NMuMG cells expressing GFP-fused C3H and HeJ Gag truncations. Anti-GFP antibody was used to probe the blot; free GFP appears at 27 kD. C) Confocal microscopy of live NMuMG cells expressing GFP-fused

C3H Gag truncations. D) Confocal microscopy of live NMuMG cells expressing GFP- fused HeJ Gag truncations.

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Figure 4.6 Chart of P-body and stress granule proteins used in this study. The protein name, site of primary localization, and known/putative functions are listed, followed by a confocal image of protein expression in fixed NMuMG cells.

Immunofluorescent staining was performed to detect myc-tagged proteins with primary antibody to Myc followed by Cy3-conjugated secondary antibody. Proteins that are found in both P-bodies (PBs) and stress granules (SGs) are marked to denote the primary localization in SGs by SG>>PB.

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Figure 4.7 Colocalization of MMTV Gag with PB and SG markers. The three left- most columns represent the expression of PB/SG markers (red) in C3H infected

NMuMG cells (Gag in green). The overlay channel shows overlap of the PB/SG protein and Gag protein signals (yellow). White arrows in the green channel indicate places where Gag protein is relocalized to larger cytoplasmic aggregates concurrent with

SG/PB expression. A white arrow in the overlay image of Dcp1a indicates the location of the inset image. Viral Gag protein is visualized with immunofluorescence using anti-

CA antibody followed by FITC- or Cy3-conjugated antibody, and Myc-tagged proteins were stained with anti-myc antibody followed by Cy3-conjugated antibody. Images of

TIA-1 expression in infected cells are false colored. Coexpression of PB/SG markers with GFP-tagged Gag proteins (C3H and HeJ) comprise the two right-most columns; only overlay images are shown, with the PB/SG markers in red and the Gag-GFP proteins in green. Immunofluorescence was used as before to detect the myc-tagged proteins.

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Figure 4.8 Examination of collected media from Gag-GFP transfected cells for the presence of virus-like particles (VLPs). All three parts of this figure represent

Western blots on the lysates and media of cells. A) Western blot of lysates and media from quail fibroblasts expressing RSV Gag-GFP, MMTV C3H Gag-GFP, MMTV HeJ

Gag-GFP, or no transfection (QT6). The blots were proved with anti-GFP antibody.

Protein concentrations of samples were not determined. B) Expression of MMTV C3H and HeJ Gag-GFP in normal NMuMG cells and in NMuMG cells stably expressing the

MMTV Env protein (NMuMG.Q4). Blots were probed with anti-CA antibody. The narrow blot on the far right shows the expression of MMTV gp52 Env protein in NMuMG.Q4 lysates. Protein concentrations of samples were not determined. C) Western blot of lysates and media collected from HeLa cells transfected with HIV Gag-GFP, RSV Gag-

GFP, and MMTV C3H Gag-GFP. Blots were probed with anti-GFP antibody to detect

Gag-GFP of all 3 species. Equal amounts of lysate protein were loaded (50 μg). Red asterisks indicate the primary Gag bands in each lane. Untransfected HeLa lysates and media were included as a negative control.

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Figure 4.9 Expression of C3H Gag-GFP in selected Gag-GFP NMuMG cells. A)

Confocal microscopy showing the presence of Gag-GFP in cells that were sorted by flow cytometry. The zoom out shows a larger number of green cells, while the zoom in shows the characteristic cytoplasmic foci of Gag-GFP expression. DAPI was used to stain nuclei of the fixed cells. B) Western blots of lysates and media collected from the selected Gag-GFP cells. Similar amounts of lysate protein samples were loaded and

C3H infected cells were used as a positive control for virus release into the media. The two images represent the same blot at a lighter (left) and a darker (right) exposure after

ECL development. The viral proteolytic cleavage products are indicated in the C3H infected cell samples. Of note, media was collected from one confluent 100 mm dish of infected cells, while media was collected and pooled from 6x 100 mm dishes of selected

Gag-GFP cells.

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Figure 4.10 Transmission electron microscopy images of stained, thin-sectioned

NMuMG cells. Selected Gag-GFP cell images are shown on the top row and C3H infected NMuMG cells are shown on the bottom. Scales are noted for each individual image. The far-right images are noted with C (cytoplasm), N (nucleus), and NM (nuclear membrane) to illustrate VLPs/virus particles adjacent to the cell nuclear membrane.

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Figure 4.11 Confocal microscopic visualization of cells transfected with C3H Gag

constructs bearing various RNA transport elements. The black and white box/line

drawings represent the promoters, genes, and RNA transport sequences of the created

DNA constructs. The gray boxes indicate the ORF/protein product. The promoter of the

cytomegalovirus (CMV) is used for all constructs, while the polyadenylated tail

sequences of either bovine growth hormone (BGH) or herpes simplex virus thymidine

kinase (HSV TK) were used. Confocal images of the constructs expressed in NMuMG

cells are shown on the right; all cells are fixed, with the GFP-less proteins stained by

immunofluorescence using anti-CA antibody and Cy3-conjugated secondary antibody.

Gag-Δ is the simplest construct, with no tags on Gag or added downstream RNA

signals. Gag-RevRE possesses the Rev response element (black knob) downstream of

the gag sequence, and is identified by the in trans expression of HIV Rev protein. Gag-

4xCTE bears 4 copies of the Mason Pfizer monkey virus constitutive transport element

(CTE, dark gray knobs) located downstream of the MMTV gag sequence. Next, the

Gag-GFP construct that is used throughout this report is shown, with the C-terminal fusion of GFP to Gag. Not shown is the small stretch of 21 nucleotides of the endogenous MMTV gag upstream sequences located just before the gag translation start site. The 5’ UTR Gag-GFP construct contains the entire 5’ untranslated region of

MMTV (R and U5), which should encompass the entirety of the MMTV RNA psi (ψ)

packaging sequence. Last, the 5’ UTR Gag-GFP RemRE construct bears the 5’ UTR of

MMTV as well as the Rem response element (RemRE, light gray knobs) just

downstream of the gag stop codon. The RemRE cooperates with the MMTV Rem

protein that is expressed in trans.

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Figure 4.12 Detection of Gag in collected lysates and media from cells transfected with the Gag constructs bearing various RNA transport elements. Western blots of cell lysates (top) and collected media (bottom) were probed with anti-CA antibody to detect the presence of Gag. Equal amounts of cell lysates were analyzed (50 μg per lane). The Gag-RevRE was expressed in the absence and in the presence of the HIV

Rev-YFP, and the 5’ UTR Gag-GFP RemRE was expressed alone or in conjunction with the MMTV Rem protein fused to mCherry. C3H infected cells were used as a positive control for the presence of Gag in lysates and in virions collected from the media; the proteolytic cleavage products of viral Gag are noted.

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Chapter 5: Overall Dissertation Discussion

The complicated and intricate signals of retroviral Gag protein trafficking and

assembly pathways are becoming increasingly appreciated. The increased knowledge

of this process sheds light on the novel trafficking routes that retroviruses use during the

production of new virus particles. This final section of the dissertation draws together

the conclusions from the data concerning the MMTV Gag protein and relates the

information to the field at large.

C3H and HeJ strains of MMTV- is there a difference in Gag?

Throughout the studies presented here, the signaling and localization of both

C3H and HeJ strains of Gag were compared. Hook et al. had reported that sequences within the CA and NC domains of MMTV Gag protein contribute to the severity of mammary tumorigenesis in mice (197,413). Strain HeJ arose from a recombination event between the more tumorigenic C3H strain and the endogenous, less tumorigenic

Mtv1 strain of MMTV (197). That same laboratory provided the initial yeast two hybrid work suggesting a difference between C3H and HeJ Gag in the ability to interact with the cellular protein, RPL9 (Figure 3.1). Twelve amino acids define the difference between the C3H and HeJ sequences, with just over half of those differences lying within the CA and NC domains, and four differences within the putative nuclear localization signals

(NLSs) of pp21 and p8 (Figure 4.1). Upon visualization of exogenous Gag-GFP fusion proteins, or endogenous Gag from C3H or HP (HeJ-like) infected cells via immunofluorescence, the cellular localization of C3H and HeJ is indistinguishable. Both

Gags form cytoplasmic foci under steady-state conditions (Figure 4.2), and are primarily distributed throughout the cytoplasm when sequential portions of the Gag C-terminus is removed (Figure 4.5). Additionally, no obvious differences were discerned by visualizing interactions of C3H- and HeJ- Gags with RPL9, either through confocal microscopy

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(Figure 3.3) or by FRET analysis (Figure 3.5). The only difference identified thus far was the preliminary cell fractionation data where C3H- but not HeJ Gag-GFP was found in the insoluble nuclear fraction, which presumably contains nucleoli (Figure 4.4).

Additional fractionation studies using cells with HeJ Gag-GFP overexpression as well as

HP infected cells will further support if a difference exists between Gag strains in localization to the nucleolus.

It is possible that the techniques employed thus far are not sensitive enough to reveal differences in C3H and HeJ Gag localization and RPL9 interaction. Perhaps at physiological expression levels, RPL9 does not interact with HeJ Gag as efficiently as with C3H, and thus does not accumulate sufficient HeJ Gag in the nucleolus to interfere with tumor suppression. Overexpression of RPL9 may saturate the system and mask the subtle differences in RPL9 interaction with different Gags. An in vitro approach to determine the relative interaction of the Gag proteins with RPL9 would be to use affinity chromatography with RPL9-conjugated beads and lysates of C3H or HP infected cells, with subsequent quantification of bound Gag. This would be an alternative to coimmunoprecipitation experiments which have proven to be difficult. However, preparation of sufficient quantities of recombinant RPL9 protein for the affinity chromatography may also be challenging, as early attempts to produce GST-tagged

RPL9 were unsuccessful. Further mapping of the RPL9 interaction site(s) within the

Gag CA domain may also reveal residue(s) important for RPL9 interactions that differ between C3H and HeJ (Figure 3.7).

Role of RPL9 in the MMTV Gag story: a link to pathogenesis?

The data presented in this dissertation provide strong evidence that MMTV Gag interacts with the RPL9 protein in the nucleolus of infected cells. In addition, the Gag interaction was mapped to amino acids 86-166 within the C-terminal half of RPL9

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(Figure 3.10 and 3.11). A bipartite NLS/NoLS motif was identified within the first 61 residues of the protein (Figure 3.11).

Is RPL9 the mechanism by which Gag contributes to carcinogenesis? The evidence is indirect, but RPL9 has been implicated as a potential tumor suppressor and increased RPL9 expression levels has been reported in a number of cancers

(26,249,251,298,451,478). Interestingly, more and more reports show that ribosomal proteins not only play a significant role in translation machinery but also possess other properties including p53 regulation (236,469,478). These extraribosomal functions are not limited to free proteins that have yet to be incorporated into ribosomes; a study of

RPL13a shows that it can leave the ribosome complex to participate in other roles (275).

While the alternate functions of RPL9 are currently not known, it is easy to speculate that

RPL9 has downstream effects on p53-related pathways considering the multitude of cellular regulation proteins that it can interact with (Table 3.2). The hypothesis is that interaction of Gag with RPL9 abrogates RPL9’s normal cellular roles in tumor suppression and results in the increased pathogenicity of tumors in infected mice. Until a way to alter the interaction of RPL9 with Gag is achieved, (such as through mutation of the RPL9 binding sites in Gag), the link of RPL9-Gag interaction to pathogenesis is undetermined. Ultimately, the goal is to create a virus with altered interaction with RPL9 which could then be used to infect mice for examination of tumor formation.

Though a link of RPL9 to the cancer phenotype seems more intuitive with the indirect evidence available, an effect of RPL9 on Gag-mediated viral assembly cannot be ruled out. From Western blot analysis of uninfected and infected cell lysates, MMTV infection does not appear to affect endogenous RPL9 expression levels (Figure 3.12).

Efforts to determine the biological role for RPL9 in MMTV assembly have been thwarted by technical problems in terms of siRNA/shRNA knockdown and transfection efficiency for RPL9 overexpression. To overcome transfection efficiency issues, approaches such

174 as retroviral transduction or nucleofection/electroporation of cells may need to be taken to achieve a system where effects of RPL9 on virus production can be examined. RPL9 is not detectable in MMTV virions, implying that RPL9-MMTV Gag interactions occur transiently within the cell (Figure 3.12).

Several other viral proteins have been demonstrated to interact with ribosomal proteins: first, a select group of ribosomal proteins, but not entire ribosomes, were found to be enriched in cells infected with the severe acute respiratory syndrome coronavirus

(SARS-CoV) and to be packaged within the virions (320,446) though neither the mechanism for nor the consequence of this is known. Additionally, the retrovirus mouse leukemia virus (MLV) was found to specifically package rRNA and ribosomes into virions when the normal signals for viral RNA packaging were disrupted (313). Second, RPS6 associates with the nonstructural protein 2 (nsp2) of the singled-stranded RNA virus

Alphavirus and is implicated in differential translation of the host and viral messages

(300). Third, the hantavirus nucleocapsid protein interacts with RPS19 of the small ribosomal subunit and is speculated to facilitate ribosome loading on capped viral mRNAs during N-mediated translation initiation (180). Finally, viral proteins interacting with ribosome components have been shown to mediate translation initiation from internal ribosome entry sites (IRESs) (331). Considering the varying uses of ribosomal proteins by other viruses, other possibilities of the involvement of RPL9 in MMTV infection that have yet to be examined include assistance with IRES initiation or alteration of translation in context of the ribosome or the large 60S subunit. The C- terminal portion of RPL9 was shown to interact with Gag in these studies, and is likely buried in the center of the large 60S subunit participating in rRNA interactions (162), making it difficult for Gag to access. The association of MMTV Gag with isolated polysomes would suggest that RPL9-Gag interactions may not only affect tumorigenesis but play additional roles in cellular translation.

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P-bodies and stress granules- a novel step in Gag assembly?

Early visualization of Gag intracellular localization revealed that, in both transfected and infected cells, Gag accumulates in the cytoplasm within discrete foci

(Figure 4.2). This was not entirely unexpected, as MMTV Gag is known as a Type B retrovirus that forms intracellular immature particles prior to transporting them to the membrane for budding. These foci were presumed to be sites of capsid assembly.

Electron microscopy of infected cells confirmed that the cytoplasm contained many immature virus particles, some of which were in the process of forming (Figure 4.10).

Upon further examination of these assembly sites in the cell, it was observed that a subset of Gag colocalizes with proteins associated with the cellular RNA granules, P- bodies (PBs) and stress granules (SGs) (Figure 4.7). The study of PBs and SGs is a fairly new, rapidly developing field and PBs/SGs have been implicated in a variety of cellular pathways and processes. The cytoplasmic granules are a collection of proteins involved in RNA storage and degradation (PBs) and stalled translation ribonucleoprotein complexes (SGs).

Currently, only one other study has reported Gag proteins in association with

PBs; the yeast retrotransposon uses PBs as a site for Gag and RNA accumulation for assembly of virus-like particles (VLPs) (28). Additional studies have implicated PBs and

SGs in virus assembly: 1) the host defense protein APOBEC3G that is packaged in retroviral virions was found to be localized to PBs (153,458); 2) HIV replication is inhibited by PB component Mov10, which is also packaged into virions (67); and 3) proteins involved with RNA-induced silencing are associated with PBs and SGs and linked to endosomal sorting complex required for transport (ESCRT) proteins that participate in virus budding (158,246). The data in this thesis contributes to the growing

PB/SG field that MMTV also associates with these cytoplasmic ribonucleoprotein complexes, which may play a novel role in the MMTV virus life cycle.

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As suggested by Beckham et al, the dynamic nature of the protein and RNA in

PB/SG complexes provides an ideal location where Gag may rendezvous with its genomic RNA, sequestered away from translation machinery, and begin the assembly process with other Gag molecules (27). Although at this point it is not know if MMTV

RNA localizes to RNA granules, a number of tools are available to study the role of PBs and SGs in the virus assembly pathway.

Nuclear and nucleolar localization signals in Gag: a link to genomic MMTV RNA packaging?

Under steady-state conditions, a small portion of Gag protein is visible in the nucleus of Gag-GFP transfected and MMTV infected cells (Figure 4.2). Additionally, fractionation of transfected cells revealed that Gag-GFP is detectable in nuclear and nucleolar extracts (Figure 4.4). Gag appears to use at least one of the 2 possible NLSs within its pp21 and p8 domains (Figure 4.1) as deletion of the p8 domain from truncated

Gag proteins fused to GFP resulted in a decrease of Gag nuclear accumulation (Figure

4.5). Also, expression of the NC domain of Gag fused to GFP revealed that NC is capable of localizing to nucleoli in the absence of other viral proteins (Figure 3.8). While the NoLS of MMTV Gag was not further mapped, the nucleolar-specific trafficking is most likely conferred by some or all of the basic amino acids that flank the Zinc-finger binding motifs of NC (Figure 4.1). Numerous other NoLSs within proteins are reported to be rich in basic amino acid residues, yet do not conform to an identifiable pattern

(130,182). Recent research in our laboratory shows that the ability of NC to traffic to nucleoli is conserved among several retroviruses (including RSV, HIV, and MLV), and may be intimately linked with the ability to selectively package genomic RNA (manuscript in preparation: Lochmann, Beyer, Ryan, and Parent).

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While localization of the MMTV RNA was beyond the scope of this project, it is possible that MMTV Gag may also use nuclear and nucleolar trafficking as a means to select its genome for viral packaging. As a complex retrovirus, MMTV uses the virally- encoded Rem protein to transport its unspliced viral RNA from the nucleus to the cytoplasm for translation. However, it is not known if this same RNA can be cotranslationally packaged, or if separate pools of RNA are maintained for packaging and translation. The next step in defining the Gag trafficking pathway needs to examine the involvement of the viral genomic RNA. A proposed model for MMTV assembly is shown in Figure 5.1 and discussed below. A role for the nucleolus is probable in the

MMTV life cycle suggested not only by the presence of a NoLS in NC but also by the visualization of Gag colocalized with RPL9 in nucleoli. Alternatively, nucleolar trafficking of Gag may contribute to pathogenesis, as the nucleolus has documented roles in cell transformation events (reviewed in (299,329).

Another interesting point to consider with MMTV is that unlike the HIV Gag/Rev system, which absolutely requires Rev-mediated viral RNA transport for Gag production

(391,393), MMTV Gag protein was expressed in a CMV-driven vector without addition of

Rem. Restriction of HIV Gag expression is mediated by a series of AU-rich inhibitory elements that span the MA and CA domains of Gag (391). Mutagenesis of these sequences allows the expression of HIV Gag in a Rev-independent manner. MMTV apparently does not follow this restriction, as expression of MMTV Gag was produced reliably with and without the presence of the Rem response element (RRE) (Figure

4.12). Examination of the MMTV Gag nucleotide sequence did reveal a slight enrichment of AU nucleotides (57%), with the pp21 domain as the most AU-rich (62% of pp21 contains AU nucleotides). Two AUUUA sequences were found within MA and pp21, which purportedly induce rapid degradation of mRNA (400). While MMTV expression occurs from the CMV-driven plasmid vectors, it is possible that AU-rich

178 regions of Gag may reduce expression to a degree, as Gag expression is not as high as that seen for other proteins expressed from the same GFP vectors (not shown).

Regardless, the expression of unaltered MMTV Gag supports that RNA transport element restriction, such as that seen with HIV Gag expression and Rev, is not a property shared by all complex retroviruses.

The requirements for Gag-mediated particle production (to be continued…)

One of the most interesting questions raised by the experiments described in this thesis is: what are the minimal components required for the production and release of

MMTV VLPs? Early experiments showed that MMTV Gag-GFP expression alone was not sufficient, nor was coexpression with Env (Figure 4.8). Despite the dogma that retroviral Gag protein is sufficient for VLP production, other studies have shown that Env is also needed for viruses like FV and MPMV (21,228,397), which form intracellular particles similar to MMTV. Several MMTV reports also suggest that Env interacts with the MA domain of Gag, which may be a critical step in the membrane targeting and release of particles (see section 2.7.8). I have yet to examine the expression of Gag without GFP in the presence of Env, but the GFP tag has not hindered the budding of other retroviral Gags, as shown in Figure 4.8C. Interestingly, overexpression of Gag-

GFP in NMuMG cells did not result in the abundant production of intracellular VLPs, as determined by electron microscopy (Figure 4.10); only a few rare examples were found in selected Gag-GFP cells, whereas numerous immature capsids were visualized in cells infected with native virus.

Attempts were also made to induce VLP formation and release by altering the pathway by which the Gag mRNA is exported to the cytoplasm (Figure 4.11).

Experiments from Swanson et al. revealed differences in Gag translation and assembly based on the nuclear export route of the mRNA (412). Regardless of the addition of the

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HIV Rev response element (RevRE), the constitutive transport element (CTE) of MPMV, the upstream untranslated region (UTR) of MMTV that contains the psi packaging signal, or the MMTV RemRE, Gag VLPs were not detected in the media of transfected cells

(Figure 4.12).

The techniques employed in this dissertation to evaluate budding of MMTV Gag are not exhaustive. Other approaches likely to yield critical information include: electron microscopy of the untagged Gag constructs to examine the ability to form immature capsids, coexpression of those same constructs with Env (in the event that GFP prevented proper particle formation in previous Env experiments), and forced membrane targeting of Gag by addition of a membrane-binding motif from the src gene to determine if budding can be induced. A study by Jouvenet et al. shows that a host defense protein, tetherin, inhibits virus particle release of a broad spectrum of viruses and can be counteracted with expression the HIV-encoded protein, Vpu (212). Recently, the lab has obtained constructs to overexpress Vpu in context of MMTV Gag. Although Vpu-like sequences have not been identified within the MMTV genome, this does not exclude the possibility that other MMTV proteins such as Env contribute a Vpu-like function in VLP release.

Fair to compare? MPMV vs. MMTV

Up to now, studies have historically used MPMV as the prototype betaretrovirus in terms of studying intracellular trafficking and assembly mechanisms, based presumably on its ease of study in comparison to MMTV (see section 2.9). However, despite baseline similarities of intracellular assembly of capsids and possession of similar CTRS sequences, experiments described in this thesis emphasize the unique aspects of MMTV that warrant independent study.

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Several inherent properties distance the two betaretroviral relatives from one another. First, MPMV and MMTV target different cell types (immune cells versus mammary epithelium) of different species (simian versus mouse) with differing pathogenic outcomes (fatal immunodeficiency syndrome and thymic atrophy versus mammary tumors and selective T-cell depletion). On a molecular level, MPMV is a simple retrovirus that uses a cis-acting constitutive transport element (CTE) to shuttle its unspliced RNA from the nucleus, while MMTV bears a trans-acting Rem RNA transport element that classifies it as a complex retrovirus. Genomically speaking, MPMV encodes one accessory protein (dUTPase) while MMTV encodes three (dUTPase, Sag, and Rem). In terms of Gag, MPMV encodes a phosphoprotein, pp24, similar to the

MMTV pp21, but MPMV Gag includes a central p12 domain as well as a C-terminal p4 domain, which are not homologous to the additional Gag domains of MMTV (p3, p8, n).

Although studies of MPMV have identified a late domain involved in budding (330) and an internal scaffold domain (ISD) in p12 responsible for Gag-Gag interactions (481),

MMTV Gag does not bear congruent sequences. Lastly, studies of the protease activity in MPMV have been shown to be very tightly coordinated with immature capsid trafficking to the membrane and particle release, whereas MMTV Gag proteolytic cleavage occurs in the cytoplasm independently of membrane targeting (479).

Collectively, there is substantial evidence to propose that MPMV and MMTV be classified in different retroviral genera (289). All together, these observations emphasize that use of MPMV as a tool for discerning general virus mechanisms should not serve as a surrogate for MMTV study, and comparisons between MPMV and MMTV should be analogous to the use of RSV or MLV as a model for understanding HIV. NMuMG cells offer an amenable, biologically relevant system in which to carry out studies on MMTV on the cellular level, promising further discovery of the elusive properties of MMTV virus replication.

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Summary and model

In total, this work presents a broad range of intracellular possibilities for MMTV

Gag trafficking and localization. Essentially, no part of the cell appears to be restricted from viral components in comparison to rigid models showing ribosome-to-assembly site-to-membrane progression of particle progression. Figure 5.1 serves to summarize the findings reported here and to speculate on the possible movements, assembly, and budding of MMTV Gag. After transcription and transport of the unspliced viral RNA into the cytoplasm by Rem, and translation of Gag on ribosomes, a subset of Gag traffics into the nucleus/nucleolus via NLSs in p8 and possibly pp21, as well as through a NoLS within the NC domain (Figure 5.1, blue box). While the purpose of nuclear entry is not clear, it may serve to collect viral genomic RNA similar to previous studies of RSV nuclear trafficking. MMTV Gag exits the nucleus via CRM1-independent signals and returns to the cytoplasm, possibly with the cargo of viral RNA or a cellular protein. This nuclear/nucleolar trafficking of Gag may also occur in conjunction with the association of

Gag with RPL9, which contains a bipartite signal for nucleolar localization (green box).

Again, the definitive purpose behind this trafficking is currently unclear but is suggestive of a role in tumor progression via interference with RPL9 tumor suppressive activities.

After the nuclear trafficking step and export into the cytoplasm, Gag next accumulates adjacent to PB and SG complexes where assembly begins using the viral

RNA as a scaffold upon which to build Gag oligomers (Figure 5.1, red box). Although

MMTV RNA has not yet been localized to these structures, RNA is included in the model in light of the documented roles of PBs/SGs as a hub of translationally repressed RNA messages. By implicating the use of RNA granules as a step in assembly, a stepwise model is proposed which begins with the translocation of viral RNA to the cytoplasm by

Rem for translation of Gag polyproteins. The Gag protein would then traffic through the nucleus to select and carry the genomic RNA to the cytoplasm for packaging. Upon

182 export to the cytoplasm, Gag may deliver the RNA to PBs/SGs where Gag molecules oligomerize and form intracellular capsids; Gag may be targeted to this location via its

CTRS or attracted by interactions with the PB/SG-associated viral RNA already present.

Once a critical concentration of Gag protein accumulates near the RNA granules with viral RNA present, assembly can commence.

Upon completion of the immature capsids, compounded myristic acid moieties work in conjunction with membrane-targeting residues of the MA domain on the outer surface of the capsids to target the capsid structure to the plasma membrane.

Interactions with the Env protein may mediate this process as well, and particles could

“hitch a ride” with Env-bearing vesicles similar to those shown previously by electron microscopy of MMTV infected cells (155,156) (Figure 5.1, purple box). Upon reaching the membrane, host defense mechanisms (such as tetherin) are defeated by inherent viral gene products (possibly through Env) to allow for budding and release to occur

(Figure 5.1, yellow box). While MMTV Gag bears the potential to participate in any or all of these suggested pathways, further work on the specific mechanisms of Gag will elucidate the details of its carefully choreographed sequence of assembly events. This dissertation is a foundation to build upon for the further exploration of how this interesting and complex assembly process occurs for MMTV.

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Figure 5.1 Summary and model of MMTV Gag intracellular possibilities. This image represents the cellular trafficking events that may occur after translation of the

Gag protein in the cytoplasm and prior to release of Gag within particles at the plasma membrane. The MMTV-encoded Rem protein (green oval) is known to facilitate the translocation of unspliced viral RNA (black line with squares on ends) from the nucleus to the cytoplasm for translation of Gag on ribosomes (peach ovals). After translation,

Gag may localize to nuclei/nucleoli via nuclear localization signals within pp21 and p8 and a nucleolar localization signal within NC (blue box) and export via unidentified

CRM1-independent signals. Alternately, Gag localizes to nucleoli via interactions with the RPL9 protein (peach oval) and is taken to nucleoli via the RPL9 bipartite NoLS

(green box). Either pathway may allow for Gag to bind to its RNA for transport out of the nucleus and into the cytoplasm for packaging. Gag, with or without viral RNA, then moves to P-body and stress granule ribonucleoprotein structures (proteins as multicolored shapes, cellular RNAs as heavy dark blue lines) in the cytoplasm, possibly through the cytoplasmic transport/retention signal within the MA domain (red box). Gag accumulates at these intracellular sites as a platform for virus assembly with viral RNA in the absence of protein translational machinery. Once spherical immature capsids are produced, they are targeted to the membrane by the collective signal of the myristic acid moieties and the membrane-binding sequences within MA on the particle surface. As

MA is known to interact with the viral Env protein, Env may facilitate the trafficking of particles to the membrane, via direct interactions at the membrane or through trafficking on Env-studded vesicles (purple box). Upon reaching the cell periphery, virus particles may have to overcome cellular defense mechanisms (red stop-signs) with virally- encoded components (X over stop-sign) in order for egress from the cell to proceed

(yellow box).

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Vita Andrea R. Beyer

EDUCATION Doctor of Philosophy in Microbiology & Immunology, 2004-2011 The Pennsylvania State University, College of Medicine, Hershey, PA Bachelor of Science in Genetic Engineering, with a Chemistry minor, 2000-2004 Cedar Crest College, Allentown, PA

HONORS AND AWARDS Tobacco Settlement Award for Graduate Research, Penn State College of Medicine (2009) College of Medicine Class of 1977 Scholarship, Penn State College of Medicine (2009) 3rd place poster presentation at The Penn State University Graduate Exhibition (2008) Graduate Student Service Award, Penn State University (2008) Louis Pasteur Prize in Microbiology, Penn State College of Medicine (2007) National Science Foundation Graduate Student Research Fellowship Recipient (2006) Karl H. Beyer, Jr., M.D.,Ph.D. Scholarship, Penn State College of Medicine (2006) Penn State University Fellowship (2004-2005) Student Ambassador for Cedar Crest College (2003-2004) Delphi Honor Society, Cedar Crest College (2003) βββ Honor Society, Cedar Crest College (2002-2004 member, President 2003-2004) Board of Associate’s Scholarship, Cedar Crest College (2001 – 2004) Dean’s List, Cedar Crest College (2000 – 2004) Robert C. Byrd Scholarship, Cedar crest College (2000-2004) Girl Scout Gold Award Scholarship, Cedar Crest College (2000-2004) Girl Scout Gold Award, Girl Scouts of the U.S.A. (2000) Keystone Girls State, Pennsylvania American Legion (1999)

TEACHING EXPERIENCE Laboratory Techniques Lunch Lecture Series-Guest Lecturer August 2010 Penn State College of Medicine Medical Microbiology Laboratory Teaching Assistant Springs 2006-2010 Dept. of Microbiology and Immunology, Penn State College of Medicine Elementary Microbiology - Guest Lecturer Fall 2009 Penn State College of Medicine and Penn State Harrisburg Completed the Penn State Course in College Teaching Spring 2009 Schreyer Institute for Teaching Excellence, Penn State, University Park Instructor for Laboratory Techniques in Biomedical Research Fall 2008 Penn State College of Medicine and Penn State Harrisburg Instructional Assistant for Biology Laboratory Fall 2001 – Spring 2004 Cedar Crest College

PUBLICATIONS Beyer, A.R., Swanson, I., Yurkovetskiy, L., Goff, S.P., Golovkina, T.V., and Parent, L.J. Interaction of Mouse Mammary Tumor Virus Gag Protein with Ribosomal Protein L9 in the Nucleolus. In preparation. Lochmann, T.L., Ryan, E., Beyer, A.R., Parent, L.J. Nucleolar Trafficking of Retroviral Gag Proteins. In preparation. Beyer, A.R., Bann, D.V., Parent, L.J. Association of MMTV Gag with P-bodies and Stress Granules. In preparation. Beyer, A.R. and Parent, L.J. Trafficking and Budding of the Anomalous MMTV Gag Protein. In preparation.