The Pennsylvania State University The Graduate School College of Medicine FROM THE RIBOSOME TO THE MEMBRANE: SUBCELLULAR TRAFFICKING OF THE ROUS SARCOMA VIRUS GAG POLYPROTEIN A Thesis in Cell and Molecular Biology by Lisa Z. Scheifele Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2004 The thesis of Lisa Z. Scheifele was reviewed and approved* by the following: Leslie J. Parent Associate Professor of Medicine, and Microbiology and Immunology Thesis Advisor Chair of Committee Rebecca C. Craven Associate Professor of Microbiology and Immunology James E. Hopper Professor of Biochemistry and Molecular Biology Craig M. Meyers Professor of Microbiology and Immunology Michael F. Verderame Associate Professor of Medicine, and Assistant Professor of Microbiology and Immunology Henry J. Donahue Baker Professor and Vice Chair for Research Professor of Cellular and Molecular Physiology Head of the Cell and Molecular Biology Graduate Program *Signatures are on file in the Graduate School iii ABSTRACT Retroviruses such as human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV) are the etiological agents of immunodeficiency diseases and cancer. We have been studying the subcellular targeting of the Gag protein of Rous sarcoma virus (RSV), one of the first simple retroviruses identified. The Gag protein directs assembly at the plasma membrane and incorporates the viral genome through association with a cis-acting packaging element in the viral RNA. Although functional domains within Gag that mediate the assembly process have been well defined, the activity of these motifs has not been well correlated with the trafficking of Gag throughout the cell during assembly. We have identified distinct subcellular targeting motifs within the Rous sarcoma virus Gag protein that may influence the retroviral assembly pathway. Within the Gag membrane-binding domain, we have identified an alpha helix that is crucial for plasma membrane targeting of Gag. When this helix is deleted, the Gag protein accumulates at intracellular membranes. Yet membrane affinity is not diminished, indicating that membrane binding and plasma membrane targeting of the RSV Gag polyprotein are genetically separable. We have also identified both a nuclear targeting signal within the N-terminal MA domain of Gag and a nuclear export signal (NES) within the p10 domain. Expression of dominant-negative nuclear pore proteins redistributes the Gag protein from a cytoplasmic and plasma membrane localization to an almost exclusively nuclear localization, confirming that Gag both enters and exits the nucleus. We have further identified the soluble receptor that mediates the cytoplasmic localization of Gag; treatment of virus-expressing cells with leptomycin B, a specific inhibitor of the CRM-1 export pathway results in sequestration of iv the Gag protein within the nucleus. Single amino-acid substitutions within a leucine-rich cluster in the p10 domain of Gag result in accumulation of the protein within the nucleus, confirming the localization of the Gag NES within the p10 region. Interestingly, we find that the NES sequence is highly conserved among avian retroviruses; when naturally occurring variations within this sequence are recreated in the RSV Gag protein, NES function is retained, while artificial substitutions predicted to retain NES function do not. We have sought to identify additional cellular proteins that influence the import and export of Gag from the nucleus by employing the powerful genetic system developed in the yeast Saccharomyces cerevisiae. Expression of Gag linked to a tandem GFP reporter reveals a localization to the cytoplasm of yeast cells with an accumulation in the nucleus not seen at steady-state in avian cells. Not only is the dwell time between the nucleus and cytoplasm altered, but the nuclear export pathway is perturbed as well; yeast cells do not show an enhanced accumulation of Gag proteins within the nucleus following leptomycin B treatment, suggesting that the Crm1 pathway may not be the predominant export pathway utilized by Gag in yeast cells. To further investigate the role of Gag nuclear trafficking in retroviral replication, we have studied the replication of viruses containing mutations in the Gag NES. These mutant Gag proteins are able to direct the budding of virions which contain the normal complement of viral polyproteins and the proper amount of viral genomic RNA. However, the virions that are produced show profound morphological defects, including amorphous and heterogeneously shaped particles, and viral cores that are elongated or granular in appearance. Despite these substantial morphological defects, the particles are able to enter new cells and undergo reverse transcription, producing 2-LTR circles, hallmarks of entry of the synthesized DNA genome into the nucleus. Although the particles are able to complete v reverse transcription, they are non-infectious. Nuclear transport of Gag may therefore crucial not only for the efficient production of viral particles at the plasma membrane, but also for the establishment of a productive infection. vi TABLE OF CONTENTS LIST OF FIGURES .....................................................................................................ix ACKNOWLEDGEMENTS.........................................................................................xii CHAPTER 1: LITERATURE REVIEW.....................................................................1 1.1 THE ROLE OF GAG IN RETROVIRAL ASSEMBLY ...............................2 1.1.1 Identification Of Rous Sarcoma Virus .................................................3 1.1.2 Characterization Of Viral Gene Products.............................................4 1.1.3 Isolation Of Reverse Transcriptase ......................................................5 1.1.4 Identification Of Human Retroviruses .................................................6 1.2 THE RETROVIRAL LIFE CYCLE...............................................................7 1.2.1 Classification Of Retroviruses..............................................................8 1.2.2 Virion Organization..............................................................................11 1.2.3 Entry .....................................................................................................14 1.2.4 Reverse Transcription...........................................................................15 1.2.5 Nuclear Transport Of The Genome And Integration ...........................18 1.2.6 Genome Organization And Transcription ............................................20 1.2.7 Retroviral Assembly.............................................................................23 1.2.8 RNA Packaging....................................................................................27 1.2.9 Viral Maturation ...................................................................................29 1.3 INTERACTION OF GAG WITH THE HOST CELL DURING ASSEMBLY..................................................................................................30 1.3.1 Transformation By Avian Retroviruses................................................31 1.3.2 Endogenous Retroviruses And Host Cell Restriction ..........................33 1.3.3 Host Cell Interactions Of The HIV Gag Polyprotein ...........................34 1.3.3.1 Host Cell Interactions Of The HIV Gag Polyprotein: Cyclophilin A..................................................................................35 1.3.3.2 Host Cell Interactions Of The HIV Gag Polyprotein: Cytoskeleton....................................................................................36 1.3.3.3 Host Cell Interactions Of The HIV Gag Polyprotein: Endosomal Sorting Proteins............................................................37 1.4 SUBCELLULAR TRAFFICKING OF THE RSV GAG POLYPROTEIN...39 1.4.1 Membrane Binding Mechanisms For RSV Gag...................................40 1.4.1.1 Membrane Binding Mechanisms Of Cellular Proteins ..............41 1.4.1.2 Membrane Binding Of The HIV Gag Polyprotein.....................45 1.4.1.3 Membrane Binding Mechanisms Of Nonmyristoylated Gag Proteins............................................................................................47 1.4.1.4 Identification Of The RSV Membrane Binding Domain ...........48 1.4.1.5 Characterization of RSV Membrane Binding Mutants ..............50 1.4.2 Nuclear Transport In Retroviral Replication........................................53 1.4.2.1 Identified Nuclear Shuttling Proteins In HIV Replication .........54 vii 1.4.2.2 Nuclear Transport In RSV Replication ......................................55 1.4.2.3 The Nuclear Pore Complex........................................................57 1.4.2.4 Nuclear Transport Signals..........................................................62 1.4.2.5 The Crm-1 Export Pathway........................................................64 1.5 OVERVIEW...................................................................................................67 CHAPTER 2: SPECIFICITY OF PLASMA MEMBRANE TARGETING BY THE ROUS SARCOMA VIRUS GAG POLYPROTEIN ...................................70 2.1 ABSTRACT ...................................................................................................71 2.2 INTRODUCTION .........................................................................................71 2.3 MATERIALS AND METHODS ...................................................................75 2.4 RESULTS.......................................................................................................78