DETERMINANTS OF INFECTIOUS HIV-1 PARTICLE PRODUCTION
Paul D Bieniasz
Aaron Diamond Aids Research Center, 455 First Ave, 6th Fl, New York, Ny 10016-9102
Grant 5R01AI050111-09 from National Institute Of Allergy And Infectious Diseases
Abstract: This is a competing renewal application in which we request support for our studies of retrovirus assembly. In the next grant period, we will address two important mechanistic questions relating to the cell biology of retroviral particle assembly. Specific Aim 1 is to understand how retroviral Gag proteins are targeted to specific sites in various cell types and the purpose of the myristoyl-switch in retarding HIV-1 Gag membrane binding. We will first refine and elaborate our biochemical and real-time live cell imaging assays of retrovirus assembly to determine the route that HIV-1 Gag takes to sites of assembly and egress in various cell types, including primary macrophages. These assays, should enable us to determine how HIV-1 Gag proteins select and move to sites of particle assembly. We will test the hypotheses that active transport pathways, or the intrinsic membrane binding properties of Gag, or both, govern Gag targeting. Additionally, we will determine the consequences for HIV-1 assembly and egress of targeting Gag to distinct cellular membranes in various cell types. Moreover we will test several hypotheses as to why HIV-1 has evolved a mechanism that inhibits membrane binding. Specific Aim 2 is to determine how HIV-1 Vpu affects the targeting of retroviral Gag proteins and promiscuously affects enveloped viral particle release. At present, the mechanism by which Vpu facilitates virus release is almost completely unclear. However our preliminary studies reveal dramatic relocalization of HIV-1 and MLV Gag proteins to the plasma membrane in the presence of Vpu. We will undertake a comprehensive mutagenesis approach to map the determinants of Vpu localization, and to determine the importance of Vpu localization and its effects on Gag localization in the stimulation of virus release virus release. We will also determine the underlying mechanism by which Vpu causes relocalisation of retroviral Gag proteins Finally we will attempt to identify cellular targets and/or cofactors of Vpu´s virus release function using tandem affinity purification. Understanding how HIV-1 and other retroviral Gag proteins select sites with the cell at which to assemble, and how viral and cellular activities influence this process, remains one of the most enigmatic problems in retrovirology. The experiments proposed herein should provide greater clarity as to how Gag proteins behave during assembly in cells
Keywords: AIDS Virus; Active Biological Transport; Active Transport; Address; Affect; Affinity Chromatography; Assay; Binding; Binding (Molecular Function); Bioassay; Biochemical; Biologic Assays; Biologic Transport, Active; Biological Assay; Biological Transport, Active; Cell membrane; Cells; Cellular Membrane; Cellular biology; Chromatography, Affinity; Consequences of HIV; Cytoplasmic Membrane; Gagging; Gene Products, gag; Genetics-Mutagenesis; Grant; HIV-1; HIV-I; HIV1; Human immunodeficiency virus 1; Immunodeficiency Virus Type 1, Human; Life; Maps; Membrane; Molecular Biology, Mutagenesis; Molecular Interaction; Mutagenesis; Pathway interactions; Plasma Membrane; Process; Production; Property; Property, LOINC Axis 2; Reflex, Pharyngeal; Retroviral Antigen gag Protein; Retroviridae; Retrovirology; Retroviruses; Route; Site; Testing; Time; Uphill Transport; Viral; Viral gag Proteins; Virus; Virus-Retrovirus; Viruses, General; affinity purification; cell biology; cell imaging; cell type; cellular imaging; cellular targeting; cofactor; experiment; experimental research; experimental study; gag Antigens; gag Gene Products; gag Polyproteins; gag Protein; group specific antigen; human T cell leukemia virus III; human T lymphotropic virus III; macrophage; membrane structure; particle; pathway; plasmalemma; research study
Project start date: 2001-04-01
Project end date: 2011-03-31
Budget start date: 1-APR-2009
Budget end date: 31-MAR-2010
5R01AI050111-09 (2009): $272835
Sponsored Links Excellgen http://Excellgen.com
Determinants Of Infectious HIV-1 Particle Production
Paul D Bieniasz, Associate Professor
Aaron Diamond Aids Research Center 455 First Ave, 6th Fl New York, Ny 100169102
Grant 5R01AI050111-07 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: This is a competing renewal application in which we request support for our studies of retrovirus assembly. In the next grant period, we will address two important mechanistic questions relating to the cell biology of retroviral particle assembly. Specific Aim 1 is to understand how retroviral Gag proteins are targeted to specific sites in various cell types and the purpose of the myristoyl-switch in retarding HIV-1 Gag membrane binding. We will first refine and elaborate our biochemical and real-time live cell imaging assays of retrovirus assembly to determine the route that HIV-1 Gag takes to sites of assembly and egress in various cell types, including primary macrophages. These assays, should enable us to determine how HIV-1 Gag proteins select and move to sites of particle assembly. We will test the hypotheses that active transport pathways, or the intrinsic membrane binding properties of Gag, or both, govern Gag targeting. Additionally, we will determine the consequences for HIV-1 assembly and egress of targeting Gag to distinct cellular membranes in various cell types. Moreover we will test several hypotheses as to why HIV-1 has evolved a mechanism that inhibits membrane binding. Specific Aim 2 is to determine how HIV-1 Vpu affects the targeting of retroviral Gag proteins and promiscuously affects enveloped viral particle release. At present, the mechanism by which Vpu facilitates virus release is almost completely unclear. However our preliminary studies reveal dramatic relocalization of HIV-1 and MLV Gag proteins to the plasma membrane in the presence of Vpu. We will undertake a comprehensive mutagenesis approach to map the determinants of Vpu localization, and to determine the importance of Vpu localization and its effects on Gag localization in the stimulation of virus release virus release. We will also determine the underlying mechanism by which Vpu causes relocalisation of retroviral Gag proteins Finally we will attempt to identify cellular targets and/or cofactors of Vpu s virus release function using tandem affinity purification. Understanding how HIV-1 and other retroviral Gag proteins select sites with the cell at which to assemble, and how viral and cellular activities influence this process, remains one of the most enigmatic problems in retrovirology. The experiments proposed herein should provide greater clarity as to how Gag proteins behave during assembly in cells.
Keywords: gag protein, particle, respiratory reflex, Retroviridae, active transport, affinity chromatography, cell, cell biology, cell membrane, cell type, cofactor, emotion, macrophage, membrane, molecular /cellular imaging, virus
Project start date: 2001-04-01
Project end date: 2011-03-31
5R01AI050111-07 (2007): $355360
5R01AI050111-05 (2005): $319200
5R01AI050111-04 (2004): $319200
5R01AI050111-03 (2003): $319200
5R01AI050111-02 (2002): $319200
Grants awarded to Paul D Bieniasz
Determinants Of Infectious HIV-1 Particle Production
Paul D Bieniasz, Associate Professor
Aaron Diamond Aids Research Center 455 First Ave, 6th Fl New York, Ny 100169102
Grant 2R01AI050111-06 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: This is a competing renewal application in which we request support for our studies of retrovirus assembly. In the next grant period, we will address two important mechanistic questions relating to the cell biology of retroviral particle assembly. Specific Aim 1 is to understand how retroviral Gag proteins are targeted to specific sites in various cell types and the purpose of the myristoyl-switch in retarding HIV-1 Gag membrane binding. We will first refine and elaborate our biochemical and real-time live cell imaging assays of retrovirus assembly to determine the route that HIV-1 Gag takes to sites of assembly and egress in various cell types, including primary macrophages. These assays, should enable us to determine how HIV-1 Gag proteins select and move to sites of particle assembly. We will test the hypotheses that active transport pathways, or the intrinsic membrane binding properties of Gag, or both, govern Gag targeting. Additionally, we will determine the consequences for HIV-1 assembly and egress of targeting Gag to distinct cellular membranes in various cell types. Moreover we will test several hypotheses as to why HIV-1 has evolved a mechanism that inhibits membrane binding. Specific Aim 2 is to determine how HIV-1 Vpu affects the targeting of retroviral Gag proteins and promiscuously affects enveloped viral particle release. At present, the mechanism by which Vpu facilitates virus release is almost completely unclear. However our preliminary studies reveal dramatic relocalization of HIV-1 and MLV Gag proteins to the plasma membrane in the presence of Vpu. We will undertake a comprehensive mutagenesis approach to map the determinants of Vpu localization, and to determine the importance of Vpu localization and its effects on Gag localization in the stimulation of virus release virus release. We will also determine the underlying mechanism by which Vpu causes relocalisation of retroviral Gag proteins Finally we will attempt to identify cellular targets and/or cofactors of Vpu s virus release function using tandem affinity purification. Understanding how HIV-1 and other retroviral Gag proteins select sites with the cell at which to assemble, and how viral and cellular activities influence this process, remains one of the most enigmatic problems in retrovirology. The experiments proposed herein should provide greater clarity as to how Gag proteins behave during assembly in cells.
Keywords: host organism interaction, human immunodeficiency virus 1, species difference, virus replication, gag protein, virus RNA, virus genetics, animal tissue, human tissue, tissue /cell culture
Project start date: 2001-04-01
Project end date: 2011-03-31
2R01AI050111-06 (2006): $360000
RETROVIRUS RESTRICTION BY TRIMA5ALPHA
Paul D Bieniasz
Department/ Educational Institution Type:
Grant 5R01AI064003-05 from National Institute Of Allergy And Infectious Diseases
Abstract: The study of endogenous cellular factors that inhibit infection by retroviruses is an emerging area of research. We were among the first to show that such factors in monkey cells can inhibit primate lentivirus infection and contribute in a major way to defining retrovirus species tropism. The recent identification of TRIM5alpha as one of this class of retrovirus resistance factors makes this an appropriate time to expand our efforts to characterize these factors. We have obtained preliminary data indicating that TRIM5alpha from humans and nonhuman primates restricts infection by a number of retroviruses, as widely divergent as MLV and HIV-1, and that HIV-1 CAcyclophilin interaction can modulate restriction factor sensitivity. Other preliminary findings suggest that TRIM5alpha may not be the only capsid-specific retrovirus restriction factor in human cells. This proposal has three specific aims Aim 1 is to determine the breadth and specificity of retrovirus inhibitory activity exhibited by a number of TRIM5alpha variants and to map determinants of specificity in CA and TRIM5alpha. This will involve the construction of chimeric and mutant human and nonhuman primate TRIM5alpha variants and the selection of HIV-1 mutants that are resistant to TRIM5alpha variants that inhibit wild-type HIV-1. As well as illuminating determinants of restriction specificity in TRIM5alpha and CA, this will provide critical control reagents for Aim 2, which is to determine the mechanism of restriction by TRIM5alpha. We will determine the subcellular localization of TRIM5alpha, attempt to demonstrate physical interaction between HIV-1 capsids and TRIM5alpha, map sequences responsible for TRIM5alpha subcellular localization, multimerization, and capsid binding and determine whether they are required for restriction. We will also determine whether proteins that bind to TRIM5alpha are required for restriction and test the hypothesis that TRIM5alpha-catalyzed ubiquitination is part of the restriction mechanism. Finally, in Aim 3 we will determine whether TRIM proteins that are closely related to TRIM5alpha exhibit retrovirus restriction activity. The successful completion of these studies could have practical implications both for AIDS animal model development, gene therapy vector development and, eventually, for the designing completely novel therapeutic antiretroviral strategies
Keywords: Acquired Immune Deficiency; Acquired Immune Deficiency Syndrome; Acquired Immuno-Deficiency Syndrome; Acquired Immunodeficiency Syndrome; AIDS; AIDS Virus; Alleles; Allelomorphs; animal model development; anti-retroviral; Anti-Retroviral Agents; antiretroviral; Antiretroviral Agents; Area; base; Binding; Binding (Molecular Function); Capsid; Capsid Proteins; Cells; coat (nveloped virus); Coat Proteins; Cyclophilins; Cyclosporin-Binding Proteins; Data; Derivation; Derivation procedure; design; designing; Development; Exhibits; gene product; Gene Therapy Vectors; Gene Transduction Agent; Gene Transduction Vectors; Genetic Screening; Genetics-Mutagenesis; HIV-1; HIV-I; HIV1; HSPC062; Human; Human immunodeficiency virus 1; human T cell leukemia virus III; human T lymphotropic virus III; human TRIM5alpha; Human, General; Immunodeficiency Virus Type 1, Human; Immunodeficiency Viruses, Primate; Immunologic Deficiency Syndrome, Acquired; Infection; interest; Investigation; Lentivirus disease; Lentivirus Infections; Lentiviruses, Primate; loss of function; Man (Taxonomy); Man, Modern; Maps; Mediating; Molecular Biology, Mutagenesis; Molecular Interaction; Monkeys; Mutagenesis; mutant; Nature; new therapeutics; next generation therapeutics; non-human primate; nonhuman primate; novel therapeutics; Primate Lentiviruses; Protein Binding; Proteins; R Factors; R Plasmids; Reagent; Research; Resistance; Resistance Factors; resistant; Retroviridae; Retroviridae disease; Retroviridae Infections; Retrovirus Infections; Retroviruses; Role; social role; Specificity; Structure; T-Cell Receptor Interacting Molecule Gene; Testing; Time; TRIM; TRIM Gene; Tropism; ubiquination; Ubiquitilation; ubiquitin conjugation; Ubiquitination; Ubiquitinoylation; Variant; Variation; Viral; Viral Coat Proteins; Viral Outer Coat Protein; Virus-Retrovirus
Project start date: 2005-01-01
Project end date: 2010-12-31
Budget start date: 1-JAN-2009
Budget end date: 31-DEC-2010
5R01AI064003-05 (2009): $418574
5R01AI064003-03 (2007): $426681
5R01AI064003-02 (2006): $439425
1R01AI064003-01 (2005): $362500
DETERMINANTS OF INFECTIOUS HIV-1 PARTICLE PRODUCTION
Paul D Bieniasz, Associate Professor
Aaron Diamond Aids Research Center 455 First Ave, 6th Fl New York, Ny 100169102
Grant 1R01AI050111-01 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: Rodent cells have provided a useful tool for the analysis of cellular factors that are important for HIV-1 entry and transcription. Indeed, the expression of certain human genes in rodent cells is able to rescue rodent-cell specific blocks in these processes. However, despite expression of all species-specific co-factors known to be required for HIV-l replication, most rodent cells remain unable to efficiently support even a single cycle of HIV- 1 replication. In fact, we have observed a profound defect in HIV particle production by many rodent cells and the few particles that are produced are significantly less infectious than those derived from human cells. In the studies detailed in this proposal, we seek to determine how viral and species-specific host-cell factors interact to mediate infectious particle production. The defective phenotype in rodent cells often correlates with a reduced level of intracellular processing of the Pr55Gag precursor, a finding which suggests that Pr5SGag targeting to the correct site of virion assembly is the underlying defect. The biochemical and microscopic imaging experiments outlined herein will directly test this hypothesis and will further examine whether this phenotype is a consequence of a failure of rodent cells to support appropriate post-translational modifications of Pr55Gag. Experiments will also be done that determine whether rodent cell derived virions exhibit any defect in protein or RNA composition. Since the matrix region of Pr55Gag contains the major determinants of subcellular localization, a genetic analysis will focus on this domain. Specifically, we will determine whether defective particle formation in rodent cells is a general property of lentiviruses and examine whether redirecting Pr5SGag to the plasma membrane of rodent cells by manipulation of matrix sequences is sufficient to restore extracellular particle formation and infectious virus production. The finding that infectious virus production by rodent cells can be substantially rescued by fusion with human cells indicates that components of the human host-cell must play a positive and specific role in this process. We will determine whether single or multiple human chromosomes are required to restore infectious virus production in the context of a rodent cell and we will perform a genetic screen designed to identify human co-factors that enhance infectious HIV-l particle production by rodent cells. These studies have the potential to reveal new targets for chemotherapeutic intervention in HIV disease and may make a rodent model of HIV infection feasible.
Keywords: host organism interaction, human immunodeficiency virus 1, species difference, virus replication, gag protein, virus RNA, virus genetics, animal tissue, human tissue, tissue /cell culture
Project start date: 2001-04-01
Project end date: 2006-03-31
1R01AI050111-01 (2001): $319200
Mechanism Of Action Of Retroviral Late-Budding Domains
Paul D Bieniasz, Associate Professor
Aaron Diamond Aids Research Center 455 First Ave, 6th Fl New York, Ny 100169102
Grant 5R01AI052774-05 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: Like other retroviruses, HIV-1 encodes a sequence within the Gag protein termed a late or L -domain that is required for the scission of a nascent virion from the virus producing cell. L-domains are functionally interchangeable among different retroviruses and act by recruiting host-cell factors. In HIV-1, this activity is provided by a short peptide sequence (PTAP) that binds directly the host-cell protein, TsglOl, and recruits it to sites of particle assembly. TsglOl is one of a group of sixteen or more proteins, termed the class E vacuolar protein sorting (VPS) factors, that are normally required for the budding of vesicles into the endosomal lumen. As part of our long term objective to understand the mechanism by which viral L-domains mediate particle budding, this proposal aims to test the hypothesis HIV-1 L-domain activity requires the same cellular activities that mediate the topologically equivalent process of endosomal vesicle budding. TsglOl itself exhibits a homo-multimerization activity and is known to bind to a second class E VPS factor, hVPS28. The experiments described herein will first determine whether these interactions are required for the HIV-1 particle budding function of TsglOl . Subsequently, the identification other cellular proteins that are associated with Tsgl 01 will be undertaken. Using biochemical yeast 2-hybrid and microscopic analyses, it will be determined whether and how they and other, already identified, class E VPS factors are recruited to sites of HIV-1 budding. Moreover, RNA duplex mediated silencing will be used to test whether these additional factors are required for L-domain mediated HIV-1 particle release. Preliminary studies indicate that a second class of retroviral L-domains, that contain a PPXY rather than a PTAP sequence motif, act by a similar mechanism to the HIV-1 L-domain. While these L-domains do not bind directly to TsglOl, they are clearly able to recruit TsglOl and hVPS28. Therefore, in some experiments, a PPXY L-domain will be included to test the hypothesis that PPXY L-domains mediate viral budding by the same mechanism as does HIV-1 , but by binding to an alternative VPS factor. Finally, a determination of whether the HIV-1 L-domain recruits a deubiqutinating activity during viral particle assembly will be undertaken. Based on findings in yeast systems, this is anticipated to be a secondary consequence of VPS factor recruitment, and is likely to explain the apparent requirement for the cellular ubiqutination machinery in retroviral particle release. Overall this project will aim to advance our understanding to the molecular mechanisms used by HIV-1 to facilitate viral budding. These studies have the potential to reveal completely novel, host-cell targets for therapeutic blockade of the replication of HIV-1, and possibly other viruses.
Keywords: DNA binding protein, gag protein, human immunodeficiency virus 1, protein protein interaction, protein structure function, virion, virus assembly, protein transport, ubiquitin, vesicle /vacuole, virus envelope, virus infection mechanism, virus replication, protein purification, protein sequence
Project start date: 2002-05-15
Project end date: 2007-04-30
5R01AI052774-05 (2006): $311699
5R01AI052774-04 (2005): $319200
5R01AI052774-03 (2004): $319200
5R01AI052774-02 (2003): $319200
Sponsored Links Excellgen http://Excellgen.com
1R01AI052774-01 (2002): $319200
LATE EVENTS IN RETROVIRUS ASSEMBLY
Paul D Bieniasz
Aaron Diamond Aids Research Center, 7th Floor, New York, Ny 10016-9102
Grant 5R01AI052774-08 from National Institute Of Allergy And Infectious Diseases
Abstract: In this competing renewal application we request support for our studies on late events in retrovirus assembly. In the previous funding cycle, we identified and characterized several cellular proteins that are recruited to sites of retrovirus assembly by viral late budding (L)-domains. These proteins are either (i) components of the class E vacuolar protein sorting (VPS) pathway that is normally involved in the manipulation of endosomes and multivesicular bodies (MVB) or (ii) ubiquitin ligases. For several of these factors we were able to demonstrate a critical role in the release of infectious retroviral particles. Several mechanistic questions remain, however, and recently we have identified additional cellular factors that seem very likely to be involved in the late stages of the construction and release of retrovirus particles, and/or endosome/MVB biogenesis. In the next grant period, we will attempt to elucidate how previously defined and newly identified cellular factors participate in the late stages of retrovirus assembly and related cellular functions by pursuing two specific aims. Specific Aim 1 is to understand how specific endosomal proteins are incorporated into HIV-1 particles and their role in generating infectious virions. Specific aim 2 is to determine the role of ubiquitin, ubiquitin ligases and novel ubiquitin ligase binding proteins, discovered during the previous funding period, in retroviral budding and trafficking of endocytosed proteins. Execution of these two aims should result in significant progress toward our long term goal of understanding how cellular and viral factors cooperate in the construction of complete, infectious retroviral particles. Understanding these events could potentially provide new opportunities to interfere in these processes for the treatment of diseases caused by HIV and other enveloped viruses. Many enveloped viruses, for example HIV-1, are responsible for life threatening infections. To spread from cell to cell they make use of cellular proteins to facilitate the release of accurately assembled virus particles. Understanding how this system works could give opportunities to intervene with targeted therapeutics
Keywords: AIDS Virus; APF-1; ATP-Dependent Proteolysis Factor 1; Acquired Immune Deficiency Syndrome Virus; Acquired Immunodeficiency Syndrome Virus; Binding Proteins; Biogenesis; Cell Function; Cell Process; Cell physiology; Cells; Cellular Function; Cellular Physiology; Cellular Process; Clathrin; Disease; Disorder; Endocytosis; Endosomes; Event; Funding; Goals; Grant; HIV; HIV-1; HIV-I; HIV1; HMG-20; HTLV-III; High Mobility Protein 20; Human Immunodeficiency Viruses; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; Human immunodeficiency virus 1; Immunodeficiency Virus Type 1, Human; Infection; LAV-HTLV-III; Life; Ligand Binding Protein; Lymphadenopathy-Associated Virus; MVB; Mediating; Multivesicular Body; NIH RFA; Origin of Life; Pathway interactions; Process; Proteins; Receptosomes; Recruitment Activity; Request for Applications; Retroviridae; Retroviruses; Role; Site; Sorting - Cell Movement; Staging; Subcellular Process; System; System, LOINC Axis 4; Ubiquitin; VPS; Vacuolar Protein Sorting; Viral; Virion; Virus; Virus Particle; Virus-HIV; Virus-Retrovirus; Viruses, General; Work; disease/disorder; gene product; human T cell leukemia virus III; human T lymphotropic virus III; novel; particle; pathway; public health relevance; recruit; social role; sorting; therapeutic target; trafficking; ubiquitin ligase
Project start date: 2002-05-15
Project end date: 2012-05-31
Budget start date: 1-JUN-2009
Budget end date: 31-MAY-2010
5R01AI052774-08 (2009): $339054
5R01AI052774-07 (2008): $396815
2R01AI052774-06A1 (2007): $404500
Paul D Bieniasz
Aaron Diamond Aids Research Center
Project start date: 2005-01-01
Project end date: 2016-01-31