Retroviruses are important etiological agents of human disease most notable causing acquired immune deficiency syndrome (AIDS). The molecular events by which progeny retroviruses are produced is poorly understood, though fundamental to the virus replication cycle. In this application for a Physician-Scientist Award, a research training program is proposed to address retrovirus assembly, while preparing the P.I. for an independent research career. The retrovirus to be studied in this program is the AIDS virus HIV. The HIV-1 Gag protein directs retroviral assembly (budding), although its molecular mechanism remains obscure. Since budding is essential for the production of infectious virions, its disruption is a potential target for antiretroviral therapy. In the Phase I research plan, three separate projects are proposed. First, an extensive deletion analysis will be performed to map the essential and nonessential regions of HIV-1 Gag. Those portions which are identified as critical to budding will be further characterized. Specifically, the domain of Gag which associates with the cytoplasmic membrane will be mapped to allow further study of the Gag-membrane interaction, which is believed to be the first step in retroviral assembly. Once membrane-associated, the Gag proteins form aggregates which provide the nucleation site for assembly. The specific domains of the Gag protein which are responsible for these Gag-Gag interactions will be identified and characterized. The second project will focus on the development of an in vitro assay to study how the HIV-1 Gag protein interacts with the cytoplasmic membrane. Using a cell-free system similar to. that used to identify the membrane receptor for p60(src) (Resh and Ling, 1990), in vitro-translated Gag proteins will be incubated with osmotically-lysed cell membrane preparations and subjected to cell fractionation studies. If specific Gag-membrane associations can be detected, we will proceed with experiments aimed at the identification of the putative cytoplasmic receptor for HIV-1 Gag.
The third aim of this proposal is a pilot project to study in vivo the cellular factors involved in HIV-1 Gag-mediated assembly. Previous studies of Gag function in yeast have demonstrated that retroviral Gag proteins associate with the cell membrane, but the budding process is inhibited, likely by the rigid cell wall. We propose to study Gag function in Dictyostelium, a wall-less lower eukaryote with a well-established genetic system. If HIV-1 Gag can mediate budding in Dictyostelium, powerful genetic analyses can be performed to elucidate the role of cell-encoded proteins in the retroviral assembly apparatus. Phase II is planned as an intense laboratory experience which will develop the candidate's independent research career. A general research plan is included for Phase II, which will be based on the results of the experiments planned in the Phase I research program.
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