Despite great advances in AIDS diagnosis and treatment, the continuing devastation of the AIDS epidemic demands continuing efforts to understand all aspects of HIV replication, and to develop new methods for its inhibition. In pursuit of these goals, we have sought to define the activities of the HIV-1 structural (Gag) proteins so as to design antivirals that interfere with these functions. The Gag proteins are attractive targets since they perform multiple roles during the virus life cycle. The proteins initially are synthesized as N-terminally myristylated precursor (PrGag) proteins that employ their N-terminal matrix (MA) domains to adhere to cellular membranes and promote the assembly of immature virions. During viral morphogenesis from immature to mature virus particles, the viral protease cleaves PrGag into its mature constituents: MA, capsid (CA), spacer peptide 1 (SP1), nucleocapsid (NC), spacer peptide 2 (SP2), and p6. Concurrently, viral RNAs and enzymes, and cellular factors associate with NC proteins to form ribonucleoprotein complexes that are encased by highly organized conical or cylindrical cores composed of CA proteins. Using our previous studies and preliminary results as a foundation, we propose to dissect the mechanisms of membrane binding and mature core formation, and to characterize methods for their inhibition. Our results will help elucidate how the HIV assembly machinery operates;and will lead to the development of Gag-targeted antivirals, and an understanding of how they work. To achieve these ends, our specific aims are as follows: 1. Elucidation of the HIV-1 CA assembly pathway and analysis of Gag-targeted inhibitor action: The mechanics of HIV-1 Gag protein oligomerization and assembly will be characterized. Nucleation and growth steps and pathway alternatives will be examined with respect to their control by structural and environmental factors. The mechanisms of action of known and predicted Gag inhibitors will be determined in vitro. Our results will foster the design of practical and potent Gag-based antivirals 2. Examination of HIV-1 Gag membrane binding characteristics and binding partner interactions: The membrane binding preferences of HIV-1 Gag proteins will be measured with regard to phospholipid headgroup and hydrocarbon tail contributions, and the influences of other membrane components. The manner by which Gag proteins organize on membranes will be determined, the effects of potential inhibitors will be evaluated, and interactions between different binding partners will be analyzed. These studies will establish a basis for the development of therapeutics against new HIV targets.
The proposed investigations are designed to characterize how the structural proteins of HIV bind to membranes and assemble to form virus particles. The mechanisms used by inhibitors to block these activities will be determined, and will help lead to the development of new antivirals for the treatment of AIDS.
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