MECHANISM OF ACTION OF HIV-1 VIF

St. Luke'S-Roosevelt Inst For Hlth Scis, New York NY

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Abstract

This is a second revised application for funds to test our hypothesis for the action of Vif in HIV-1 replication, based upon studies during HIV-1 infection of human cells and activity in models. We have supplied the requested additional data on acute infection of cells with Vif-negative HIV-1, phenotype of multiple Vif-negative clones, and construction and function of chimeric HIV/FIV Vifs. Based on these and earlier results the investigator proposes that Vif transiently inhibits protease (PR) in HIV-1 infected cells to prevent premature activation of the enzyme and that this inhibition is relieved during virion assembly. The sum of these events ensures the orderly processing and packing of virion components and the infectivity of virus particles. This Program will test this hypothesis by elucidation of the biochemical pathway of Vif function during infection by HIV-1 and other lentiviruses. The Specific Aims are: 1) To determine the timecourse and sites of interaction of Vif during its control of activation of PR in HIV-1 infected cells. We shall determine the sequential protein-protein interactions of Vif during the course of infection. 2) To determine the requirement for the relief of PR inhibition by Vif for production of infectious virions. 3) To determine the complementary activities of Vif and PR upon processing in production of infectious virions. 4) To define the conservation of the Vif-PR interaction among lentiviruses. 5) To define the distinction between permissive and nonpermissive cells on the basis of PR regulation by Vif. The bulk of the proposed studies will be conducted during HIV-1 infection of human cells, including acutely infected PBL, as suggested by Reviewers. The rate of synthesis, movement through the cell, and interaction with other proteins will be determined for Vif, Gag, and Gag-Pol through pulse-chase analysis and cellular fractionation. Subconstructs of Vif will be created in inducible vectors to identify functional domains. Chimeric HIV-1/FIV Vifs will be constructed to demarcate the regions of common function we have identified. These studies will define the mechanism of action of a required HIV-1 protein and provide the basis for the development of antiviral agents.