This application is to determine the mechanism of Vif function in HIV-1 replication. Additional data has been supplied on acute infection of cells with Vif-negative HIV-1, physical structure of Vif-positive and Vif-negative virions, function of chimeric HIV-FIV Vifs, and inhibition of inhibitor-resistant protease (PR) by Vif. Based on these and earlier results it is proposed that Vif transiently inhibits 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 may ensure the orderly processing and packing of virion components and the infectivity of virus particles. This proposal 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 time course and sites of interaction of Vif during its control of activation of PR in HIV-1 infected cells. 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 the production of infectious virions. 4) to define the conservation of the Vif-PR interaction among lentiviruses. 5) to define the distinction between permissive and non-permissive 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. 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 identified. These studies will attempt to define the mechanism of action of a required HIV-1 protein and provide the basis for the development of antiviral agents.
| Lesner, Adam; Kartvelishvili, Alex; Lesniak, Jakob et al. (2004) Monoubiquitinated histone H1B is required for antiviral protection in CD4(+)T cells resistant to HIV-1. Biochemistry 43:16203-11 |
