The HIV Vif protein is required during virus replication to overcome the antiviral activity of APOBEC3G (A3G) and APOBEC3F (ASF), cytidine deaminases that induce G to A hypermutation in newly synthesized viral DNA. In the absence of Vif, A3G and ASF incorporation into virions renders HIV non-infectious. Vif overcomes the antiviral activity of A3G by targeting it for destruction by the ubiquitin-proteasome pathway. Vif binds directly to A3G and forms an SCF-like E3 ubiquitin ligase complex with Cullin 5 (Cul5), Elongin B (EloB), and Elongin C (EloC) through a novel SOCS-box that binds EloC. Thus, Vif plays a role similar to that of F-box proteins, mediating substrate binding and specificity by serving as the bridge that recruits A3G to a Cul5 E3. Vif-induced proteasomal degradation of ASF is likely to occur through a similar mechanism. Vif binding to A3G and ASF and Vif-mediated degradation of these restriction factors are novel targets for drug development. In preliminary studies, we developed TRF and FRET assays for Vif-A3G binding and cell based assays for Vif-mediated degradation of A3G. Using these assays, we identified Vif Mabs and peptides that inhibit Vif-ASG binding and small molecules that increase A3G levels in the presence of Vif. The overall goal of this application is to develop novel screening assays to identify inhibitors of HIV Vif and its interactions with A3G. The studies will develop two complementary screening assays that will identify inhibitors with different mechanisms of action and demonstrate their suitability for high-throughput screens (HTS) using diverse collections of approximately 2,000 compounds.. Aim 1 will develop, optimize, and validate for HTS suitability a FRET-based assay to identify inhibitors of Vif-APOBECSG binding. To complement this approach, Aim 2 will develop, optimize, and validate for HTS suitability a cell-based screen to identify inhibitors of Vif-mediated degradation of APOBEC3G. Aim 3 will further characterize and test the best hits identified in FRET- and cell-based screens to identify those that inhibit Vif-dependent replication of HIV and narrow down their mechanism of action. By this multidisciplinary approach, these studies will provide novel insights into biological processes relevant for Vif function during HIV replication and will also facilitate antiviral drug discovery for HIV/AIDS. [unreadable] [unreadable] [unreadable] [unreadable]