Vif is essential to the replication of primate lentiviruses such as HIV-1 and SIV in vivo. In the last two years, a considerable amount of insight has been gained into the mechanism by which Vif supports viral replication. Vif counteracts the antiviral effects of the cytidine deaminases apobec 3G and 3F. To inhibit viral replication, these cytidine deaminases must be packaged within virions where they edit the minus strand of nascent viral cDNA in the target cell. Vif counteracts the antiviral activity of apobec 3G and 3F by promoting their proteasomal degradation to the extent that there are insufficient levels for packaging into virions. The overall goal of this project is to identify and develop Vif antagonists that can be used to assess the antiviral impact of Vif antagonism in vivo. Project 2 will prioritize Vif antagonists that emerge from the inhibitor screen of Project 1 on the basis of antiviral activity and specificity. Since the most favorable Vif antagonists will be evaluated in the macaque model of SIV pathogenesis and SIV-induced encephalitis, antiviral activity will be evaluated in cells that are relevant to CNS infection namely, primary macrophages. Project 2 will also assess mechanisms of inhibitor escape in vitro and in vivo. The major activities of Project 2 include: Evaluation of antiviral activity and specificity in cells that express apobec (non-permissive cells) and cells that do not express apobec (permissive cells). Evaluation of antiviral activity in CNS-relevant target cells in vitro, namely, primary macrophages. Identification of the mechanism of Vif antagonism by assessing impact of Vif antagonists on virion encapsidation of apobec and on apobec-mediated editing of viral cDNA. Identification of mutations in Vif that confer inhibitor resistance in vitro and in vivo. Collectively, these studies will allow us to identify the most promising lead Vif antagonists and prioritize the selection of inhibitors that will be evaluated in an experimental model of SIV replication and SIV-induced encephalitis in macaques.