The objective of this proposal is to identify the function of HIV-1 Vif and determine the role of Vif in the virus life cycle. The long-term objective of these studies is to identify new molecular mechanisms in the HIV-1 virus life cycle which could potentially serve as targets for antiviral therapy. Vif is potentially an excellent target for antiviral therapy since its function is essential for HIV-1 replication in peripheral blood T lymphocytes and monocyte/macrophages. There are currently no assay to screen drugs which inhibit Vif, since its mechanism of action and active sites are unknown. Vif-defective viruses enter cells normally but are defective in their ability to complete proviral DNA synthesis. Recently, we have shown that Vif-defective virions show an abnormality in the structure of the virion core. More significantly, we have examined the effect of Vif on the endogenous reverse transcriptase reaction in vitro and have identified a major defect in the ability of vif-defective virions to synthesize viral DNA using endogenous viral RNA as a template. These results are significant because they indicate that a component of the virion core, such as the viral RNA genome or one of the gag or gag-pol gene products, is likely to be an important target for Vif function. The endogenous reverse transcriptase reaction and other molecular approaches will be used to identify the mechanism of the effect of Vif on proviral DNA synthesis. These in vitro studies will be complemented by studies in cell lines and primary cells to determine biological significance during early events in vivo. Studies on the role of Vif in virus assembly and maturation will include examination of the effect of Vif on the viral RNA genome and viral RNA-nucleocapsid interactions and the possibility of a direct interaction between Vif and Gag during virus production. Vif mutants in an infectious molecular clone will be used to determine the biological significance of Vif phenotypes during virus replication and to integrate data from the different sections of this proposal. Additional insights into the biochemistry and cell biology of Vif resulting from other ongoing studies in the laboratory will be used to assess new phenotypes of Vif as the project progresses. These studies may lead to the identification of mechanisms relevant ot Vif function which could potentially serve as targets for therapeutic intervention. The broad range of studies on several critical steps in the retrovirus life cycle, including reverse transcription, formation of the DNA provirus, and the organization of the viral RNA genome in the virion, might also provide insights which may be relevant for the development of retroviral vectors.
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