Productive infection of lymphocytes and macrophages derived from human blood, as well as a number of T cell lines, by the pathogenic retrovirus human immunodeficiency virus type-1 (HIV-1) requires expression of the virally encoded Vif protein (an acronym for Virion Infectivity Factor). To date, relatively little is understood regarding the mechanism(s) by which Vif exerts its profound effect on the viral life cycle. Broadly speaking, the objectives of the research outlined here are to define the role of Vif in HIV-1 replication and to determine the molecular basis for this activity. The proposed experiments are divided into four sections. One, stable cell lines (constitutive and inducible) that produce high levels of viral particles in the presence and absence of Vif and a transient assay system for monitoring Vif activity will both be developed. These reagents and assays, together with expression vectors and Vif specific antibodies previously developed in this laboratory, will then be exploited throughout the course of this project. Two, a variety of experimental approaches and techniques will be utilized to determine the stage(s) of the HIV-1 life cycle that is modulated by Vif and to identify the underlying biochemical basis for this effect. To these ends, a thorough compositional and phenotypic analysis of virions synthesized in the presence and absence of Vif will be undertaken. Three, and in parallel with the above, the critical amino acid residues and essential domains that are required for Vif activity will be defined and delineated following the introduction of a series of scanning missense and deletion mutations into the HIV-1 vif gene. Four, revertant viruses that have acquired the ability to replicate in the absence of Vif in cells that are otherwise non-permissive will be characterized; it is likely that the nature of these mutation(s) will help to identify the proposed virally encoded target (or substrate) of Vif. Taken together, it is anticipated that these studies will provide novel information pertaining to the function of this essential viral gene. Increasing our understanding of this aspect HIV-1 replication could suggest novel strategies and targets for the treatment and prevention of HIV-1 infections.
