The overall goal of this revised grant application is to investigate the function and mechanism of action of vif in HIV- l infection of peripheral blood lymphocytes (PBL). vif has been considered an accessory (nonessential) gene for HIV-l infection in T cell lines, but its function is essential for stable HIV-l replication in PBL. Through the analysis of paired vif-positive and negative isogeneic HIV-l clones we have shown that the vif-negative virus replication is distinguished by inefficient viral DNA synthesis which results in impaired viral production in T cell lines and abortive infection in PBL. We hypothesize that in PBL vif is absolutely required for the proper assembly and/or maturation of progeny virus and that vif-negative virions produced in PBL are non-infectious due to defects in virus entry, internalization, or reverse transcription. To test this hypothesis we propose: l) To examine the physical and biochemical properties of vif -negative HIV- l produced in PBL; 2) To characterize the biologic dysfunctions of PBL-derived vif-negative HIV- related to defective viral DNA synthesis and abortive infection of PBL; 3) To investigate the mechanism of action of vif in HIV- l infection in PBL by studies of: a) subcellular localization of vif; b) role of vif in virion assembly; c) vif association with viral proteins; 4) To determine the functional domains of vif by mutagenesis; and 5) To analyze the structure and function of vif genes present in HIV- l infected persons. The proposed studies will employ paired vif-positive and vif-negative isogeneic HIV- l clones, vaccinia virus-based gag-pol and vif expression vectors, and retroviral vif expression vectors, to achieve HIV- l infection and efficient expression of specific viral products in normal PBL. vif-negative HIV- l will be produced in PBL through efficient single- cycle infection with a SupT l derived vif mutant HIV-l. vif-negative virions from PBL will be characterized by biophysical and biochemical methods, and tested for their binding, virus-cell fusion (by membrane fluorescence dequenching), viral DNA synthesis and integration (by PCR). Viral protein composition will be studied by radioimmunoprecipitation; vif-viral protein interaction by cross-linking and Western blotting; functional domains of vif by site directed mutagenesis and complementation assays. vif gene sequences will be amplified directly from PBL of HIV- positive patients by PCR, cloned, sequenced, and functionally evaluated. These studies will provide the fundamental knowledge regrading the control of HIV-l infection in PBL and will contribute the design of antiviral therapy to block HIV- l infection through vif.
