The objective of this proposal is to characterize using the techniques of molecular virology the process of infection enhancement by HIV-1 Nef. A more complete mechanistic description of this process may facilitate the development of Nef as a target for therapeutic intervention in AIDS. The research design and methods are predicated on the following findings and hypotheses. Nef induces a modification of the virion that enhances infectivity. This modification is inclusion of Nef protein in the virion. Virions that are modified by Nef synthesize DNA with enhanced efficiency following entry into cells. Virion-associated Nef is modified by the viral specific protease to release a 20kd, C-terminal fragment from the full-length proteins; this fragment may be the active isoform of Nef in recipient cells. To test and elaborate this model, a variety of Nef mutants will be constructed and examined with respect to viral infectivity. These Nef-mutant-viruses will also be analyzed with respect to incorporation of Nef protein into virions, virion cores, and preintegration complexes. Mutational analysis will be used to determine whether proteolytic processing of Nef is required for enhancement of infectivity. If so, then they will determine whether the 20kd proteolytic fragment is present in sub-virion complexes and/or acts in trans in recipient cells. Genetic analysis will be used to determine whether serine residues in p17 are the viral targets for Nef action.
