CAPSID-PROTEIN INTERACTIONS IMPORTANT FOR HIV ASSEMBLY

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The aim of the proposed research is to define regions of the Gag precursor and the mature p24 that are critical for assembly of the infectious virion. To this end, regions of protein-protein interaction, or regions that affect the formation or stability of interacting surfaces in these proteins will be identified. Regions of protein-protein interaction will be defined by direct and indirect approaches. Direct biochemical approaches will utilize purified recombinant Gag and p24 proteins as substrates for bifunctional chemical crosslinking agents. Interacting regions will be isolated following proteolysis of crosslinked proteins, separation of fragments by SDS-PAGE or gel filtration and identification of linked fragments by N- and C-terminal amino acid analyses. Oligomeric p24 purified from infectious virions will be analyzed in parallel to determine the authenticity of the interaction defined with the recombinant protein. An indirect genetic approach will be used to identify the amino acid residues that are critical for Gag multimerization. This assay uses hybrid proteins whose interactions result in reconstitution of the activity of the GAL4 transcriptional activator. Gag-Gag interactions lead to transcription of a reporter gene, GAL-lacZ, which contains a binding site for GAL4. Critical sequences involved in multimerization will be defined by mutagenesis. Interacting domains identified using these direct and indirect approaches will be evaluated for their potential to interfere with wild-type particle formation by determining the effects of mutation in these regions on particle assembly in mammalian cells in culture. The long-range goal of these studies is l) design of peptide that can compete for interacting sites on caps id subunits and thereby block particle assembly; 2) design of agents that can interfere with capsid uncoating.