CAPSID-PROTEIN INTERACTIONS IMPORTANT FOR HIV ASSEMBLY

拟议研究的目的是确定Gag前体的区域 和成熟的p24，它们对感染性病毒的组装至关重要。 病毒体。 为此，蛋白质-蛋白质相互作用的区域，或区域 影响相互作用表面的形成或稳定性， 蛋白质将被识别。 蛋白质-蛋白质相互作用的区域将 通过直接和间接的方法来定义。 直接生化 方法将利用纯化的重组Gag和p24蛋白， 用于双官能化学交联剂的基材。 交互 在交联蛋白质的蛋白水解之后分离区域， 通过SDS-PAGE或凝胶过滤分离片段并鉴定 通过N-和C-末端氨基酸分析连接片段。低聚 将平行分析从感染性病毒体纯化的p24， 确定与重组体定义的相互作用的真实性 蛋白 间接遗传学方法将用于鉴定氨基酸 对Gag多聚化至关重要的酸残基。 该测定使用 杂合蛋白，其相互作用导致 GAL 4转录激活因子。 Gag-Gag相互作用导致 转录的报告基因，GAL-lacZ，其中含有结合 GAL 4的网站。 涉及多聚化的关键序列将是 通过诱变来定义。使用这些直接识别的交互域 间接方法将被评估其潜在的干扰 通过确定突变的影响， 在这些区域中，培养的哺乳动物细胞中的颗粒组装。 的 这些研究的长期目标是l）设计可以竞争 对于帽ID亚基上的相互作用位点， 组装; 2）设计可以干扰衣壳脱壳的试剂。

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.