SUPPORT FOR VIROLOGICAL/BIOCHEMICAL IDENTIFICATION OF INHIBITORS OF HIV-1 KINASES

我们实验室的研究集中在控制HIV-1的因素上 传染性，特别是病毒决定因素， 病毒核酸从病毒进入点向宿主的转运 细胞核 HIV-1 gag MA蛋白是一种主要的结构病毒体蛋白， 是核蛋白逆转录复合物的组分， 便于病毒核酸的核运输。 此外，gag mA 豆蔻酰化是将gag多聚蛋白转运到 用于掺入成熟病毒体的膜。 因此，gag MA表现出 膜和核靶向功能。 我们已经确定 gag MA的磷酸化通过以下方式调节这些相反的靶向功能： 促进逆转录复合物从 靶细胞膜，从而允许复合物进行核 导入. 阻止gag MA磷酸化的激酶抑制剂抑制 逆转录复合物易位到宿主细胞核 从而防止靶细胞的有效感染。 的对象 病毒学和试剂开发部分将支持以下工作： 开发使gag MA磷酸化的激酶的特异性抑制剂， 分别克隆这些激酶。 此外，该部门还将开展 激酶抑制剂和转显性激酶突变体的机制分析。 病毒学和试剂支持组件的具体目标包括： 目的1：鉴定合适的gag MA磷酸化底物和激酶 来源，将用于努力纯化和克隆所涉及的 激酶。 这些试剂还将用于衍生 用于高通量筛选抑制化合物的体外测定 体外激酶活性。 目的2：检测铅激酶抑制剂的抗病毒特性及其 类似物在体外进行，并确认其抑制机制。 目的3：检查反式显性的抗病毒特性和特异性 使gag MA磷酸化的细胞激酶的突变体。

Research in our laboratory has focused on factors which govern HIV-1 infectivity and, in particular, viral determinants which facilitate transport of viral nucleic acids from the point of virus entry to the host cell nucleus. The HIV-1 gag MA protein, a major structural virion protein, is a component of the nucleoprotein reverse transcription complex and facilities nuclear transport of viral nucleic acids. In addition, gag mA myristoylation is required for transport of gag polyproteins to the membrane for incorporation into maturing virions. Thus, gag MA exhibits both membrane and nuclear targeting functions. We have determined that phosphorylation of gag MA regulates these opposing targeting functions by facilitating dissociation of reverse transcription complexes from the target cell membrane thereby allowing the complex to undergo nuclear import. Kinase inhibitors which prevent phosphorylation of gag MA inhibit translocation of the reverse transcription complex to the host cell nucleus thereby preventing efficient infection of the target cell. The object of the virology and reagent development component is to support efforts to develop specific inhibitors of kinases which phosphorylate gag MA and to clone these kinases respectively. In addition, this component will conduct mechanistic analysis of kinase inhibitors and transdominant kinase mutants. Specific aims of the virology and reagent support components include: Aim 1: Identify suitable gag MA phosphorylation substrates and kinase sources which will be used in efforts to purify and clone the involved kinases. These reagents will additionally be used for derivation of in vitro assays for high throughput screening of compounds which inhibit kinase activity in vitro. Aim 2: Examine antiviral properties of lead kinase inhibitors and their analogs in vitro and confirm their mechanism of inhibition. Aim 3: Examine antiviral properties and specificities of transdominant mutants of the cellular kinases that phosphorylate gag MA.