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

我们实验室的研究重点是控制HIV-1病毒的因素 传染性，特别是病毒决定因素，使 病毒核酸从病毒入口点到宿主的运输 细胞核。HIV-1 Gag MA蛋白是一种主要的结构病毒蛋白， 是核蛋白逆转录复合体的一个组成部分， 病毒核酸的核运输设施。此外，搞笑马云 肉豆蔻酰化是GAG多蛋白运输到 用于掺入成熟病毒粒子的膜。因此，GAG MA展示了 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 促进反转录复合体从 靶细胞膜，从而允许复合体经历核 导入。阻止GAG-MA磷酸化的激酶抑制剂抑制 反转录复合体向宿主细胞核的移位 从而防止对目标细胞的有效感染。的目的 病毒学和试剂开发部分是为了支持 开发特异性的磷酸化GAG MA和TO的酶抑制剂 分别克隆这些激酶。此外，该构成部分将进行 激酶抑制剂和转显性激酶突变体的机理分析。 病毒学和试剂支持部分的具体目标包括： 目的1：确定合适的GAG MA磷酸化底物和激酶 将用于提纯和克隆涉案人员的来源 激活剂。这些试剂还将用于衍生In 高通量筛选抑制活性化合物的体外检测方法 体外激活酶活性。 目的2：检测铅激酶抑制剂的抗病毒特性及其 并证实了它们的抑制机制。 目的3：检查易位基因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.