Subunit Assembly and Substrate Interactions in HIV-1 RT

这项提议的目标是研究HIV-1逆转过程中蛋白质-配体和蛋白质-蛋白质的相互作用 转录酶(RT)。作为HIV感染的关键参与者，RT是大多数艾滋病人的分子靶标 毒品。研究的配体包括五个非核苷类逆转录酶抑制剂(NNRTI)以及 正常的底物。除了野生型外，研究的RTS还将包括最多5个抗药性突变体。这个 酶将单链基因组RNA复制到双链DNA前病毒中，这是 随后通过HIV整合酶整合到宿主染色体中。RT依赖RNA和DMA DNA聚合酶和核糖核酸酶H活性。生物活性酶是p66和p51的异源二聚体。 亚单位。P51亚基由p66亚基通过HIV蛋白酶衍生而来。该提案适用于生化 和生物物理技术，包括强大的特定部位荧光技术，以基础 研究提出的抑制机制和蛋白质构象，可能揭示新的靶点 抗病毒治疗。该实验室是目前为数不多的使用溶液生物物理技术研究RT的实验室之一。 具体来说，我们建议： 1.鉴定与RT结合的抑制物。 2.测定阻聚剂对DMA聚合各步骤的抑制作用。 3.研究抑制剂对RT亚基和组装的构象和动力学的影响。

The goal of this proposal is to investigate protein-ligand and protein-protein interactions in HIV-1 reverse transcriptase (RT). Being a key player in HIV infection, RT is the molecular target of the majority of AIDS drugs. Ligands studied will include five nonnucleoside reverse transcriptase inhbitiors (NNRTI) as well as normal substrates. RTs studied will include up to five drug resistance mutants in addition to wild type. The enzyme copies the single-stranded genomic RNA into a double-stranded DMAprovirus, which is subsequently integrated into the host chromosome by HIV integrase. RT has RNA- and DMA-dependent DMA polymerase and RNase H activities. The biologically active enzyme is a heterodimer of p66 and p51 subunits. The p51 subunit is derived from p66 subunit by HIV protease. The proposal applies biochemical and biophysical techniques, including powerful site-specific fluorescence techniques, to fundamental investigations of proposed inhibition mechanisms and protein conformations that may reveal new targets for antiviral therapy. This laboratory is presently one of few using solution biophysical techniques to study RT. Specifically, we propose to: 1. Characterize inhibitor binding to RT. 2. Determine inhibitor effects on individual steps in DMA polymerization. 3. Investigate inhibitor effects on conformation and dynamics of RT subunits and assembly.