Subunit Assembly and Substrate Interactions in HIV-1 RT

HIV-1 RT 中的亚基组装和底物相互作用

• 批准号: 7105246
• 负责人: MARY D BARKLEY
• 金额: $ 28.86万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-15 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7105246
• 关键词: DNA replication; RNA directed DNA polymerase; drug resistance; efavirenz; enzyme activity; enzyme mechanism; enzyme structure; fluorescence resonance energy transfer; human immunodeficiency virus 1; inhibitor /antagonist; mutant; nevirapine; pharmacokinetics; protein protein interaction; reverse transcriptase inhibitors; virus protein

DESCRIPTION (provided by applicant): 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 DMA provirus, 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.

描述（由申请人提供）：本提案的目标是研究蛋白质-配体和 HIV-1逆转录酶（RT）中的蛋白质-蛋白质相互作用。RT是HIV感染的关键分子，是大多数艾滋病药物的分子靶点。所研究的配体将包括五种非核苷逆转录酶抑制剂（NNRTI）以及正常底物。除了野生型之外，研究的RT将包括多达5种耐药突变体。该酶将单链基因组RNA复制到双链DNA前病毒中，随后通过HIV整合酶整合到宿主染色体中。RT具有RNA和DMA依赖性DMA聚合酶和RNase H活性。生物活性酶是p66和p51亚基的异源二聚体。p51亚基由HIV蛋白酶从p66亚基衍生而来。该提案应用生物化学和生物物理技术，包括强大的位点特异性荧光技术，对可能揭示抗病毒治疗新靶点的拟议抑制机制和蛋白质构象进行基础研究。该实验室是目前为数不多的使用溶液生物物理技术来研究RT的实验室之一。表征抑制剂与RT的结合。测定阻聚剂对DMA聚合中各个步骤的影响。3.研究抑制剂对RT亚基构象和动力学的影响。