Mechanism/inhibition of RNA binding functions of HIV Rev

HIV Rev RNA 结合功能的机制/抑制

• 批准号: 6821914
• 负责人: David P MILLAR
• 金额: $ 21.37万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6821914
• 关键词: antiviral agents; chemical binding; combinatorial chemistry; fluorescence resonance energy transfer; genetic regulatory element; human immunodeficiency virus; inhibitor /antagonist; messenger RNA; molecular assembly /self assembly; transcription factor; virus protein; virus replication

The overall goal of this program component is to elucidate the mechanism by which HIV Rev binds to the Rev Response Element and then assembles an oligomeric ribonucleoprotein (RNP) complex that mediates nuclear export of the viral mRNA. In parallel with the mechanistic studies, we will develop novel fluorescence-based assays that will be used to screen compound libraries for inhibitors of the RNA-binding and oligomerization functions of Rev. We will also perform biophysical studies of compounds identified in these screens to elucidate their mechanism of action. This project will lead to a detailed understanding of the kinetic pathway and cooperativity of oligomeric RNP complex assembly. In addition to providing information on basic Rev function, an understanding of the assembly mechanism will contribute to efforts to create small molecules that disrupt proper assembly of the RNP compex and thereby prevent export of the viral mRNA from the nucleus. The Specific Aims are: (1) Elucidate the mechanism of multimeric Rev assembly on the RRE. (2) Characterize RNA conformational changes induced during multimeric Rev-RRE complex assembly. (3) Develop fluorescence-based screening assays to identify small molecules that interfere with Rev-RRE binding or Rev-Rev multimerization. (4) Establish the mechanism of inhibition of small molecules selected from the fluorescence-based screens. A novel single-molecule fluorescence imaging technique will be used to observe spontaneous assembly of multimeric Rev complexes on a critical fragment of the RRE, revealing the sequence of events and the molecular basis for cooperative assembly. Fluorescence resonance energy transfer (FRET) methods will be used to probe the structure of the RRE fragment at each stage of complex assembly, in both ensemble and single-molecule experiments. These spectroscopic methods will be adapted for use in screening compound libraries generated in other program components, allowing for specific selection of molecules that disrupt the initial binding of Rev to the RRE or the subsequent oligomerization steps. This project will support the drug discovery effort and help to identify interesting inhibitor-target complexes for structural characterization in other program components.

该项目的总体目标是阐明HIV Rev与Rev反应元件结合，然后组装介导病毒mRNA核输出的寡聚核糖核蛋白（RNP）复合物的机制。在平行的机制研究，我们将开发新的荧光为基础的测定，将用于筛选化合物库的RNA结合和寡聚化功能的Rev的抑制剂，我们还将进行生物物理研究的化合物在这些屏幕中确定，以阐明其作用机制。该项目将导致对寡聚RNP复合物组装的动力学途径和协同性的详细了解。除了提供有关基本Rev功能的信息外，了解装配机制将有助于 产生破坏RNP复合体的正确组装的小分子，从而阻止病毒mRNA从细胞核输出。具体目的是：（1）阐明多聚体Rev在RRE上的组装机制。(2)表征多聚体Rev-RRE复合物组装期间诱导的RNA构象变化。(3)开发基于荧光的筛选试验，以鉴定干扰Rev-RRE结合或Rev-Rev多聚化的小分子。(4)建立从基于荧光的筛选中选择的小分子的抑制机制。一种新的单分子荧光成像技术将被用来观察多聚Rev复合物的RRE的关键片段的自发组装，揭示了事件的顺序和合作组装的分子基础。 荧光共振能量转移（FRET）方法将被用来探测在复杂的组件的每个阶段的RRE片段的结构，在合奏和单分子实验。这些光谱方法将适用于筛选在其他程序组件中生成的化合物文库，从而允许特异性选择破坏Rev与RRE的初始结合或后续寡聚化步骤的分子。该项目将支持药物发现工作，并帮助确定有趣的靶标复合物，用于其他项目组成部分的结构表征。