Mechanism/inhibition of RNA binding functions of HIV Rev

该程序组件的总体目标是阐明HIV Rev与REV反应元件结合的机制，然后组装一个寡聚核糖核蛋白(RNP)复合体，介导病毒mRNA的核输出。在机理研究的同时，我们将开发新的基于荧光的分析方法，用于筛选REV的RNA结合和寡聚功能抑制剂的化合物文库。我们还将对这些筛选中确定的化合物进行生物物理研究，以阐明它们的作用机制。这个项目将导致对低聚RNP复合体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，对组装机制的了解还将有助于努力 产生小分子，扰乱RNP复合体的正确组装，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复合体组装过程中的RNA构象变化。(3)建立基于荧光的筛选方法，以确定干扰REV-RRE结合或REV-REV多聚体的小分子。(4)建立荧光筛选小分子的抑制机制。一种新颖的单分子荧光成像技术将被用来观察多聚体REV络合物在RRE关键片段上的自发组装，揭示事件的顺序和合作组装的分子基础。 在系综和单分子实验中，将使用荧光共振能量转移(FRET)方法来探测复杂组装每个阶段的RRE片段的结构。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，允许特定选择破坏REV与RRE的初始结合或后续齐聚步骤的分子。该项目将支持药物发现工作，并帮助识别感兴趣的抑制剂-目标复合体，以用于其他计划组成部分的结构表征。

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.