Structure of Oligomeric HIV Rev-RRE Complexes

HIV-1病毒mRNA的核输出是由调节因子Rev.的活性介导的。 REV功能的重要性在于自我关联的能力。在REV作为单体结合到其主峰之后 亲和力RRE结合位点，蛋白质-蛋白质和蛋白质-RNA协同作用的组合 调节二级结合部位的占据，导致低聚REV-RRE复合体的形成。仅限 低聚REV-RRE络合物具有核出口能力。该提案概述了结构研究，使用 液态核磁共振方法与生化方法相结合将揭示详细的 将多个版本拷贝组装到RRE上的性质。 1)REV和REV突变体在无RRE和存在RRE的情况下自结合的特征。雷夫 在液态核磁共振测量所需的浓度下在溶液中燃烧。缓冲器的筛选 将进行溶液中REV的结构分析。我们想要识别寡聚 适用于高分辨率核磁共振的REV缺陷型突变体。我们将验证最初的高亲和力相互作用 使用RRE茎环IIB，并将确定PAGE和ITC分析形成的络合物的化学计量比。 2)基于核磁共振方法研究低聚REV/RRE络合物在溶液中的结构。我们 计划解决Rev二聚体的结构，洞察Rev强烈的自我联想倾向。 将对来自茎II的不同目标RRE结构进行研究。我们会 试图确定与RRE茎环IIB结合的REV突变体(单体/二聚体)的结构 更大的RRE变体(茎环II)。 3)以配体结构为中心表征REV-RRE-抑制剂相互作用的结构特征。 我们将尝试确定与REV和/或RRE结合的小分子抑制剂的结构 使用现代核磁共振方法。我们希望使用5-氟嘧啶取代的RRE RNA在定向 利用~(19)F核磁共振进行筛选。基于三维结构，我们试图提供一种引线结构 用于合理开发和合成效价更高的缓蚀剂。

The nuclear export of HIV-1 viral mRNA is mediated by the activity of the regulator Rev. A feature of central importance of Rev function is the ability to self-associate. After Rev binds as a monomer to its primary high affinity RRE-binding site, a combination of cooperative protein-protein and protein-RNA interactions mediate the occupation of secondary binding sites resulting in oligomeric Rev-RRE complexes. Only oligomeric Rev-RRE complexes are nuclear export-competent. This proposal outlines structural studies using liquid state NMR methodology in combination with biochemical approaches that will reveal the detailed nature of the assembly of multiple Rev copies onto the RRE. 1) Characterization of Rev and Rev mutants self-association in the absence and presence of RRE. Rev fibrillizes in solution at concentrations required for liquid state NMR measurements. A screening of buffers facilitating structural analysis of Rev in solution will be performed. We want to identify oligomerization deficiant Rev mutants suitable for high-resolution NMR. We will verify the initial high affinity interaction with RRE stem-loop IIB and will determine stoichiometry of complexes formed by PAGE and ITC assays. 2) Structural Investigation of oligomeric Rev/RRE complexes in solution based on NMR methodology. We plan to solve the structure of Rev dimers providing insight into Rev's strong tendency to self-associate. Structural investigations of different target RRE constructs derived from stem II will be carried out. We will attempt to determine the structure of Rev mutants (monomeric/dimeric) bound to RRE stem-loop IIB and larger RRE variants (stem-loop II). 3) Characterization of structural features of Rev-RRE-inhibitor interactions focussing on ligand structures. We will attempt to determine the structure of the small molecule inhibitors, bound to either Rev and/or RRE using modem NMR methodology. We want to use 5-Fluoropyrimidine substituted RRE RNAs in directed screens utilizing 19F NMR. Based on the three-dimensional structure, we attempt to provide a lead structure for the rational development and synthesis of inhibitors with improved potency.