Structure-Based Studies of RNA Binding Proteins from HIV

描述(由申请人提供)：名为SL1的35个碱基的RNA茎环， 位于HIV-1基因组5‘端附近，调节几个至关重要的功能 用于病毒组装，因此是抗病毒治疗的潜在新靶点。通过 SL1与HIV Gag蛋白的核衣壳(NCp7)部分结合，形成部分 将基因组RNA定位为病毒粒子的包装信号。之间的联系 SL1环在一对H1V RNA中(形成一个初始的“接吻环”复合体， 然后重新折叠成线性双链)也启动了基因组二聚化 由NCp7绑定促进的过程，对于完全 传染性。SL1的所有生物学活性都依赖于它的三维结构 结构。在之前的资助期间，我们使用了核磁 用核磁共振波谱研究截短的23碱基SL1的结构 它的接吻环和线性导数形成了第一个这样的结构 以解决任何逆转录病毒的问题。使用异核标记和 多维核磁共振，我们现在提出解决相应的结构 全长、可信的SL1，它包含以下附加功能 在生物学上很重要。通过测定SL1单体、接吻环和 线性结构，我们试图理解其显著的效率 SL1二聚体和稳定每一个连续的 构象。我们还将确定络合物的结构和动力学 当HIV NCp7蛋白与每个RNA构象结合时形成的，揭示了 NCp7参与的分子接触及其促进机制 SL1和其他核酸的折叠。我们的结构的有效性 然后将通过SL1和NCp7的定向突变来测试洞察力。 实时核磁共振将用于分析发生在以下位置的事件序列 在体外，当SL1二聚化，然后线性化时，SL1中的单个碱基。在……里面 此外，我们还将解决NCp7与异源， 单体RNA配体(适配子)，与SL1结合亲和力更高， 确定导致其紧密绑定的功能，并可在 合理的药物设计。这些研究的结果将为 发现针对SL1/NCp7相互作用的新的抗病毒药物。

DESCRIPTION (provided by the applicant): A 35-base RNA stem-loop called SL1, located near the 5' end of the HIV-1 genome, mediates several functions crucial for viral assembly, and so is a potential new target for antiviral therapy. By binding the nucleocapsid (NCp7) portion of the HIV Gag protein, SL1 forms part of the packaging signal that targets genomic RNA into virions. Contact between SL1 loops in a pair of H1V RNAs (forming an initial "kissing-loop" complex that then refolds into a linear duplex) also initiates genomic dimerization - a process that is facilitated by NCp7 binding and is essential for full infectivity. All biological activities of SL1 depend on its three-dimensional structure. During the previous funded period, we used nuclear magnetic resonance (NMR) spectroscopy to solve the structure of a truncated, 23-base SL1 derivative in both its kissing-loop and linear forms the first such structures to be solved for any retrovirus. Using heteronuclear labeling and multidimensional NMR, we now propose to solve the corresponding structures of full-length, authentic SL1, which contains additional features that are biologically important. By determining the SL1 monomer, kissing-loop, and linear structures, we seek to understand the remarkable efficiency with which SL1 dimerizes and the chemical features that stabilize each successive conformation. We will also determine the structures and dynamics of complexes formed when HIV NCp7 protein binds each RNA conformer, revealing the exact molecular contacts involved and the mechanism by which NCp7 facilitates refolding of SL1 and other nucleic acids. The validity of our structural insights will then be tested through targeted mutagenesis of SL1 and NCp7. Real-time NMR will be used to dissect the sequence of events that occur at individual bases in SL1 as it dimerizes and then linearizes in vitro. In addition, we will solve the complex formed by NCp7 with a heterologous, monomeric RNA ligand (aptamer) that binds with higher affinity than SL1, to identify features that account for its tight binding and could be exploited in rational drug design. Results of these studies will provide a basis for discovering new antivirals that target the SL1/NCp7 interaction.