STRUCTURE BASED STUDIES OF RNA BINDING PROTEINS FROM HIV

(改编自申请人的摘要)一个35个碱基的RNA茎环，称为 位于HIV基因组5‘端附近的SL-1介导了几种 对病毒组装至关重要的功能，因此是潜在的靶标 抗病毒治疗。通过结合HIV Gag蛋白，SL1形成了 包装信号以基因组RNA为靶点形成病毒粒子。之间的联系 一对HIV RNA中的SL1元件(通过最初的“接吻环” 然后转化为线性双链的复合体)也启动了基因组 二聚化--一个由GAG推动的过程，对于 完全的传染性。SL1的所有生物学活性都依赖于它的三个- 维度结构，其确切性质尚不清楚。PIS实验室 已经使用核磁共振光谱学检查了其 亲吻环和线性双链构象。他们取得了优异的成绩 RNA上的核磁共振波谱。他们提出要解决 单体和两种二聚体形式的这种RNA，从而获得第一个高- 逆转录病毒二聚体界面的分辨率图像。在这些指导下 结构，他们将系统地诱变SL1以映射分子 稳定每个构象状态所需的接触。他们还提议 使用实时核磁共振动力学研究来研究事件的顺序 SL1二聚化。异核标记核糖核酸的多维核磁共振研究 用于解决全长SL1模拟的结构，该结构完全 能胜任缝隙结合和二聚反应。对该RNA的研究 与未标记的GAG蛋白的复合体将揭示 GAG识别及其促进SL1介导的机制 二聚化。这些研究的结果可能会为理性的 针对SL1/GAG相互作用的新型抗病毒药物的设计。

(Adapted from the applicant's abstract) A 35-base RNA stem-loop called SL-1, located near the 5 ' end of the HIV genome, mediates several functions crucial for viral assembly, and so is a potential target for antiviral therapy. By binding the HIV Gag protein, SL1 forms part of the packaging signal that targets genomic RNA into virions. Contact between SL1 elements in a pair of HIV RNAs (through an initial "kissing-loop" complex which then converts to a linear duplex) also initiates genomic dimerization--a process that is facilitated by Gag and is essential for full infectivity. All biological activities of SL1 depend upon its three- dimensional structure, whose exact nature is unknown. The PIs laboratory has used NMR spectroscopy to examine a truncated form of SL1 in its kissing-loop and linear duplex conformations. They have obtained excellent NMR spectra on the RNA. They propose to solve the structures of the monomer and both dimer forms of this RNA, and so to obtain the first high- resolution images of a retroviral dimer interface. Guided by these structures, they will systematically mutagenize SL1 to map the molecular contacts needed to stabilize each conformational state. They also propose to use real-time NMR kinetic studies to study the sequence of events in SL1 dimerization. Multidimensional NMR on heteronuclear labeled RNA will be used to solve the structure of a full-length SL1 analogue that is fully competent for Gag binding as well as dimerization. Studies of this RNA in complex with unlabeled Gag protein will reveal the structural basis for Gag recognition and the mechanism by which Gag facilitates SL1-mediated dimerization. Results of these studies may provide a basis for rational design of new antivirals that target the SL1/Gag interaction.