Allosteric regulation of a viral RNA-dependent RNA polymerase

 描述（由申请方提供）：RNA病毒是现有和新出现的人类病原体中最大的一类，可引起危及生命的胃肠道、呼吸道、出血性和神经系统疾病。尽管RNA病毒在其基因组序列、病毒体结构、组织嗜性和病理表现方面可能差异很大，但它们都编码一种称为RNA依赖性RNA聚合酶（RdRp）的专门酶。RdRp对病毒复制至关重要，因为它介导病毒RNA合成的所有阶段。然而，病毒RdRp的活性必须在感染过程中受到调节，以便RNA合成可以与病毒生命周期中的其他步骤协调。在许多情况下，这种调节是由效应蛋白与RdRp表面上的变构位点的结合介导的，所述变构位点在物理上不同于活性位点。变构位点的效应蛋白的结合诱导一系列的分子内构象变化的RdRp，最终在活性位点调节酶的功能。这个建议的总体目标是获得机制的洞察变构RdRp调节轮状病毒作为一个结构和功能易于处理的实验系统。轮状病毒是一种双链RNA病毒，可引起幼儿严重胃肠炎。轮状病毒RdRp（VP 1）的活性要求其直接与核壳效应蛋白（VP 2）接合。然而，关于（i）哪些表面暴露的VP 1残基包含变构活化位点和（ii）哪些掩埋的VP 1残基将变构信号从表面传递到活性位点存在关键的知识缺口。提出了两个综合但独立的具体目标，以帮助缩小这些知识差距。在目标1中，将使用结构指导的功能获得性生化方法来绘制包含VP 1变构激活位点的精确残基。具体来说，嵌合和点 突变体VP 1蛋白将被工程化并测试它们在同源和非同源VP 2存在下介导体外RNA合成的能力。在目标2中，将采用计算机氨基酸共变异分析和分子动力学模拟来鉴定可能传递变构信号的掩埋的VP 1残基。然后使用突变体VP 1蛋白和体外RNA合成试验验证这些残基。在完成这项工作后，预计将产生自激活的VP 1突变体，并将确定参与VP 1变构激活的精确氨基酸残基。这项提议是创新的，因为它使用基于序列和结构-功能的方法来研究VP 1的酶活性如何被VP 2调节的原始想法。这项工作意义重大，因为它将揭示轮状病毒RdRp与其他致病RNA病毒共有的特征，这反过来可能会促进变构抗病毒药物的开发，以治疗和预防病毒性疾病。

 DESCRIPTION (provided by applicant): RNA viruses represent the largest class of existing and emerging human pathogens, causing life-threatening gastrointestinal, respiratory, hemorrhagic, and neurological diseases. Although RNA viruses can vary widely in their genome sequences, virion architectures, tissue tropism, and pathological manifestations, they all encode a specialized enzyme called an RNA-dependent RNA polymerase (RdRp). The RdRp is critical for viral replication, as it mediates all stages of viral RNA synthesis. Yet, the activity of the vral RdRp must be regulated during infection so that RNA synthesis can be coordinated with other steps in the viral lifecycle. In many cases, such regulation is mediated by the binding of an effector protein to an allosteric site on the RdRp surface, which is physically distinct from the active site. Binding of the allosteric site by the effector protein induces a series of intra-molecular conformational changes in the RdRp that culminate at the active site to modulate enzyme function. The overall objective of this proposal is to gain mechanistic insight into allosteric RdRp regulation using rotavirus as a structurally- and functionally-tractable experimental system. Rotavirus is a double-stranded RNA virus that causes severe gastroenteritis in young children. The activity of the rotavirus RdRp (VP1) requires that it be directly engaged by the core shell effector protein (VP2). However, key gaps in knowledge exist about (i) which surface-exposed VP1 residues comprise the allosteric activation site and (ii) which buried VP1 residues transmit the allosteric signal from the surface to the active site. Two integrated, yet independent, specific aims are proposed to help close these gaps in knowledge. In Aim 1, a structure-guided, gain-of-function biochemical approach will be used to map the precise residues that comprise the VP1 allosteric activation site. Specifically, chimeric and point mutant VP1 proteins will be engineered and tested for their capacity to mediate in vitro RNA synthesis in the presence of cognate and non-cognate VP2. In Aim 2, in silico amino acid co-variation analysis and molecular dynamic simulations will be employed to identify buried VP1 residues that may transmit the allosteric signal. These residues will then be validated using mutant VP1 proteins and in vitro RNA synthesis assays. Upon completion of this work, it is expected that an auto-activated VP1 mutant will have been created, and the precise amino acid residues involved in VP1 allosteric activation will have been defined. This proposal is innovative because it uses sequence-based and structure-function methodologies to investigate original ideas about how the enzymatic activity of VP1 is regulated by VP2. The work is significant because it will reveal features of the rotavirus RdRp that are shared with those of other pathogenic RNA viruses, which may in-turn foster the development of allosteric antiviral drugs to treat and prevent viral diseases.