Allosteric regulation of a viral RNA-dependent RNA polymerase

 描述(申请人提供)：RNA病毒是现存的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、呼吸系统、出血性和神经系统疾病。尽管RNA病毒在基因组序列、病毒粒子结构、组织嗜性和病理表现上可能有很大的差异，但它们都编码一种称为RNA依赖的RNA聚合酶(RdRp)的特殊酶。RdRp对病毒复制至关重要，因为它调节病毒RNA合成的所有阶段。然而，病毒RdRp的活性必须在感染期间进行调节，以便RNA合成能够与病毒生命周期的其他步骤相协调。在许多情况下，这种调节是通过将效应蛋白与RdRp表面的变构位点结合而介导的，该变构位点在物理上与活性部位不同。效应蛋白与变构位点的结合导致RdRp在分子内发生一系列构象变化，最终在活性部位达到调节酶功能的目的。这项提议的总体目标是利用轮状病毒作为结构和功能上易于处理的实验系统来获得对变构RdRp调控的机械性洞察。轮状病毒是一种双链RNA病毒，会导致幼儿严重的胃肠炎。轮状病毒RdRp(VP1)的活性需要与核壳效应蛋白(VP2)直接结合。然而，关于(I)哪些表面暴露的VP1残基构成变构激活位点，以及(Ii)哪些埋藏的VP1残基将变构信号从表面传递到活性部位，存在着关键的知识空白。提出了两个综合但独立的具体目标，以帮助弥合这些知识差距。在目标1中，将使用结构指导的、功能获得的生化方法来绘制组成VP1变构激活位点的精确残基。具体地说，嵌合体和点 突变的VP1蛋白将被设计并测试它们在同源和非同源VP2存在的情况下调节体外RNA合成的能力。在目标2中，将利用氨基酸协变分析和分子动力学模拟来识别可能传递变构信号的埋藏的VP1残基。然后将使用突变的VP1蛋白和体外RNA合成试验来验证这些残基。在这项工作完成后，预计将创建一个自动激活的VP1突变体，并定义参与VP1变构激活的准确氨基酸残基。这一建议是创新的，因为它使用基于序列和结构功能的方法来调查关于VP1的酶活性如何由VP2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的特征，这些特征与其他致病RNA病毒的特征相同，可能反过来促进变构抗病毒药物的开发，以治疗和预防病毒疾病。

 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.