Allosteric regulation of a viral RNA-dependent RNA polymerase

病毒RNA依赖性RNA聚合酶的变构调节

基本信息

批准号：
8952493
负责人：
Sarah Marie McDonald Esstman
金额：
$ 24.15万
依托单位：
VIRGINIA POLYTECHNIC INST AND ST UNIV
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-05-01 至 2017-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8952493
关键词：
Active Sites Algorithms Allosteric Regulation Allosteric Site Amino Acid Sequence Amino Acids Antiviral Agents Architecture Base Sequence Binding Biochemical Biological Assay Birds C-terminal Catalysis Cells Child Communication Computer Simulation Coupling Data Dengue Virus Development Double Stranded RNA Virus Drug Targeting Ebola virus Economic Burden Engineering Enzymes Event Exhibits Fingers Fostering Foundations Gastroenteritis Genes Hepatitis C virus Human Human poliovirus In Vitro Infection Influenza A virus Knowledge Laboratories Life Maps Mediating Medical Economics Methodology Molecular Motion Mutagenesis Mutation N-terminal Norovirus Nucleotides Pathway interactions Polioviruses Polymerase Process Protein Binding Proteins RNA RNA Viruses RNA chemical synthesis RNA-Directed RNA Polymerase Recombinants Regulation Research Resolution Roentgen Rays Rotavirus Sequence Alignment Series Signal Pathway Signal Transduction Site Site-Directed Mutagenesis Societies Specificity Staging Structure Surface System Testing Thumb structure Time Variant Viral Virion Virus Diseases Work base design gain of function gastrointestinal genome sequencing inhibitor/antagonist innovation insight molecular dynamics mutant nervous system disorder pathogen phosphodiester prevent public health relevance respiratory tissue tropism viral RNA virtual

项目摘要

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.

描述（由申请人提供）：RNA 病毒代表最大一类现有和新兴的人类病原体，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 病毒的基因组序列、病毒体结构、组织趋向性可能存在很大差异。和病理表现，它们都编码一种称为 RNA 依赖性 RNA 聚合酶 (RdRp) 的特殊酶。RdRp 对病毒复制至关重要，因为它介导病毒 RNA 的所有阶段。然而，vral 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相协调，在许多情况下，这种调节是通过效应蛋白与变构位点的结合来介导的。 RdRp 表面在物理上与活性位点不同，效应蛋白与变构位点的结合诱导了 RdRp 中的一系列分子内构象变化，最终在活性位点处达到顶峰。该提案的总体目标是使用轮状病毒作为结构和功能上易于处理的实验系统来了解变构 RdRp 调节的机制，轮状病毒是一种双链 RNA 病毒，可导致幼儿严重胃肠炎。轮状病毒 RdRp (VP1) 需要它直接与核壳效应蛋白 (VP2) 结合，但是，关于 (i) 表面暴露的知识存在关键空白。 VP1 残基包含变构激活位点，并且 (ii) 埋藏的 VP1 残基将变构信号从表面传递到活性位点。在目标 1 中，提出了两个综合但独立的具体目标。结构引导的功能获得性生化方法将用于绘制构成 VP1 变构激活位点的精确残基图谱，特别是嵌合和点。突变体 VP1 蛋白将被设计并测试其在同源和非同源 VP2 存在的情况下介导体外 RNA 合成的能力。在目标 2 中，将采用计算机氨基酸共变分析和分子动力学模拟来识别埋藏的。然后，将使用突变型 VP1 蛋白和体外 RNA 分析合成来验证可能传递变构信号的 VP1 残基。 VP1 突变体将被创建，并且涉及 VP1 变构激活的精确氨基酸残基将被定义。该提案是创新的，因为它使用基于序列和结构功能的方法来研究有关 VP1 酶活性的原始想法。这项工作意义重大，因为它将揭示轮状病毒 RdRp 与其他致病性 RNA 病毒共有的特征，这可能反过来促进变构抗病毒药物的开发，以治疗和治疗。预防病毒性疾病。