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：
9060290
负责人：
Sarah Marie McDonald Esstman
金额：
$ 20.13万
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/9060290
关键词：
Active Sites Algorithms Allosteric Regulation Allosteric Site Amino Acid Sequence Amino Acids Antiviral Agents Architecture Base Sequence Binding Binding Proteins 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 Polymerase Process 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 Virus Replication 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（由适用提供）：RNA病毒代表了现有和新兴的人类病原体，导致威胁生命的胃肠道，呼吸道，出血和神经系统疾病。尽管RNA病毒在其基因组序列，病毒架构，组织疗法和病理表现方面的变化差异很大，但它们都编码了一种称为RNA依赖性RNA聚合酶（RDRP）的专用酶。 RDRP对于病毒复制至关重要，因为它介导了病毒RNA合成的所有阶段。然而，必须在感染过程中调节Vral RDRP的活性，以便可以与病毒生命周期中的其他步骤协调RNA合成。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，RDRP表面与活性位点物理上不同。效应蛋白对变构位点的结合诱导RDRP中的一系列分子内构象变化，这些变化最终在活性位点上调节酶功能。该提案的总体目的是使用轮状病毒作为结构性和功能可吸引的实验系统来获得对变构RDRP调节的机械洞察力。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）的活性要求其直接由核心壳效应蛋白（VP2）参与。但是，关于（i）哪些表面暴露的VP1保留了变构激活位点和（ii）的知识存在关键差距，该位点保留了构建VP1的变构激活位点，保留了VP1保留的，将变构信号从表面传输到活性位点。提出了两个集成但独立的特定目标，以帮助缩小知识中的这些差距。在AIM 1中，将使用一种结构引导的功能生化方法来绘制构成VP1变构激活位点的精确残差。具体而言，嵌合和点突变体VP1蛋白将经过设计和测试，以在存在和非同源VP2的情况下介导体外RNA合成的能力。在AIM 2中，将使用氨基酸共同变化分析和分子动态模拟来识别可能传递变构信号的构建VP1残差。然后，这些残留物将使用突变体VP1蛋白和体外RNA合成测定法对这些残差进行验证。这项工作完成后，预计将创建自动激活的VP1突变体，并且将定义与VP1变构激活有关的确切氨基酸保留。该提案具有创新性，因为它使用基于序列的结构 - 功能方法来研究有关VP1的酶促活性如何受VP2调节的原始思想。这项工作很重要，因为它将揭示与其他致病性RNA病毒共享的轮状病毒RDRP的特征，这些病毒可能会促进变构抗病毒药物的发展以治疗和预防病毒疾病。