RNA-dependent RNA Polymerase

RNA依赖性RNA聚合酶

• 批准号: 10430269
• 负责人: Jamie Jon Arnold
• 金额: $ 40.43万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10430269
• 关键词: Achievement; Active Sites; Amino Acids; Antiviral Agents; Attenuated; Biochemical; Biological; Biological Assay; Biological Models; Biological Process; Biology; Biophysics; Bypass; Cells; Complex; DNA; Defect; Development; Ebola virus; Enterovirus; Enterovirus 68; Evolution; Frequencies; Genetic; Genetic Drift; Genetic Recombination; Genetic Transcription; Genome; Goals; Guanine; Human poliovirus; In Vitro; Individual; Knowledge; Laboratories; Link; Mediating; Modeling; Modification; Mutation; Nucleotides; Oxides; Pharmacology; Polymerase; Positioning Attribute; Process; Property; Public Health; RNA; RNA Virus Infections; RNA Viruses; RNA-Directed RNA Polymerase; SARS coronavirus; Speed; Testing; Viral; Viral Pathogenesis; Viral Proteins; Virion; Work; base; biophysical analysis; biophysical properties; epitranscriptome; genetic manipulation; in vitro Assay; in vivo; inhibitor; insight; mutant; novel; prevent; reconstitution; sugar; vaccine development; viral RNA; viral fitness

Abstract The RNA-dependent RNA polymerase (RdRp) drives viral evolution by mediating both genetic drift (mutation) and genetic/antigenic shift (recombination) of RNA viruses. RdRp speed and fidelity contribute to the rate of mutation and formation of the mutant swarm, a well-established determinant of viral fitness and pathogenesis. For more than two decades, we have used the RdRp from poliovirus (PV) as a model system to elucidate fundamental biochemical and biophysical principles governing speed and fidelity of nucleotide addition. A major conclusion of this work is that both speed and fidelity are controlled by the dynamics of a conserved array of amino acid residues in the active site. Importantly, these dynamics can be manipulated genetically or exploited pharmacologically, thus contributing to the creation of attenuated strains and antiviral agents. RNA recombination in PV occurs by a template-switching mechanism, a process in which the RdRp initiates elongation on one template (donor) but completes elongation on a different template (acceptor). PV RdRp is sufficient to catalyze template switching in vitro. However, the trigger(s) and mechanism of template switching remain largely unknown. Using a novel cell-based assay for PV RNA recombination, the Evans laboratory observed a direct correlation between RdRp infidelity and the frequency of RNA recombination. RdRp misincorporation frequency as a biochemical property governing template switching was not expected but motivated our foray into the study of RNA recombination. Over the past five years, we have established an experimental paradigm to elucidate the mechanism of RdRp-catalyzed RNA recombination, discover biochemical and biophysical properties and structural determinants of the RdRp governing RNA recombination, and reveal the biological consequences of perturbations to the mechanism and/or efficiency of RNA recombination. Here, we will utilize our experimental paradigm to achieve the following: (1) Link structural determinants of the RdRp to elementary steps and mechanisms of RNA recombination; (2) Investigate the impact of RNA modifications on elongation and recombination by the RdRp; and (3) Reveal the mechanism and biological function of forced-copy-choice RNA recombination.

抽象的 RNA依赖性RNA聚合酶（RDRP）通过介导两个遗传漂移（突变）驱动病毒进化 RNA病毒的遗传/抗原转移（重组）。 RDRP速度和保真度有助于 突变群的突变和形成，这是病毒适应性和发病机理的公认决定因素。 二十多年来，我们一直使用Poliovirus（PV）的RDRP作为模型系统来阐明 基本的生化和生物物理原理，负责添加核苷酸的速度和保真度。专业 这项工作的结论是，速度和保真度都由保守的阵列的动态控制 活性位点中的氨基酸残基。重要的是，可以通过遗传或利用这些动力来操纵这些动力 在药理上，从而有助于导致菌株和抗病毒药物的产生。 PV中的RNA重组是通过模板切换机制发生的，RDRP启动了一个过程 在一个模板上伸长（供体），但在另一个模板上完成伸长率（受体）。 PV RDRP是 足以在体外催化模板切换。但是，模板切换的触发器和机制 在很大程度上未知。 Evans实验室使用新型基于细胞的测定法进行PV RNA重组 观察到RDRP不忠与RNA重组频率之间的直接相关性。 RDRP 不期望的是，构成频率是生化属性的管理模板切换，但 激发了我们进入RNA重组的研究。在过去的五年中，我们建立了一个 实验范式以阐明RDRP催化的RNA重组机制，发现 RNA重组的RDRP的生化和生物物理特性以及结构决定因素， 并揭示扰动对RNA机制和/或效率的生物学后果 重组。 在这里，我们将利用我们的实验范式来实现以下操作：（1）链接的结构决定因素 RDRP到RNA重组的基本步骤和机制； （2）研究RNA的影响 RDRP对伸长和重组的修改； （3）揭示机制和生物学 强制选择RNA重组的功能。