Watching Conformational Rearrangements in Poliovirus RNA-Dependent RNA Polymerase

观察脊髓灰质炎病毒 RNA 依赖性 RNA 聚合酶的构象重排

• 批准号: 8631819
• 负责人: David Douglas Boehr
• 金额: $ 37.12万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-15 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8631819
• 关键词: Active Sites; Amino Acids; Antiviral Agents; Applications Grants; Attenuated; Attenuated Live Virus Vaccine; Bacteriophages; Base Pairing; Binding; Bioavailable; Biology; Catalysis; Clinical Trials; DNA Sequence Rearrangement; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Development; Diphosphates; Ebola virus; Enzymatic Biochemistry; Enzymes; Equilibrium; Exhibits; Foundations; Genome; Grant; Health; Hepatitis C; Hepatitis C virus; Human; Human poliovirus; Kinetics; Laboratories; Lead; Link; Molecular; Molecular Conformation; Monitor; Motion; Mutation; NMR Spectroscopy; Nature; Nuclear Magnetic Resonance; Nucleic Acids; Nucleosides; Nucleotides; Phenotype; Polioviruses; Polymerase; Positioning Attribute; Process; Proteins; Publications; RNA; RNA Viruses; RNA-Directed RNA Polymerase; Reaction; Research Personnel; Ribose; Site; Solutions; Structure; System; Testing; Thermodynamics; Vaccines; Viral; Virulence; Virus; Work; X-Ray Crystallography; anti-hepatitis C; base; design; meetings; member; millisecond; mutant; novel; novel vaccines; nucleoside analog; nucleoside triphosphate; pathogen; prototype; public health relevance; resistance mechanism; small molecule; viral RNA

Project Summary The RNA genomes of important human pathogens such as poliovirus, heptatitis C and ebola virus are replicated by virally encoded RNA-dependent RNA polymerases (RdRp), an establish anti-viral target. The molecular mechanisms of RdRp function will only be understood once we have both characterized its accessible structural states and delineated the transitions between these states as RdRp progresses through its catalytic cycle. This information would be critical for predicting fidelity-altering mutations that alter this process, and/or for designing small molecule modulators of RdRp function that would interfere with the structural transitions. Crystal structures, by themselves, have been unable to capture the full range of structural rearrangements necessary for RdRp function, and give no information about the timescale of the conformational fluctuations. For example, active-site remote mutations that change RdRp fidelity and virus biology, do not lead to substantial structural differences, but rather, they change RdRp protein fluctuations over multiple timescales. In this grant application, we propose to use solution-state nuclear magnetic resonance (NMR) to "watch" the conformational rearrangements in an archetypal RdRp (in our case, from poliovirus) throughout its nucleotide addition cycle, contrast these conformational dynamics between wild-type and low/high fidelity-mutant RdRps, and delineate the molecular mechanisms of a novel class of nucleoside analogs, which include members under clinical trials. We predict that the fidelity-mutations and the nature of the incoming nucleotide ('correct' or 'incorrect' Watson-Crick base-pair) will change the kinetics and/or thermodynamics of structural rearrangements critical for RdRp function. We also propose that the fidelity- altering, remote-site mutations exert their effects through a long-range, amino acid network. We will delineate this network through kinetic and NMR studies of selected mutants, including mutants derived from the Sabin 1 vaccine strain (i.e. a clinically-used, orally bioavailable vaccine strain for poliovirus). We predict that RdRp mutations contribute to the Sabin attenuated phenotype through altering RdRp fidelity. Understanding the interactions for coordinating the structural rearrangements in RdRp will allow us to predict amino acid changes that interfere with these motions and alter polymerase function. Such mutations in RdRp would be predicted to change polymerase fidelity, and therefore may serve as the basis of novel vaccine strains. Small molecules may also be used to perturb the structural rearrangements in RdRps; our studies will serve as a basis for illuminating the poorly understand mechanisms of actions for these compounds. Structure and dynamics are highly conserved among RdRps, so these concepts will be applicable to RNA viruses in general.

项目概要 脊髓灰质炎病毒、丙型肝炎病毒和埃博拉病毒等重要人类病原体的 RNA 基因组 由病毒编码的RNA依赖性RNA聚合酶（RdRp）复制，这是一个既定的抗病毒靶标。这 只有当我们都表征了 RdRp 功能的分子机制后，我们才能了解其功能 可访问的结构状态并描述了随着 RdRp 的进展这些状态之间的转变 它的催化循环。该信息对于预测改变保真度的突变至关重要 过程，和/或设计 RdRp 功能的小分子调节剂，该调节剂会干扰 结构转变。晶体结构本身无法捕获结构的全部范围 RdRp 函数所需的重新排列，并且没有提供有关时间尺度的信息 构象波动。例如，改变 RdRp 保真度的活性位点远程突变和病毒 生物学，不会导致实质性的结构差异，而是改变 RdRp 蛋白的波动 多个时间尺度。在本次拨款申请中，我们建议使用溶液态核磁共振 （NMR）“观察”原型 RdRp 中的构象重排（在我们的例子中，来自脊髓灰质炎病毒） 在整个核苷酸添加周期中，对比野生型和野生型之间的构象动力学 低/高保真突变 RdRps，并描述一类新型核苷的分子机制 类似物，其中包括正在进行临床试验的成员。我们预测保真度突变和本质 输入的核苷酸（“正确”或“不正确”沃森-克里克碱基对）将改变动力学和/或 对 RdRp 功能至关重要的结构重排的热力学。我们还建议保真度- 改变远程位点的突变通过远程氨基酸网络发挥作用。我们将划定 该网络通过对选定突变体（包括来自 Sabin 1 的突变体）的动力学和 NMR 研究 疫苗株（即临床使用的、口服生物可利用的脊髓灰质炎病毒疫苗株）。我们预测 RdRp 突变通过改变 RdRp 保真度导致 Sabin 减弱表型。了解 协调 RdRp 结构重排的相互作用将使我们能够预测氨基酸变化 干扰这些运动并改变聚合酶功能。 RdRp 中的此类突变预计会 改变聚合酶的保真度，因此可以作为新型疫苗株的基础。小分子 也可用于扰乱 RdRps 中的结构重排；我们的研究将作为基础 阐明了人们对这些化合物的作用机制知之甚少。结构和动力学是 RdRps 之间高度保守，因此这些概念一般适用于 RNA 病毒。