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: 9098572
• 负责人: David Douglas Boehr
• 金额: $ 37.1万
• 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/9098572
• 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-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; Pathogenesis; Phenotype; Polymerase; Positioning Attribute; Process; Proteins; Publications; RNA; RNA Viruses; RNA-Directed RNA Polymerase; Reaction; Research Personnel; Ribose; Site; Structure; System; Testing; Therapeutic; 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; resistance mechanism; small molecule; viral RNA

DESCRIPTION (provided by applicant): The RNA genomes of important human pathogens such as poliovirus, hepatitis C and ebola virus are replicated by virally encoded RNA-dependent RNA polymerases (RdRp), an established 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 thi 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 understood 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.

描述（由申请人提供）：重要人类病原体（例如脊髓灰质炎病毒、丙型肝炎和埃博拉病毒）的 RNA 基因组通过病毒编码的 RNA 依赖性 RNA 聚合酶（RdRp）进行复制，RdRp 是一种已确定的抗病毒靶点。只有当我们表征了 RdRp 的可接近的结构状态并描述了 RdRp 在其催化循环中进展时这些状态之间的转变时，我们才能理解 RdRp 功能的分子机制。该信息对于预测改变该过程的保真度改变突变和/或设计干扰结构转变的 RdRp 功能小分子调节剂至关重要。晶体结构本身无法捕获 RdRp 功能所需的全部结构重排，并且无法提供有关构象波动时间尺度的信息。例如，改变 RdRp 保真度和病毒生物学的活性位点远程突变不会导致实质性的结构差异，而是会改变 RdRp 蛋白在多个时间尺度上的波动。在这项拨款申请中，我们建议使用溶液态核磁共振（NMR）来“观察”原型 RdRp（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的构象重排，对比野生型 RdRp 和野生型 RdRp 之间的构象动力学。类型和低/高保真突变 RdRps，并描述一类新型核苷类似物的分子机制，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传入核苷酸的性质（“正确”或“不正确”Watson-Crick 碱基对）将改变对 RdRp 功能至关重要的结构重排的动力学和/或热力学。我们还提出，改变保真度的远程位点突变通过长程氨基酸网络发挥作用。我们将通过对选定突变体的动力学和核磁共振研究来描绘这个网络，包括来自 Sabin 1 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）的突变体。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干扰这些运动并改变聚合酶功能的氨基酸变化。 RdRp 中的此类突变预计会改变聚合酶保真度，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；我们的研究将作为阐明这些化合物鲜为人知的作用机制的基础。 RdRps 的结构和动力学高度保守，因此这些概念一般适用于 RNA 病毒。