Watching Conformational Rearrangements in Poliovirus RNA-Dependent RNA Polymerase

项目摘要 重要的人类病原体如脊髓灰质炎病毒、丙型肝炎和埃博拉病毒的RNA基因组是 通过病毒编码的RNA依赖的RNA聚合酶(RdRp)复制，建立了抗病毒的靶点。这个 RdRp功能的分子机制只有在我们都表征了它的特征之后才能被理解 可访问的结构状态，并描绘了随着RdRp的进展这些状态之间的转换 它的催化循环。这一信息对于预测改变这一点的保真度改变的突变至关重要。 方法，和/或设计RdRp功能的小分子调制器，该小分子调制器将干扰 结构转型。晶体结构本身一直无法捕捉到结构的全部范围 RdRp函数所需的重新排列，但未提供有关 构象起伏。例如，改变RdRp保真度和病毒的活性部位远程突变 生物学上，不会导致实质性的结构差异，相反，它们会改变RdRp蛋白质的波动 多个时标。在这项赠款申请中，我们建议使用溶液状态核磁共振 (核磁共振)观察原型RdRp中的构象重排(在我们的案例中，来自脊髓灰质炎病毒) 在其整个核苷酸加成循环中，对比野生型和 低/高保真突变体RdRps，并阐明了一类新的核苷的分子机制 类似物，包括正在进行临床试验的成员。我们预测，保真度突变和 进入的核苷酸(正确的或不正确的沃森-克里克碱基对)将改变动力学和/或 结构重排的热力学对RdRp函数至关重要。我们还建议，保真度- 发生改变的远程位点突变通过一个长距离的氨基酸网络发挥作用。我们将描绘出 通过对所选突变体的动力学和核磁共振研究，包括来自Sabin 1的突变体 疫苗株(即临床上使用的、口服生物可用的脊髓灰质炎病毒疫苗株)。我们预测RdRp 突变通过改变RdRp的保真度而导致Sabin的表型减弱。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的变化 干扰这些运动并改变聚合酶功能的物质。RdRp中的这种突变将被预测为 改变聚合酶的保真度，因此可以作为新疫苗株的基础。小分子 也可能被用来扰乱RdRp中的结构重排；我们的研究将作为 阐明了这些化合物鲜为人知的作用机制。结构和动力学是 在RdRp中高度保守，因此这些概念将适用于一般的RNA病毒。

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.