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、丙型肝炎病毒和埃博拉病毒的RNA基因组是 通过病毒编码的RNA依赖性RNA聚合酶（RdRp）复制，这是一种已建立的抗病毒靶点。的 RdRp功能的分子机制只有在我们都表征了其 可访问的结构状态，并描绘了这些状态之间的过渡，因为RdRp的进展， 催化循环。这一信息对于预测改变生育能力的突变至关重要， 方法，和/或用于设计会干扰细胞的RdRp功能的小分子调节剂。 结构转型。晶体结构，本身，已经无法捕捉的全部范围的结构 RdRp功能所需的重排，并没有给出有关时间尺度的信息。 构象波动例如，改变RdRp保真度和病毒的活性位点远程突变， 生物学，不会导致实质性的结构差异，而是，它们改变了RdRp蛋白质的波动， 多个时间尺度。在这项拨款申请中，我们建议使用溶液态核磁共振 (NMR)“观察”原型RdRp的构象重排（在我们的例子中，来自脊髓灰质炎病毒） 在整个核苷酸添加循环中，对比野生型和 低/高纯度突变RdRps，并描绘了一类新的核苷 类似物，其中包括正在进行临床试验的成员。我们预测，突变和性质的 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 RdRp功能的关键结构重排热力学。我们还建议忠诚- 改变，远程位点突变通过远程氨基酸网络发挥其作用。我们将描绘 通过对选定的突变体的动力学和NMR研究，包括来自Sabin 1的突变体， 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保真度而促成Sabin减毒表型。了解 协调RdRp中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。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 也可以用来扰乱RdRps的结构重排;我们的研究将作为基础， 阐明了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。

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.