Watching Conformational Rearrangements in Poliovirus RNA-Dependent RNA Polymerase

描述(申请人提供)：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依赖的RNA聚合酶(RdRp)复制，RdRp是一种既定的抗病毒靶标。只有当我们表征了RdRp的可接近的结构状态并描述了RdRp在催化循环中这些状态之间的转变时，我们才能理解RdRp功能的分子机制。这些信息对于预测改变这一过程的保真度变化的突变和/或设计干扰结构转变的RdRp功能的小分子调节剂至关重要。晶体结构本身不能捕捉RdRp功能所需的全部结构重排，也不能提供关于构象涨落的时间尺度的信息。例如，改变RdRp保真度和病毒生物学的活性部位远程突变并不会导致实质性的结构差异，相反，它们会改变RdRp蛋白在多个时间尺度上的波动。在这项拨款申请中，我们建议使用溶液状态核磁共振(NMR)来观察原型RdRp(在我们的案例中，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，对比野生型和低/高保真突变RdRp之间的构象动力学，并描述一类新型核苷类似物的分子机制，其中包括临床试验中的成员。我们预测保真度突变和传入核苷酸的性质(‘正确’或‘不正确’沃森-克里克 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一个长距离的氨基酸网络发挥作用。我们将通过对选定的突变体进行动力学和核磁共振研究来描绘这一网络，包括来自Sabin 1疫苗株(即一种临床使用的口服生物可用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的保真度而导致Sabin减弱的表型。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的氨基酸变化并改变聚合酶功能。据预测，RdRp的这种突变会改变聚合酶的保真度，因此可能成为新疫苗株的基础。小分子也可能被用来扰乱RdRp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。RdRp的结构和动力学是高度保守的，因此这些概念将适用于一般的RNA病毒。

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.