Watching Conformational Rearrangements in Poliovirus RNA-Dependent RNA Polymerase

描述（由申请人提供）：重要人类病原体（如脊髓灰质炎病毒、丙型肝炎病毒和埃博拉病毒）的RNA基因组通过病毒编码的RNA依赖性RNA聚合酶（RdRp）复制，RdRp是一种已确立的抗病毒靶标。RdRp功能的分子机制只有在我们表征了其可接近的结构状态并描绘了RdRp通过其催化循环进展时这些状态之间的转换后才能理解。该信息对于预测改变该过程的改变遗传性的突变和/或设计将干扰结构转变的RdRp功能的小分子调节剂将是至关重要的。晶体结构，本身，已无法捕捉的RdRp功能所需的全方位的结构重排，并没有给出有关的时间尺度的构象波动的信息。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，而是在多个时间尺度上改变RdRp蛋白质的波动。在这项资助申请中，我们建议使用溶液状态核磁共振（NMR）来“观察”原型RdRp（在我们的情况下，从脊髓灰质炎病毒）在整个核苷酸添加周期的构象重排，对比野生型和低/高亲和力突变RdRps之间的这些构象动力学，并描绘一类新的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，突变和性质的传入核苷酸（'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，改变生育率的远程位点突变通过长距离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NMR研究来描述该网络，包括来自Sabin 1疫苗株（即临床使用的口服生物可利用脊髓灰质炎病毒疫苗株）的突变体。我们预测，RdRp突变有助于通过改变RdRp保真度的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干扰这些运动并改变聚合酶功能的氨基酸变化。预计RdRp中的此类突变会改变聚合酶的保真度，因此可以作为新型疫苗株的基础。小分子也可用于干扰RdRps的结构重排;我们的研究将作为阐明这些化合物的作用机制的基础。结构和动力学在RdRps之间是高度保守的，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.