RNA-dependent RNA Polymerase

 描述(申请人提供)：使用脊髓灰质炎病毒(PV)及其依赖RNA的RNA聚合酶(RdRp)3Dpol作为我们的主要模型系统，我们、我们的合作者和其他微微核糖体病毒学家提出了一个非常令人信服的理由，证明RdRp的保真度和相应的病毒种群的遗传多样性是病毒致病和毒力的贡献者。这 使用PV模型建立的病毒遗传多样性和病毒适合度之间的关系现在已经在各种病毒家族中得到证实。此外，我们还证明了核苷酸添加的速度是可以调节的，并有助于病毒遗传多样性和病毒适合性。因此，推动我们研究的首要假设是，控制RdRp速度和保真度的机制可以分别在基因和化学上针对减毒病毒和抗病毒药物的开发。然而，如果要充分利用调节的RdRp功能的预防和治疗潜力，则需要了解控制RdRp速度和保真度的物理机制(S)。这个应用程序的一个目标是测试和开发一种新的核苷酸选择模型，我们预计这种模型可以应用于任何RdRp。重组是病毒进化的主要因素，导致疫苗耐药株的出现和病毒宿主范围的扩大。不幸的是，我们对RNA病毒重组的分子机制缺乏足够的了解，无法阻止这一过程阻碍疫苗的开发和长期疗效。因此，对重组的理解具有潜在的广泛的实用价值。例如，对重组机制的详细了解可以建立可用于制定抑制重组和/或设计重组缺陷疫苗毒株的策略的原则。该应用程序的第二个目标是使用PV模型创建关于RNA病毒重组机制的基础知识。在下一个资助期，我们的主要目标将是：(1)确定RdRp掺入保真度的活性位点决定因素，可作为病毒减弱的靶点；(2)利用RdRp新生碱基对结合口袋的混杂作用进行抗病毒治疗；(3)阐明光伏RdRp进行模板切换/链转移的机制，并建立其与光伏病毒在细胞中的RNA重组的相关性。拟议的研究将创造重要的知识，有助于制定治疗和预防RNA病毒感染的战略，包括目前令人关切的病毒：埃博拉病毒、肠道病毒D68和中东呼吸综合征冠状病毒。

 DESCRIPTION (provided by applicant): Using poliovirus (PV) and its RNA-dependent RNA polymerase (RdRp), 3Dpol, as our primary model system, we, our collaborators and other picorna virologists have made a very compelling case for RdRp fidelity and the corresponding genetic diversity of the viral population as contributors to viral pathogenesis and virulence. This relationship between viral genetic diversity and viral fitness established using the PV model has now been demonstrated for a variety of virus families. In addition, we have demonstrated that the rate of nucleotide addition can be tuned and contributes to viral genetic diversity and viral fitness. Therefore, the overarching hypothesis driving our studies is that mechanisms governing RdRp speed and fidelity can be targeted genetically and chemically for development of attenuated viruses and antiviral agents, respectively. If one is to harness the full therapeutic an prophylactic potential of modulated RdRp function, however, knowledge of the physical mechanism(s) controlling RdRp speed and fidelity is needed. One goal of this application is to test and to exploit a new model for nucleotide selection that we anticipate can be applied to any RdRp. Recombination is a major contributor to viral evolution, leading to the emergence of vaccine-resistant strains and expansion of virus host range. Unfortunately, we lack sufficient knowledge of the molecular mechanism of RNA virus recombination to prevent this process from thwarting vaccine development and long- term efficacy. Therefore, an understanding of recombination is of potentially broad, practical value. For example, detailed knowledge of the mechanism of recombination may establish principles that can be exploited for development of strategies to suppress recombination and/or design of recombination-deficient vaccine strains. A second goal of this application is to create fundamental knowledge on the mechanism of RNA virus recombination using the PV model. During the next funding period, our central objectives will be: (1) Identify active-site determinants of RdRp incorporation fidelity that can be targeted for viral attenuation; (2) Exploit the promiscuity of the RdRp nascent base pair-binding pocket for antiviral therapy; (3) Elucidate a mechanism for template switching/strand transfer by PV RdRp and establish its relevance to RNA recombination by PV in cells. The proposed studies will create important knowledge that should contribute to development of strategies to treat and to prevent RNA viral infections, including those of current concern: Ebola virus, enterovirus D68, and Middle Eastern respiratory syndrome coronavirus.