RNA-dependent RNA Polymerase

描述（由申请人提供）：这是一份研究RNA病毒复制型聚合酶（RNA依赖性RNA聚合酶（RdRp））核苷酸掺入的机制和保真度的资助续期申请。在过去的十年里，世界目睹了SARS的出现，西尼罗河脑炎的传播，以及对全球流感大流行的恐惧。这些疾病是由RNA病毒引起的。此外，故意释放RNA病毒作为恐怖武器或制剂的威胁也大大增加。该研究计划的长期目标是通过靶向RdRp来开发治疗和/或预防RNA病毒感染的策略。我们的程序采用了一个原型的RNA病毒，脊髓灰质炎病毒（PV），和它的RdRp（3Dpol）作为我们的模型系统。上一个资助期致力于探究3Dpol催化的核苷酸转移的化学机制，阐明3Dpol掺入保真度的结构基础，以及开发用于解决3Dpol与引物模板和核苷酸复合物的晶体结构的工具。我们在实现所有目标方面取得了显著进展。我们对核苷酸转移的化学机制有了新的认识。我们发现了RdRp掺入保真度和发病机制之间的联系。我们发现了RdRp动力学和掺入保真度之间的联系。总之，我们的研究导致了一个非常具有挑衅性的假设，即RdRp掺入保真度是抗病毒和疫苗开发的目标，将在下一个资助期内详细说明。我们将追求以下具体目标：（1）阐明一般酸在聚合酶功能中的额外作用;（2）确定聚合酶保真度的新决定因素和机制;（3）建立RdRp的动力学-功能关系。 公共卫生相关性：RNA病毒是对美国公共卫生的现有和新出现的威胁。本申请目标的实现将为疫苗和抑制剂的开发提供新的靶点和机制，以预防和治疗RNA病毒感染。

DESCRIPTION (provided by applicant): This is an application for renewal of a grant to study to the mechanism and fidelity of nucleotide incorporation by the replicative polymerase of RNA viruses, the RNA-dependent RNA polymerase (RdRp). Over the past ten years, the world has witnessed the emergence of SARS, the spread of West Nile encephalitis, and the fear of a global flu pandemic. These diseases are caused by RNA viruses. In addition, the threat of intentional release of RNA viruses as weapons or agents of terror has increased substantially. The long-term goal of this research program is to develop strategies to treat and/or prevent RNA virus infection by targeting the RdRp. Our program has employed a prototypical RNA virus, poliovirus (PV), and its RdRp (3Dpol) as our model system. The previous funding period was devoted to the interrogation of the chemical mechanism for 3Dpol- catalyzed nucleotidyl transfer, elucidation of the structural basis for 3Dpol incorporation fidelity, and development of the tools to solve a crystal structure for 3Dpol in complex with primed template and nucleotide. We have made outstanding progress towards completion of all our aims. We have obtained new insight into the chemical mechanism for nucleotidyl transfer. We discovered a link between RdRp incorporation fidelity and pathogenesis. We discovered a connection between RdRp dynamics and incorporation fidelity. Together, our studies lead to the very provocative hypothesis that RdRp incorporation fidelity is a target for antiviral and vaccine development that will be elaborated during the next funding period. We will pursue the following specific aims: (1) Elucidate additional roles for the general acid in polymerase function; (2) Identify novel determinants and mechanisms of polymerase fidelity; and (3) Establish dynamics-function relationships for the RdRp. PUBLIC HEALTH RELEVANCE: RNA viruses represent an existing and emerging threat to US public health. Achievement of the goals of the application will provide novel targets and mechanisms for development of vaccines and inhibitors to prevent and to treat infections by RNA viruses.