RNA Processing in Non-Segmented Minus-Strand RNA Viruses

描述（由申请人提供）：这是一份申请，旨在更新研究负链RNA病毒中RNA合成的资助。非分段负链（NNS）RNA病毒包括一些最重要的人类病原体，这些病原体是对美国公共卫生的主要持续威胁。为了对抗这些病原体，我们需要将抗病毒药物和疫苗结合起来。我们的长期目标是了解水泡性口炎病毒（VSV）的复制机制，NNS RNA病毒的原型，功能。VSV是这种研究的理想选择，因为它是唯一的NNS RNA病毒，其稳健的转录可以在体外从纯化的重组组分重建。RNA合成机制的催化核心是241 kDa的大聚合酶蛋白（L），其含有RNA依赖性RNA聚合酶（RdRP）、为mRNA加帽的多核糖核苷酸转移酶（PRNTase）和双特异性mRNA帽甲基转移酶（MTase）。在mRNA合成过程中，这些活性相互协调，使得新生mRNA被加帽、甲基化和聚腺苷酸化。虽然L含有RNA合成的所有酶活性，但它需要一个29 kDa的磷蛋白（P）来桥接L和完全覆盖基因组RNA模板的核衣壳蛋白（N）之间的相互作用。在上一个资助期，我们开发了体外试验，以单独研究mRNA帽添加的每个步骤，而不依赖于正在进行的转录。这些测定与强大的反向遗传系统相结合，允许对病毒RNA合成机制中的致命突变进行机制分析。我们已经使用这些测定来提供mRNA帽添加的每个步骤的不同酶活性在L内定位的地图。这些研究使我们假设L含有独立的功能结构域，其活性通过将L组装到具有N-RNA模板的L-P复合物的RNA合成机器中来协调。理解NNS RNA病毒的RNA合成机制的一个主要差距是缺乏L。在下一个资助期间，我们将使用电子显微镜，X射线晶体学和聚合酶功能的体外测定，以提供独特的结构和功能的见解VSV的RNA合成机制。我们将：（i）确定VSV聚合酶复合物的功能组织;（ii）确定VSV聚合酶的三维结构;和（iii）探测L.这项研究的成功完成将提供NNS RNA病毒聚合酶的结构，以及对这种RNA合成机器功能的新机制见解，这可能有助于抗病毒治疗和候选疫苗的合理设计。

DESCRIPTION (provided by applicant): This is an application to renew a grant to study RNA synthesis in minus-strand RNA viruses. Nonsegmented negative-strand (NNS) RNA viruses include some of the most significant human pathogens that are a major ongoing threat to US public health. To combat those agents, we need a combination of antiviral drugs and vaccines. Our long-term objective is to understand the mechanisms by which the replication machinery of vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, functions. VSV is the ideal choice for such studies because it is the only NNS RNA virus for which robust transcription can be reconstituted in vitro from purified recombinant components. The catalytic core of the RNA synthesis machinery is a 241 kDa large polymerase protein (L) that contains an RNA dependent RNA polymerase (RdRP), a polyribonucleotidyltransferase (PRNTase) that caps the mRNA, and a dual specificity mRNA cap methyltransferase (MTase). During mRNA synthesis, those activities are coordinated so that the nascent mRNA is capped, methylated and polyadenylated. Although L contains all the enzymatic activities for RNA synthesis, it requires a 29 kDa phosphoprotein (P) that bridges interactions between L and the nucleocapsid protein (N) that completely coats the genomic RNA template. In the last grant period, we developed in vitro assays to separately study each of the steps of mRNA cap addition independent of ongoing transcription. Those assays, combined with a powerful reverse genetic system allow mechanistic analysis of lethal mutations in the viral RNA synthesis machinery. We have used those assays to provide a map of where the different enzymatic activities for each step of mRNA cap addition are localized within L. Those studies lead us to the hypothesis that L contains independent functional domains whose activities are coordinated by the assembly of L into the RNA synthesis machine of the L-P complex with the N-RNA template. A major gap to understanding the mechanisms by which the RNA synthesis machinery of NNS RNA viruses function is the absence of structural information for L. During the next funding period, we will use electron microscopy, X-ray crystallography and in vitro assays of polymerase function to provide unique structural and functional insights into the RNA synthesis machinery of VSV. We will: (i) determine the functional organization of the VSV polymerase complex; (ii) determine the three dimensional structure of the VSV polymerase, and (iii) probe the relationship between the mRNA capping and RNA synthesis activities of L. The successful completion of this study will provide a structure of the polymerase of an NNS RNA virus as well as new mechanistic insights into the function of this RNA synthesis machine that may help in the rational design of antiviral therapeutics and candidate vaccines.