RNA Processing in Non-Segmented Minus-Strand RNA Viruses

说明（由申请人提供）：本申请为研究负链RNA病毒的RNA合成而更新拨款。非分段负链（NNS） RNA病毒包括一些最重要的人类病原体，它们是对美国公共卫生的主要持续威胁。为了对抗这些病原体，我们需要抗病毒药物和疫苗的结合。我们的长期目标是了解水疱性口炎病毒（VSV）的复制机制，这是NNS RNA病毒的一种原型。VSV是此类研究的理想选择，因为它是唯一一种可以在体外从纯化的重组成分中重组出稳健转录的NNS RNA病毒。RNA合成机制的催化核心是一个241 kDa的大聚合酶蛋白(L)，它包含一个RNA依赖的RNA聚合酶（RdRP）、一个覆盖mRNA的多核糖核苷基转移酶（PRNTase）和一个双特异性mRNA帽甲基转移酶（MTase）。在mRNA合成过程中，这些活动是协调的，因此新生的mRNA被盖住，甲基化和聚腺苷化。尽管L含有RNA合成的所有酶活性，但它需要一个29 kDa的磷酸化蛋白(P)来连接L和完全包裹基因组RNA模板的核衣壳蛋白(N)之间的相互作用。在上一个资助期，我们开发了体外实验，分别研究mRNA帽添加的每个步骤，独立于正在进行的转录。这些检测与强大的反向遗传系统相结合，可以对病毒RNA合成机制中的致命突变进行机制分析。我们使用这些分析提供了一个地图，显示mRNA帽添加的每个步骤的不同酶活性在L中定位的位置。这些研究使我们得出这样的假设，即L包含独立的功能域，其活性通过L组装到带有N-RNA模板的L- p复合物的RNA合成机中来协调。了解NNS RNA病毒RNA合成机制的主要空白是缺乏l的结构信息。在下一个资助期内，我们将使用电子显微镜，x射线晶体学和体外聚合酶功能分析来提供VSV RNA合成机制的独特结构和功能见解。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，（iii）探测L. mRNA盖层与RNA合成活性之间的关系。本研究的成功完成将提供NNS RNA病毒聚合酶的结构，以及对该RNA合成机器功能的新的机制见解，这可能有助于合理设计抗病毒治疗药物和候选疫苗。

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.