RNA Processing in Non-Segmented Minus-Strand RNA Viruses

描述(申请人提供)：这是一个申请续期研究负链RNA病毒的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，对美国公共卫生构成持续的重大威胁。为了对抗这些病原体，我们需要抗病毒药物和疫苗的组合。我们的长期目标是了解水疱性口炎病毒(VSV)的复制机制，它是NNS RNA病毒的原型。VSV是此类研究的理想选择，因为它是唯一一种可以从纯化的重组成分在体外重建强劲转录的NNS RNA病毒。核糖核酸合成机制的催化核心是一个241 kDa的大聚合酶蛋白(L)，它包含一个依赖核糖核酸的核糖核酸聚合酶(RdRP)、一个覆盖该基因的多聚核糖核酸基转移酶(PRNTase)和一个双重特异性的mRNA帽甲基转移酶(MTase)。在信使核糖核酸的合成过程中，这些活动被协调，使得新生信使核糖核酸被封顶、甲基化和多腺化。尽管L具有合成核糖核酸的所有酶活性，但它需要一个29 kDa的磷酸蛋白(P)来连接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。在上一次授予期间，我们开发了体外试验，以独立于正在进行的转录来单独研究mRNA帽添加的每一步。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械分析。我们使用这些分析提供了一张图，说明每一步不同的酶活性在L内的定位。这些研究导致我们假设L含有独立的功能结构域，其活性是通过L组装到L-P复合体的RNA合成机器中与N-RNA模板相协调的。了解NNS RNA病毒的RNA合成机制的一个主要差距是缺乏L的结构信息。在下一个资助期，我们将使用电子显微镜、X射线结晶学和体外聚合酶功能分析，以提供对VSV的RNA合成机制的独特结构和功能的见解。我们将：(I)确定VSV聚合酶复合体的功能组织；(Ii)确定VSV聚合酶的三维结构，以及(Iii)探索RNA封顶与L的RNA合成活性之间的关系。这项研究的成功完成将提供NNS RNA病毒的聚合酶的结构以及对这种RNA合成机器的功能的新的机制见解，这可能有助于抗病毒治疗和候选疫苗的合理设计。 公共卫生相关性：非分段负链核糖核酸病毒的L聚合酶蛋白含有依赖核糖核酸的核糖核酸聚合活性，以及一组高度保守和不寻常的酶活性，增加了信使核糖核酸的帽子结构。了解如何控制这些活动以确保病毒基因组的表达具有内在的意义，并有可能影响抗病毒药物和减毒活疫苗的开发。在这里，我们将获得一个原型NNS RNA病毒，水泡性口炎病毒的这种蛋白质的结构和功能的见解。

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. PUBLIC HEALTH RELEVANCE: The L polymerase protein of nonsegmented negative-strand (NNS) RNA viruses contains an RNA dependent RNA polymerization activity as well as a set of highly conserved and unusual enzymatic activities that add an mRNA cap structure. Understanding how those activities are controlled to ensure that the viral genome is expressed is of intrinsic interest and has the potential to impact the development of antiviral drugs and live attenuated vaccines. Here we will obtain structural and functional insights into this protein for a prototype NNS RNA virus, vesicular stomatitis virus.