RNA Processing in Non-Segmented Minus-Strand RNA Viruses

 描述（由申请人提供）：本申请是为了更新研究负链RNA病毒中RNA合成的资助。非节段负义（NNS）RNA病毒包括一些最重要的人类病原体，这些病原体对美国公共卫生构成持续威胁。对于麻疹、腮腺炎和狂犬病，有许可的疫苗，但对于大多数NNS RNA病毒，没有疫苗，也没有抗病毒药物。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大蛋白（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RNA模板的核衣壳蛋白（N）之间的相互作用。在上一个资助期间，我们获得了对NNS RNA病毒聚合酶复合体的第一个结构见解，并开发了独特的工具和试剂，使我们能够获得VSV聚合酶复合体的原子级模型。我们的基本假设是，L在RNA聚合、mRNA帽加成和帽甲基化中的催化活性（其位于结构上分离的结构域中）通过P和模板相关N的存在来协调，以在mRNA合成期间调节它们的活性，并在组装期间通过与病毒基质蛋白（M）形成复合物来下调它们。理解RNA合成机制的一个主要差距是目前对L.我们手头有一个可解释的密度图的VSV L-P复杂的冷冻EM。在下一个资助期间，我们将继续使用冷冻电子显微镜（EM），负染色EM，X射线晶体学，聚合酶的体外生物化学和分子病毒学，以提供独特的结构和功能的见解VSV聚合酶在RNA合成和组装的不同阶段。我们将：（i）确定VSV聚合酶复合物的完整分子模型;（ii）确定模板相关的N蛋白如何从RNA中置换;和（iii）确定M蛋白下调聚合酶活性的机制。这项工作的成功完成将提供一个原子水平的结构的NNS RNA病毒聚合酶复合物和新的机制的见解，这个RNA合成机器在转录，复制和组装过程中的功能和调节。这些结果可能有助于合理减毒其他相关的致病性人类病毒疫苗的目的，并为抗病毒治疗的发展。

 DESCRIPTION (provided by applicant): This application is to renew a grant to study RNA synthesis in minus-strand RNA viruses. Nonsegmented negative-sense (NNS) RNA viruses include some of the most significant human pathogens that are an ongoing threat to US public health. For measles, mumps and rabies there are licensed vaccines, but for most NNS RNA viruses there are no vaccines and no antiviral drugs. 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 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 RNA template. In the last grant period, we obtained the first structural insights into the polymerase complex of an NNS RNA virus and developed unique tools and reagents that will permit us to obtain an atomic-level model of the VSV polymerase complex. Our underlying hypothesis is that the catalytic activities of L in RNA polymerization, mRNA cap addition and cap methylation which reside within structurally separate domains are coordinated by the presence of the P and the template associated N to regulate their activities during mRNA synthesis, and to downregulate them during assembly by complex formation with the viral matrix protein (M). A major gap in understanding the machinery of RNA synthesis is the current resolution of structures of L. We have in hand an interpretable density map of a VSV L-P complex from cryo EM. During the next funding period, we will continue to use cryo electron microscopy (EM), negative-stain EM, X-ray crystallography, in vitro biochemistry of polymerase and molecular virology to provide unique structural and functional insights into the VSV polymerase during distinct stages of RNA synthesis and assembly. We will: (i) determine a complete molecular model of the VSV polymerase complex; (ii) determine how the template associated N protein is displaced from the RNA, and (iii) determine the mechanism by which M protein downregulates polymerase activity. The successful completion of this work will provide an atomic-level structure of an NNS RNA virus polymerase complex and new mechanistic insights into the function and regulation of this RNA synthesis machine during transcription, replication and assembly. Those results may help in the rational attenuation of other related pathogenic human viruses for vaccine purposes, and for the development of antiviral therapeutics.