RNA Processing in Non-Segmented Minus-Strand RNA Viruses

 描述(申请人提供)：本申请旨在续期研究负链RNA病毒中RNA合成的资助。非分段负义(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RNA模板的核衣壳蛋白(N)之间的相互作用。在上一次资助期间，我们获得了对NNS RNA病毒聚合酶复合体的第一次结构洞察，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。我们的假设是，L在RNA聚合、mRNA帽加成和帽甲基化过程中的催化活性是通过P和模板相关N的存在来协调的，以调节它们在mRNA合成过程中的活性，并在组装过程中通过与病毒基质蛋白(M)形成络合物来下调它们。了解RNA合成机制的一个主要差距是目前对L的结构的分辨。我们手头有一张来自低温EM的VSV L-P复合体的可解释密度图。在下一个资助期间，我们将继续使用低温电子显微镜(EM)、负染色EM、X射线结晶学、聚合酶的体外生物化学和分子病毒学，以提供对VSV聚合酶在RNA合成和组装的不同阶段的独特结构和功能的见解。我们将：(I)确定VSV聚合酶复合体的完整分子模型；(Ii)确定模板相关N蛋白如何从RNA中移位；以及(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NNS RNA病毒聚合酶复合体的原子级结构，并对这种RNA合成机器在转录、复制和组装过程中的功能和调节提供新的机械性见解。这些结果可能有助于疫苗用途的其他相关致病人类病毒的合理减毒，以及抗病毒治疗药物的开发。

 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.