RNA Processing in Non-Segmented Minus-Strand RNA Viruses

非分段负链 RNA 病毒中的 RNA 加工

• 批准号: 8067072
• 负责人: Sean PJ Whelan
• 金额: $ 41.38万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8067072
• 关键词: Amino Acids; Antiviral Agents; Attenuated Live Virus Vaccine; Biochemical; Biological Assay; Biology; Catalytic Domain; Cells; Characteristics; Complex; Crystallization; Data; Development; Electron Microscopy; Ensure; Enzymes; Failure; Funding; Gene Expression; Genetic; Genetic Transcription; Genome; Genomics; Glycine decarboxylase; Grant; Human; Image; In Vitro; Individual; Infection; Lead; Maps; Measures; Messenger RNA; Methylation; Methyltransferase; Mutation; Negative Staining; Nucleocapsid; Nucleocapsid Proteins; Nucleotides; Phosphoproteins; Poly A; Polyadenylation; Polymerase; Proteins; Proteolysis; Public Health; RNA; RNA Processing; RNA Viruses; RNA chemical synthesis; RNA-Directed RNA Polymerase; Recombinants; Resolution; Role; Series; Specificity; Staging; Structure; System; Testing; Therapeutic; Vaccines; Vesicular stomatitis Indiana virus; Viral Genome; Work; X-Ray Crystallography; appendage; combat; design; in vitro Assay; insight; interest; mRNA capping; member; particle; pathogen; polymerization; positional cloning; premature; prevent; prototype; reconstitution; reconstruction; research study; three dimensional structure; vaccine candidate; vesicular stomatitis virus L protein; viral RNA

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.

描述（由申请人提供）：这是一种申请，以续签赠款来研究负链RNA病毒中的RNA合成。未分段的负链（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威胁。为了打击这些药物，我们需要抗病毒药物和疫苗的组合。我们的长期目的是了解囊泡气孔病毒（VSV）的复制机制，即NNS RNA病毒的原型，功能。 VSV是此类研究的理想选择，因为它是唯一可以从纯化的重组成分中重新构成鲁棒转录的NNS RNA病毒。 RNA合成机械的催化核心是241 kDa大型聚合酶蛋白（L），其中包含RNA依赖性RNA聚合酶（RDRP），一种polyribiboineucletidylansferase（pRNTAPE），该酶（PRNTASE）coct of MRNA和双重特异性mRNA CAP甲基转移酶（MIRNA）。在mRNA合成过程中，这些活性是协调的，因此新生的mRNA被封顶，甲基化和聚腺苷酸化。尽管L包含RNA合成的所有酶促活性，但它需要29 kDa磷酸蛋白（P），该磷酸蛋白（P）将L与Nucleocapsid蛋白（N）之间的相互作用桥接起来，该蛋白（N）完全覆盖了基因组RNA模板。在最后一个赠款期间，我们开发了体外测定法，以分别研究添加mRNA CAP的每个步骤，而与持续的转录无关。这些测定与强大的反向遗传系统结合使用，可以对病毒RNA合成机制中致命突变进行机械分析。我们已经使用这些测定法提供了一个图的图，介绍了MRNA CAP添加的每个步骤的不同酶促活性位于L中。这些研究使我们提出了以下假说：L包含L-P-P复合物与N-RNA模板的LNA合成机中的活性协调的独立功能结构域。理解NNS RNA病毒功能的RNA合成机制的主要差距是L。LNA病毒功能的缺乏。在下一个资金期间，我们将使用电子显微镜，X射线晶体学和多聚合酶的体外测定功能来提供独特的结构和功能，以提供独特的结构和功能，使RNA综合机器VSV vsv vsv of vsv。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. L.的mRNA封盖和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，以及该rna合成机器的功能，可以帮助您的RNA合成及其prisation theTir and rations Desigriir的功能。