mRNA synthesis and capping in nonsegmented negative strand RNA viruses

非节段负链 RNA 病毒中 mRNA 的合成和加帽

• 批准号: 8604362
• 负责人: Tomoaki Ogino
• 金额: $ 35.66万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8604362
• 关键词: Active Sites; Amino Acids; Biochemical; Biogenesis; Biological Assay; Biology; Cells; Complex; Cultured Cells; DNA-Directed RNA Polymerase; Development; Drug Targeting; Exhibits; Family; Future; Genetic Transcription; Genetic Translation; Goals; Growth; Guanosine; Guanosine Triphosphate; Health; Human; In Vitro; Knowledge; Lead; Length; Life; Life Cycle Stages; Maps; Measles; Mediating; Messenger RNA; Modeling; Molecular; Mononegavirales; Mumps; Mutation; Parainfluenza; Paramyxovirus; Pharmaceutical Preparations; Play; Production; Proteins; Proteomics; Publishing; RNA; RNA Caps; RNA Viruses; RNA chemical synthesis; RNA triphosphatase; RNA-Directed RNA Polymerase; Rabies; Reaction; Recombinants; Research; Rhabdoviridae; Role; Sendai virus; Site-Directed Mutagenesis; Specificity; Structure; Study models; System; Testing; Vesicular stomatitis Indiana virus; Viral; aminoadipoyl-cysteinyl-allylglycine; analog; drug discovery; enzyme activity; frontier; high throughput screening; in vitro activity; inhibitor/antagonist; insight; killings; mRNA Precursor; mRNA Stability; mRNA capping; mRNA guanylyltransferase; novel; novel strategies; pathogen; polyadenylated messenger RNA; prototype; respiratory; small molecule; small molecule libraries; vesicular stomatitis virus L protein; viral RNA

DESCRIPTION (provided by applicant): Nonsegmented negative strand (NNS) RNA viruses include many life-threatening human pathogens, such as rabies, measles, and Ebola. However, there is presently no effective drug against NNS RNA viruses. NNS RNA viruses possess conserved RNA-dependent RNA polymerase L proteins that may catalyze all enzymatic activities required for the synthesis of 5'-capped and 3'-polyadenylated mRNAs. Despite the fact that L proteins play multiple essential roles in viral life cycles, the precise functions of these L proteins in mRNA biogenesis as well as their domain structures remain elusive. Our long-term goals are to define the structure and function of L proteins and to develop anti-NNS RNA viral agents against them. To analyze the roles of L proteins in mRNA biogenesis, we have established in vitro mRNA synthesis and capping assay systems with the recombinant L protein of vesicular stomatitis virus (VSV), a prototypic model NNS RNA virus. By using our in vitro systems, we have discovered that the mechanism of mRNA capping mediated by the VSV L protein is fundamentally different from that of eukaryotic host cells. Since the cap structure is required for mRNA translation and stability in eukaryotic host cells, selective killing of NNS RNA viruses could be achieved by targeting their unique capping activities. Importantly, we have found that a conserved active site motif required for the capping activity of the VSV L protein is essential for efficient production of full-length VSV mRNAs in vitro as well as VSV growth in cultured cells. These findings lead us to hypothesize that NNS RNA viral L proteins are modular mRNA-synthesizing proteins having novel mRNA capping domains as potential drug targets. The hypothesis will be rigorously tested by the following Specific Aims: (1) to define the roles of the NNS RNA viral L proteins in mRNA capping and (2) to find small molecule inhibitors against rhabdoviral mRNA capping. In Aim 1, we will identify active sites and domains for mRNA capping in the VSV L protein, elucidate the mechanism of co-transcriptional capping of VSV mRNAs, and study the mechanisms of mRNA capping by other NNS RNA viral L proteins. In Aim 2, we will search for guanosine analogs that inhibit the formation of the functional cap structure by the VSV L protein, and establish high-throughput screening systems to screen small molecule libraries for non-nucleoside capping inhibitors. We will use biochemical, proteomic, and molecular virological approaches to achieve these Aims. Collectively, our proposed studies will expand the frontier of our fundamental knowledge about the unique mechanisms of mRNA biogenesis in NNS RNA viruses. We expect that detailed characterization of NNS RNA viral L proteins will provide new insight into their evolutionary origin and molecular diversification. Furthermore, information derived from our research has the potential to guide future development of new anti-NNS RNA viral agents that specifically inhibit cap-forming activities of these L proteins.

描述（由申请人提供）：未分段的阴性链（NNS）RNA病毒包括许多威胁生命的人类病原体，例如狂犬病，麻疹和埃博拉病毒。但是，目前尚无针对NNS RNA病毒的有效药物。 NNS RNA病毒具有保守的RNA依赖性RNA聚合酶L蛋白，可催化合成5'Papped和3'-聚乙酰化mRNA所需的所有酶活性。尽管L蛋白在病毒生命周期中起多个重要作用，但这些L蛋白在mRNA生物发生以及它们的结构结构中的精确功能仍然难以捉摸。我们的长期目标是定义L蛋白的结构和功能，并针对它们开发抗NNS RNA病毒剂。为了分析L蛋白在mRNA生物发生中的作用，我们已经用囊泡口腔炎病毒（VSV）的重组L蛋白（一种原型型Model Mote NNS RNA RNA病毒）建立了体外mRNA合成和封盖测定系统。通过使用我们的体外系统，我们发现由VSV L蛋白介导的mRNA限制机理与真核宿主细胞的根本不同。由于真核宿主细胞中mRNA翻译和稳定性所需的帽结构，因此可以通过靶向其独特的封盖活性来选择性杀死NNS RNA病毒。重要的是，我们发现VSV L蛋白上限活性所需的保守活性位点基序对于在体外有效产生全长VSV mRNA以及培养细胞中VSV生长至关重要。这些发现使我们假设NNS RNA病毒L蛋白是具有新型mRNA限值结构域作为潜在药物靶标的模块化mRNA合成蛋白。该假设将通过以下特定目的进行严格检验：（1）定义NNS RNA病毒L蛋白在mRNA限制中的作用，以及（2）找到针对色齿mRNA限额的小分子抑制剂。在AIM 1中，我们将确定VSV L蛋白中mRNA封盖的活性位点和域，阐明VSV mRNA共转录封盖的机理，并研究其他NNS RNA RNA病毒lna病毒L蛋白的mRNA限额的机制。在AIM 2中，我们将搜索VSV L蛋白抑制功能帽结构的形成的鸟嘌呤类似物，并建立高通量筛选系统以筛选小分子库，以供非核苷限值抑制剂。我们将使用生化，蛋白质组学和分子病毒学方法来实现这些目标。总的来说，我们提出的研究将扩大我们关于NNS RNA病毒中mRNA生物发生独特机制的基本知识的领域。我们预计NNS RNA病毒L蛋白的详细表征将为它们的进化起源和分子多样化提供新的见解。此外，从我们的研究中得出的信息具有指导新的抗NNS RNA病毒剂的未来开发，这些抗NNS RNA病毒剂特别抑制了这些L蛋白的cap形成活性。