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

基本信息

批准号：
8415505
负责人：
Tomoaki Ogino
金额：
$ 4.75万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-02-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8415505
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

项目摘要

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）：非节段负链（NNS）RNA病毒包括许多危及生命的人类病原体，例如狂犬病、麻疹和埃博拉病毒。然而，目前尚无针对NNS RNA病毒的有效药物。 NNS RNA 病毒拥有保守的 RNA 依赖性 RNA 聚合酶 L 蛋白，可催化合成 5'-加帽和 3'-聚腺苷酸化 mRNA 所需的所有酶活性。尽管 L 蛋白在病毒生命周期中发挥着多种重要作用，但这些 L 蛋白在 mRNA 生物发生中的精确功能及其结构域仍然难以捉摸。我们的长期目标是确定 L 蛋白的结构和功能，并开发针对它们的抗 NNS RNA 病毒制剂。为了分析L蛋白在mRNA生物发生中的作用，我们利用水疱性口炎病毒（VSV）（一种原型模型NNS RNA病毒）的重组L蛋白建立了体外mRNA合成和加帽测定系统。通过使用我们的体外系统，我们发现VSV L蛋白介导的mRNA加帽机制与真核宿主细胞的机制有根本的不同。由于帽结构是真核宿主细胞中 mRNA 翻译和稳定性所必需的，因此可以通过靶向 NNS RNA 病毒独特的帽活性来选择性杀死 NNS RNA 病毒。重要的是，我们发现 VSV L 蛋白加帽活性所需的保守活性位点基序对于体外全长 VSV mRNA 的有效生产以及培养细胞中 VSV 的生长至关重要。这些发现使我们推测 NNS RNA 病毒 L 蛋白是模块化 mRNA 合成蛋白，具有新的 mRNA 帽结构域作为潜在的药物靶点。该假设将通过以下具体目标进行严格检验：(1) 定义 NNS RNA 病毒 L 蛋白在 mRNA 加帽中的作用；(2) 寻找针对弹状病毒 mRNA 加帽的小分子抑制剂。在目标1中，我们将鉴定VSV L蛋白中mRNA加帽的活性位点和结构域，阐明VSV mRNA的共转录加帽机制，并研究其他NNS RNA病毒L蛋白的mRNA加帽机制。在目标2中，我们将寻找抑制VSV L蛋白形成功能帽结构的鸟苷类似物，并建立高通量筛选系统来筛选非核苷帽抑制剂的小分子文库。我们将使用生物化学、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展我们关于 NNS RNA 病毒中 mRNA 生物发生的独特机制的基础知识的前沿。我们期望 NNS RNA 病毒 L 蛋白的详细表征将为了解其进化起源和分子多样性提供新的见解。此外，从我们的研究中获得的信息有可能指导未来新型抗 NNS RNA 病毒制剂的开发，这些病毒制剂特异性抑制这些 L 蛋白的帽子形成活性。