mRNA synthesis and capping in nonsegmented negative strand RNA viruses

描述（由申请人提供）：非分段负链（NNS） RNA病毒包括许多危及生命的人类病原体，如狂犬病、麻疹和埃博拉病毒。然而，目前还没有针对NNS RNA病毒的有效药物。NNS RNA病毒具有保守的RNA依赖RNA聚合酶L蛋白，可催化合成5'-cap和3'-聚腺苷化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生物发生中的确切功能及其结构域仍然是难以捉摸的。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，我们建立了水疱性口炎病毒（VSV）的重组L蛋白体外mRNA合成和封盖检测系统。通过我们的体外系统，我们发现VSV L蛋白介导的mRNA capping机制与真核宿主细胞有本质的不同。由于帽盖结构是真核宿主细胞mRNA翻译和稳定性所必需的，因此可以通过靶向NNS RNA病毒独特的帽盖活性来选择性杀伤NNS RNA病毒。重要的是，我们发现了一个保守的活性位点基序，这是VSV L蛋白capping活性所必需的，对于有效地在体外产生全长VSV mrna以及在培养细胞中VSV生长是必不可少的。这些发现使我们假设NNS RNA病毒L蛋白是模块化mRNA合成蛋白，具有新的mRNA capping结构域作为潜在的药物靶点。这一假设将通过以下具体目标得到严格的检验：(1)确定NNS RNA病毒L蛋白在mRNA capping中的作用；(2)找到针对横纹肌病毒mRNA capping的小分子抑制剂。在Aim 1中，我们将确定VSV L蛋白中mRNA capping的活性位点和结构域，阐明VSV mRNA的共转录capping机制，并研究其他NNS RNA病毒L蛋白的mRNA capping机制。在Aim 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盖帽抑制剂的小分子文库。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对NNS RNA病毒mRNA生物发生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开发新的抗nns RNA病毒药物，特异性地抑制这些L蛋白的帽形形成活性。

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.