mRNA synthesis and capping in nonsegmented negative strand RNA viruses

描述(申请人提供)：非分段负链(NNS)RNA病毒包括许多威胁人类生命的病原体，如狂犬病、麻疹和埃博拉。然而，目前还没有有效的药物来对抗NNS RNA病毒。NNSRNA病毒具有保守的依赖于RNA的RNA聚合酶L蛋白，该蛋白可催化合成5‘-帽和3’-多腺苷基化的mRNAs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蛋白在基因生物合成中的确切功能及其结构域结构仍不清楚。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NNSRNA病毒制剂。为了分析L蛋白在基因生物合成中的作用，我们以水疱性口炎病毒的重组L蛋白为模板，建立了体外基因合成和封帽实验体系。利用我们的体外实验系统，我们发现VSV L蛋白介导的基因封顶机制与真核宿主细胞的机制有本质的不同。由于帽结构是真核宿主细胞中mRNA翻译和稳定所必需的，因此可以通过靶向NNS RNA病毒独特的帽结构活性来实现对其的选择性杀伤。重要的是，我们发现VSV L蛋白的封端活性所需的保守活性位点基序对于VSV全长mRNAs的体外高效生产以及VSV在培养细胞中的生长是必不可少的。这些发现使我们推测，NNSRNA病毒L蛋白是一种模块化的信使核糖核酸合成蛋白，具有新的信使核糖核酸封闭区作为潜在的药物靶点。这一假说将通过以下具体目标进行严格的检验：(1)确定NNSRNA病毒L蛋白在基因封顶中的作用；(2)寻找抗横纹病毒基因封顶的小分子抑制剂。在目标1中，我们将确定VSV L蛋白中基因封顶的活性部位和结构域，阐明VSVmRNAs共转录封顶的机制，并研究其他NNSRNA病毒L蛋白封顶的机制。在目标2中，我们将寻找能够抑制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封顶抑制剂的小分子文库。我们将使用生化、蛋白质组和分子病毒学方法来实现这些目标。总而言之，我们提出的研究将扩大我们关于NNS RNA病毒中mRNA生物发生的独特机制的基础知识的前沿。我们预计，对NNSRNA病毒L蛋白的详细描述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新的抗NNSRNA病毒制剂的开发，这些病毒制剂可以特异性地抑制这些L蛋白的帽子形成活性。

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.