mRNA synthesis and capping in nonsegmented negative strand RNA viruses

描述（由申请方提供）：非节段负链（NNS）RNA病毒包括许多危及生命的人类病原体，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依赖性RNA聚合酶L蛋白，其可催化合成5 '-加帽和3'-聚腺苷酸化mRNA所需的所有酶活性。尽管L蛋白在病毒的生命周期中扮演着重要的角色，但这些L蛋白在mRNA生物发生中的确切功能以及它们的结构域结构仍然难以捉摸。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病毒剂。为了分析L蛋白在mRNA生物合成中的作用，我们建立了水泡性口炎病毒（VSV）的重组L蛋白的体外mRNA合成和加帽分析系统，VSV是NNS RNA病毒的原型模型。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根本不同。由于帽结构是真核宿主细胞中mRNA翻译和稳定性所必需的，因此可以通过靶向其独特的加帽活性来实现NNS RNA病毒的选择性杀伤。重要的是，我们发现VSV L蛋白的加帽活性所需的保守活性位点基序对于体外有效生产全长VSV mRNA以及VSV在培养细胞中的生长是必不可少的。这些发现使我们假设NNS RNA病毒L蛋白是具有新的mRNA加帽结构域作为潜在药物靶点的模块化mRNA合成蛋白。该假设将通过以下特定目的进行严格检验：（1）确定NNS RNA病毒L蛋白在mRNA加帽中的作用，以及（2）找到针对弹状病毒mRNA加帽的小分子抑制剂。在目的1中，我们将确定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蛋白形成功能性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加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的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病毒中mRNA生物合成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，特异性抑制这些L蛋白的帽形成活性。

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.