Structure and Regulation of The Respiratory Syncytial Virus Polymerase

非分段负义（NNS）RNA病毒包括许多致病性最强、有时甚至致命的病毒，例如麻疹、狂犬病、埃博拉病毒和呼吸道合胞病毒（RSV）。不幸的是，没有有效的疫苗或抗病毒疗法可用于预防或治疗RSV和许多其他NNS RNA病毒的感染。因此，迫切需要确定NNS RNA病毒的RNA合成的结构和分子基础以及它们之间的任何差异。我们的长期目标是了解NNS RNA病毒的RNA合成机制，并促进抗病毒药物的开发。本申请的总体目标是阐明RSV聚合酶的分子机制，并提供RSV RNA合成的功能和结构见解。我们的基本假设是RNA聚合、帽加成和帽甲基化的催化活性存在于RSV L蛋白内，并且L需要与其辅因子P和M2-1的动态组装以在RNA合成期间协调这些活性。该项目的基本原理是，了解RSV RNA合成的机制可能提供强有力的科学框架，从而可以开发研究相关NNS RNA病毒的新策略。为了检验中心假设，我们将定义RSV L蛋白的功能组织，并使用冷冻EM确定RSV聚合酶的结构。我们还将确定M2-1对RSV转录的调控机制。这些结果预计将产生比RSV更广泛的影响，因为NNS RNA病毒之间RNA合成机器的共享策略意味着我们的发现将与该顺序的所有成员相关。申请人认为，该拟议研究计划具有创新性，因为拟议研究将为深入研究NNS RNA病毒的新型酶活性奠定坚实的基础，并定义RSV L蛋白的新型结构和生化特征，以及提供关于RSV RNA合成调控的新见解。拟议的研究具有重要意义，因为它利用了包括单粒子冷冻EM在内的跨学科方法的力量，为有效地可视化RSV RNA合成的关键阶段开辟了新的视野。最终，这些知识有可能为合理设计新型抗病毒药物提供新的机会，以治疗RSV和相关NNS RNA病毒引起的毁灭性疾病。

Non-segmented negative-sense (NNS) RNA viruses include many of our most pathogenic and sometimes deadly viruses, such as measles, rabies, Ebola, and respiratory syncytial virus (RSV). Unfortunately, no effective vaccine or antiviral therapy is available to prevent or treat infection by RSV and many other NNS RNA viruses. Therefore, there is a critical need to define the structural and molecular basis of RNA synthesis of NNS RNA viruses and any differences between them. Our long-term goal is to understand the RNA synthesis machinery of NNS RNA viruses and facilitate the development of antiviral drugs. Our overall objectives in this application are to elucidate the molecular mechanisms of the RSV polymerase and provide functional and structural insights into RSV RNA synthesis. Our underlying hypothesis is that the catalytic activities of RNA polymerization, cap addition, and cap methylation reside within the RSV L protein, and L requires a dynamic assembly with its cofactors P and M2-1 to coordinate these activities during RNA synthesis. The rationale for this project is that understanding the mechanism of the RSV RNA synthesis is likely to offer strong scientific frameworks whereby new strategies to investigate related NNS RNA viruses can be developed. To test the central hypothesis, we will define the functional organization of the RSV L protein and determine the structure of the RSV polymerase using cryo-EM. We will also define the regulatory mechanisms of the RSV transcription by M2-1. These results are expected to have a broader impact beyond RSV because the shared strategies of RNA synthesis machines among NNS RNA viruses imply that our findings will be relevant to all members of this order. This proposed research program is innovative, in the applicant’s opinion, because the proposed research will lay solid foundations for in-depth mechanistic studies of the novel enzymatic activities of NNS RNA viruses and define novel structural and biochemical features of the RSV L protein as well as provide novel insights on the regulation of RSV RNA synthesis. The proposed research is significant because it leverages the power of interdisciplinary approaches that include single particle cryo-EM to open new horizons for visualizing key stages of RSV RNA synthesis effectively. Ultimately, such knowledge has the potential of offering new opportunities for the rational design of novel antiviral drugs to treat the devastating diseases that RSV and related NNS RNA viruses cause.