Structure and Regulation of The Respiratory Syncytial Virus Polymerase

项目摘要 非分段负义(NNS)RNA病毒包括我们许多最具致病性的，有时甚至是致命的 麻疹、狂犬病、埃博拉和呼吸道合胞病毒(RSV)等病毒。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，迫切需要确定合成NNS RNA的结构和分子基础 病毒以及它们之间的任何差异。我们的长期目标是了解RNA合成机制 研究NNS RNA病毒，并促进抗病毒药物的开发。我们在此应用程序中的总体目标 旨在阐明RSV聚合酶的分子机制，并提供功能和结构方面的见解 以合成呼吸道合胞病毒RNA。我们的基本假设是，RNA聚合的催化活性 加法和帽甲基化位于呼吸道合胞病毒L蛋白内，L需要与其动态组装 辅因子P和M2-1在RNA合成过程中协调这些活动。这个项目的基本原理是 了解RSV RNA合成的机制可能会提供强有力的科学框架，从而 可以开发新的策略来研究相关的NNS RNA病毒。为了检验中心假设，我们将 确定呼吸道合胞病毒L蛋白的功能组织和确定呼吸道合胞病毒聚合酶的结构 冷冻机。我们还将定义M2-1对RSV转录的调控机制。这些结果是 预计将产生比RSV更广泛的影响，因为RNA合成机器的共享策略 在NNS中，RNA病毒暗示我们的发现将与这一目的所有成员相关。这项建议 在申请人看来，研究计划是创新的，因为拟议的研究将奠定坚实的基础 深入研究NNS RNA病毒新的酶活性的基础和定义 呼吸道合胞病毒L蛋白的新结构和生化特征以及对调控的新见解 呼吸道合胞病毒RNA的合成。这项拟议的研究具有重要意义，因为它利用了跨学科的力量 包括单粒子冷冻-EM在内的方法为可视化RSV RNA的关键阶段开辟了新的视野 有效地进行综合。归根结底，这样的知识有可能为理性的人提供新的机会 设计新的抗病毒药物来治疗RSV和相关的NNS RNA病毒引起的破坏性疾病。

PROJECT ABSTRACT 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 robust 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.