Dissecting catalytic and regulatory functions of nonsegmented negative strandRNA viral polymerases

狂犬病毒（RABV）、人呼吸道合胞病毒（HRSV）、人副流感病毒3型（HPIV3）和埃博拉病毒（EBOV）等非节段负链RNA病毒对人类健康构成持续威胁，但目前尚无针对大多数NNS RNA病毒性疾病的预防和/或治疗对策。NNS RNA病毒具有一种多功能RNA依赖RNA聚合酶（RdRp）大(L)蛋白，该蛋白催化病毒RNA生物发生所需的所有酶促反应（例如RNA合成、5 ' -capping、鸟嘌呤- n7 -和核糖-2 ' - o位的甲基化、3 ' -聚腺苷化）。L蛋白的所有这些酶活性都是独特的，具有潜在的药物价值。我们的目标是阐明RNA合成和L蛋白加工的分子机制，并开发针对它们的抗病毒药物。为了研究L蛋白在RNA生物合成中的作用，我们开发了一些用于原型水疱性口炎病毒（VSV）和RABV的体外横纹肌病毒RNA合成和加工系统。利用这些系统，我们发现横纹肌病毒L蛋白用一种新的GDP多核糖核苷酸转移酶（PRNTase, EC 2.7.7.88）结构域催化非常规的mRNA capping。然而，mRNA前盖帽和甲基化与mRNA链延伸耦合的机制仍然很大程度上未知。此外，L蛋白的病毒特异性功能尚未被研究。我们假设L蛋白通过进化保守的元件催化常见的酶促反应，但通过多种元件表现出病毒特异性功能。这一假设将通过以下具体目标得到严格的验证：阐明(1)横纹肌病毒L蛋白共转录mRNA成熟的机制，(2)肺炎病毒和副粘病毒L蛋白形成帽状结构的机制，以及(3)丝状病毒L蛋白转录和复制的机制。在Aim 1中，我们将确定VSV L蛋白在mRNA链延伸过程中的前mRNA加工的时间和顺序，从而建立横纹肌病毒和其他NNS RNA病毒的L蛋白共转录mRNA成熟的新模型。在Aim 2中，我们将揭示HRSV和HPIV3 L蛋白的PRNTase和甲基转移酶结构域在mRNA帽形成中的共同和多样化的功能。在Aim 3中，我们将提供一种新的EBOV L蛋白基因组复制模型，该模型与其他NNS RNA病毒L蛋白的基因组复制模型有显著不同。总的来说，拟议的研究将为了解多样化的NNS L蛋白如何与其同源辅因子蛋白一起进行RNA合成和加工的每个步骤开辟新的前沿。我们的研究将为未来开发针对这些重要NNS RNA病毒独特L结构域的抗病毒药物提供基础。

Non-segmented negative strand (NNS) RNA viruses, such as rabies virus (RABV), human respiratory syncytial virus (HRSV), human parainfluenza virus type 3 (HPIV3), and Ebola virus (EBOV), pose continuing threats to human health, but there are currently no established prophylactic and/or therapeutic countermeasures against most NNS RNA viral diseases. NNS RNA viruses possess a multifunctional RNA-dependent RNA polymerase (RdRp) large (L) protein, which catalyzes all enzymatic reactions required for viral RNA biogenesis (e.g., RNA synthesis, 5′-capping, cap methylation at the guanine-N7- and ribose-2′-O-positions, 3′-polyadenylation). All these enzymatic activities of the L proteins are unique and potentially druggable. Our goals are to elucidate the molecular mechanisms of RNA synthesis and processing with the L proteins and to develop anti-viral agents against them. To dissect the roles of the L proteins in RNA biosynthesis, we have developed a number of in vitro rhabdoviral RNA synthesis and processing systems for prototypic vesicular stomatitis virus (VSV) and RABV. Using these systems, we discovered that rhabdoviral L proteins catalyze unconventional mRNA capping with a novel GDP polyribonucleotidyltransferase (PRNTase, EC 2.7.7.88) domain. However, the mechanisms of pre- mRNA capping and methylation coupled to mRNA chain elongation remain largely unknown. Furthermore, virus- specific functions of L proteins have not been studied. We hypothesize that L proteins catalyze common enzymatic reactions via evolutionary conserved elements, but manifest virus-specific functions via diversified elements. This hypothesis will be rigorously tested by the following specific aims: to elucidate the mechanisms of (1) co-transcriptional mRNA maturation by rhabdoviral L proteins, (2) cap formation by pneumoviral and paramyxoviral L proteins, and (3) transcription and replication by filoviral L proteins. In Aim 1, we will determine the timing and order of pre-mRNA processing by the VSV L protein during mRNA chain elongation, leading to a new model of co-transcriptional mRNA maturation by L proteins of rhabdoviruses and, by extension, other NNS RNA viruses. In Aim 2, we will reveal common and diversified functions of putative PRNTase and methyltransferase domains of the HRSV and HPIV3 L proteins in mRNA cap formation. In Aim 3, we will provide a novel model for genome replication by the EBOV L protein that is significantly different from that by other NNS RNA viral L proteins. Collectively, the proposed studies will open up a new frontier in understanding how diversified NNS L proteins carry out each step of RNA synthesis and processing together with their cognate co- factor proteins. Our studies will provide foundations for the future development of antiviral agents targeting unique L domains of these significant NNS RNA viruses.