Dissecting catalytic and regulatory functions of nonsegmented negative strandRNA viral polymerases

非节段负链（NNS）RNA病毒，如狂犬病病毒（RABV）、人呼吸道合胞病毒（HRSV）、人副流感病毒3型（HPIV 3）和埃博拉病毒（EBOV），对人类健康构成持续威胁，但目前还没有针对大多数NNS RNA病毒疾病的既定预防和/或治疗对策。NNS RNA病毒具有多功能RNA依赖性RNA聚合酶（RdRp）大（L）蛋白，其催化病毒RNA生物合成所需的所有酶促反应（例如，RNA合成，5′-加帽，鸟嘌呤-N7-和核糖-2 ′-O-位置的帽甲基化，3′-聚腺苷酸化）。L蛋白的所有这些酶活性都是独特的，并且可能是可药用的。我们的目标是阐明RNA合成和加工的分子机制与L蛋白，并开发抗病毒药物。为了剖析L蛋白在RNA生物合成中的作用，我们已经开发了一些体外弹状病毒RNA合成和加工系统的原型水泡性口炎病毒（VSV）和RABV。使用这些系统，我们发现弹状病毒L蛋白用新的GDP多核糖核苷酸转移酶（PRNTase，EC 2.7.7.88）结构域催化非常规mRNA加帽。然而，前mRNA加帽和甲基化与mRNA链延长的机制在很大程度上仍然未知。此外，L蛋白的病毒特异性功能尚未研究.我们假设L蛋白通过进化保守元件催化常见的酶促反应，但通过多样化的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进行严格检验：阐明（1）弹状病毒L蛋白的共转录mRNA成熟机制，（2）肺病毒和副粘病毒L蛋白的帽形成机制，以及（3）丝状病毒L蛋白的转录和复制机制。在目的1中，我们将确定的时间和顺序的前mRNA处理的VSV L蛋白在mRNA链延伸，导致一个新的模式的共同转录mRNA成熟的L蛋白的弹状病毒，并通过扩展，其他NNS RNA病毒。在目标2中，我们将揭示HRSV和HPIV 3 L蛋白的推定PRNTase和甲基转移酶结构域在mRNA帽形成中的共同和多样功能。在目标3中，我们将提供一种新的模型，基因组复制的EBOV L蛋白，这是显着不同的其他NNS RNA病毒L蛋白。总的来说，提出的研究将开辟一个新的前沿，了解多样化的NNS L蛋白如何进行RNA合成和加工的每一步与它们的同源辅因子蛋白。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.