Mechanism of Flavivirus RNA Capping

大约三分之二的世界人口面临感染35种虫媒病毒中至少一种的风险。 黄病毒已知会引起人类疾病。目前几乎没有疫苗，也没有可用的治疗方法 治疗黄病毒感染的患者，如登革热，寨卡和西尼罗河病毒，尽管严重的 它们每年在全球造成的发病率和死亡率。改进疫苗和治疗方法的发展 为了预防和治疗黄病毒感染， 严重的人类病原体用来复制它们的基因组。黄病毒基因组的RNA加帽已经收到 由于其在维持病毒感染中的关键作用， RNA稳定性、控制病毒蛋白翻译和先天免疫逃避。然而， 了解黄病毒RNA在感染过程中是如何被加帽的。因此，本提案将界定如何 黄病毒在感染过程中给它们的RNA基因组加帽，并评估加帽如何影响先天免疫逃避。 1)NS5 RNA鸟苷酰转移酶是一种新的黄病毒酶，与NS5 RNA没有结构或序列相似性。 任何其它已知的核苷酸转移酶。目前尚不清楚这种重要的病毒酶 功能，这是我们理解黄病毒RNA复制的关键差距。我们使用的是 组合诱变和病毒复制实验以确定黄病毒NS5的活性位点 鸟苷酰转移酶，提供了这种独特的病毒复制酶的第一次深入表征。 2)黄病毒基因组的5'非翻译区（UTR）含有保守序列和结构特征。 已知参与RNA复制的元件，但它们在RNA加帽中的作用从未被评估过。我们 将使用一系列突变的5' UTR RNA来测试NS5介导的RNA加帽的特异性，以确定5' UTR RNA如何与NS5结合。 UTR末端序列和茎环A结构影响结合和加帽效率。3)降解 通过细胞RNA降解途径对黄病毒基因组的抑制导致RNA降解和RNAi 途径，改变对感染的免疫反应。我们希望验证一个有趣的假设， 加帽效率可能受到病毒的策略性调节，以产生改变感染的非编码RNA 动力学有趣的是，我们最近发现， 并且可以在受感染的细胞中包含多达三分之一的病毒RNA。这种高水平的无帽RNA 很可能是由RNA衰变因子处理的，并导致高水平的小sfRNA， 以前拮抗RNAi和干扰素反应（除其他外）。因此，我们将研究， NS5加帽效率如何影响病毒RNA的产生，细胞中未加帽病毒RNA的命运，以及 这些脱帽RNA的产物影响黄病毒感染的动力学。总体而言，该项目将 显着提高了我们对黄病毒复制机制的理解，我们可以利用这些机制来抗病毒 和疫苗开发，并将提供关于黄病毒如何引起疾病的重要新信息。

Approximately 2/3rd of the world population is at risk of infection by at least one of the 35 insect-borne flaviviruses known to cause disease in humans. There are currently few vaccines and no therapeutics available to treat patients infected by flaviviruses such as Dengue, Zika, and West Nile viruses despite the severe morbidity and mortality they cause globally each year. The development of improved vaccines and therapeutics to prevent and treat flavivirus infections requires improved knowledge of the molecular mechanisms these serious human pathogens use to replicate their genomes. RNA capping of flavivirus genomes has received increasing attention over the last decade as an antiviral drug target due to its critical roles in maintaining viral RNA stability, controlling viral protein translation, and innate immune evasion. There is, however, not much known about how flavivirus RNAs are capped during infection. Therefore, this proposal will define how flaviviruses cap their RNA genomes during infection and evaluate how capping affects innate immune evasion. 1) The NS5 RNA guanylyltransferase is a novel flavivirus enzyme with no structural or sequence similarities to any other known nucleotidyltransferase enzyme. It is currently unknown how this important viral enzyme functions, which is a critical gap in our understanding of flavivirus RNA replication. We are using a combination mutagenesis and viral replication experiments to define the active site of the flavivirus NS5 guanylyltransferase, providing the first in-depth characterization of this unique viral replication enzyme. 2) The 5' untranslated region (UTR) of the flavivirus genome contains conserved sequence and structural elements known to be involved in RNA replication, but their role in RNA capping has never been assessed. We will use a series of mutated 5' UTR RNAs to test the specificity of NS5-mediated RNA capping to define how 5' UTR terminal sequences and the stem-loop A structure affect binding and capping efficiency. 3) Degradation of flavivirus genomes by the cellular RNA decay pathway results in the inhibition of RNA decay and RNAi pathways, altering the immune response to infection. We wish to test the intriguing hypothesis that viral capping efficiency may be strategically regulated by viruses to produce non-coding RNAs that alter infection dynamics. Interestingly, we have recently found that uncapped viral RNAs are incorporated into virus particles and may comprise up to a third of viral RNAs in an infected cell. This surprisingly high level of uncapped RNA is likely processed by RNA decay factors and leads to high levels of a small sfRNA which has been shown previously to antagonize RNAi and interferon responses (among other things). We will examine, therefore, how NS5 capping efficiency affects viral RNA production, the fate of uncapped viral RNAs in cells, and how the products of these uncapped RNAs influence the dynamics of a flavivirus infection. Overall, this project will significantly advance our understanding of flavivirus replication mechanisms that we can exploit for antiviral and vaccine development, and will provide critical new information about how flaviviruses cause disease.