Mechanism of Flavivirus RNA Capping

大约三分之二的世界人口面临着被35种虫媒中的至少一种感染的危险 已知会导致人类疾病的黄病毒。目前几乎没有疫苗，也没有治疗方法。 治疗感染黄病毒的患者，如登革热、寨卡病毒和西尼罗河病毒，尽管严重 它们每年在全球范围内造成的发病率和死亡率。改良疫苗和治疗方法的发展 要预防和治疗黄病毒感染，需要更好地了解这些分子机制 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封顶已收到 在过去的十年里，由于其在维持病毒方面的关键作用，作为抗病毒药物的靶点越来越受到关注 RNA的稳定性，控制病毒蛋白的翻译，以及先天免疫逃避。然而，没有太多 知道黄病毒RNA在感染过程中是如何被封顶的。因此，这项提案将定义如何 黄病毒在感染期间对其RNA基因组进行封顶，并评估封顶如何影响先天免疫逃避。 1)NS5 RNA鸟苷酸转移酶是一种新的黄病毒酶，其结构或序列与 任何其他已知的核苷酸转移酶。目前尚不清楚这种重要的病毒酶是如何 功能，这是我们在理解黄病毒RNA复制方面的一个关键差距。我们使用的是 联合诱变和病毒复制实验确定黄病毒NS5的活性部位 鸟苷酸转移酶，首次对这种独特的病毒复制酶进行了深入的表征。 2)黄病毒基因组5‘端非翻译区含有保守的序列和结构 已知参与RNA复制的元件，但它们在RNA封顶中的作用从未被评估过。我们 将使用一系列突变的5‘非编码区RNA来测试NS5介导的RNA封顶的特异性，以定义5’端 UTR末端序列和茎环A结构影响结合和封端效率。3)降级 通过细胞RNA衰变途径抑制黄病毒基因组导致RNA衰变和RNAi的抑制 改变对感染的免疫反应。我们希望测试一个有趣的假设，病毒 封顶效率可能由病毒战略性地调节，以产生改变感染的非编码RNA 动力学。有趣的是，我们最近发现无上限的病毒RNA被结合到病毒颗粒中 并可能包含受感染细胞中高达三分之一的病毒RNA。这种令人惊讶的高水平的无上限RNA 可能是由RNA衰变因子处理的，并导致高水平的小sfRNA，这已经被证明 以前对抗RNAi和干扰素反应(除其他外)。因此，我们将研究， NS5的封端效率如何影响病毒RNA的生产，未封顶的病毒RNA在细胞中的命运，以及NS5如何 这些无帽RNA的产物会影响黄病毒感染的动态。总体而言，该项目将 极大地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，并将提供有关黄病毒如何致病的关键新信息。

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.