Mechanisms of viral RNA maturation by co-opting cellular exonucleases

项目总结 黄病毒是单链阳性RNA病毒，包括危险的人类病原体，如 登革热、西尼罗河、黄热病、寨卡病毒和许多其他疾病。在感染期间，这些病毒产生一组非 编码被称为亚基因组黄病毒RNA(SfRNAs)的RNA，它与细胞蛋白质相互作用，操纵 细胞环境，包括抑制抗病毒反应。SfRNA与细胞病变直接相关 和致病后果，以及不能产生sfRNA的病毒被减弱，激励努力 了解xrRNA的产生机制。SfRNA是当细胞内的5‘à3’外切核糖核酸酶(in 具体地，Xrn1)过程地降解病毒基因组RNA，但随后在特定结构的RNA处停止 病毒3‘端非编码区中的元件称为外切核酸酶抗性RNAs(XrRNAs)。通过解决问题的结构 通过x射线结晶学和生物化学、生物物理学和病毒学相结合，我们发现了多种xrrna 显示了xrRNA折叠成一个独特的环状拓扑结构，它创建了一个机械地阻止外切核糖核酸酶 不能通过。此外，我们利用我们的发现对xrRNA进行了分类，并找到了新的例子 与非编码和编码RNA相关联。这些成功现在定义了几个新的问题。第一, XrRNA经常被发现在多个拷贝中，它们的功能以某种方式耦合在一起，但 这种偶联的结构基础，以及打破这种偶联对sfRNA形成和病毒的影响 感染动力学，目前尚不清楚。第二，虽然我们对几类xrRNA有很好的了解， 我们还没有解决扁虱传播的黄病毒的xrRNA的结构，它似乎具有有趣的和 独一无二的特性。第三，虽然我们已经发现了许多xrRNA的新例子，但似乎有很多 还有更多未被发现的基因，我们也不了解不同类别的xrrrna之间的关系。 从进化上讲。考虑到折叠的严格限制，这些结构如何在3-D中多样化和进化？ 在这里，我们建议从三个目标来回答这些问题，采用一种结合生物化学、x- 射线结晶学、低温EM、病毒学和体外选择与计算工具相结合。这项研究 这里所描述的将有助于对广谱的一个重要的分子过程的重要的基本知识 对病毒性疾病的适用性，从发现一种机制到将其作为目标的必要步骤 治疗性干预。

PROJECT SUMMARY Flaviviruses are single-stranded positive-sense RNA viruses that include dangerous human pathogens like dengue, West Nile, Yellow Fever, Zika, and many others. During infection, these viruses produce a set of non- coding RNAs called ‘subgenomic flavivirus RNAs’ (sfRNAs) that interact with cellular proteins to manipulate the cellular environment, to include inhibiting the antiviral response. sfRNAs have been directly linked to cytopathic and pathogenic outcomes, and viruses that cannot produce sfRNAs are attenuated, motivating efforts to understand the mechanism of xrRNA production. sfRNAs are made when cellular 5’à3’ exoribonucleases (in particular, Xrn1) processively degrade the viral genomic RNA but then halt at specifically structured RNA elements in the viral 3’ UTR called exoribonuclease resistant RNAs (xrRNAs). By solving the structures of multiple xrRNAs by x-ray crystallography and combining this with biochemistry, biophysics, and virology, we showed that xrRNAs fold into a unique ring-like topology that creates a mechanical block the exoribonuclease cannot pass through. Furthermore, we used our discoveries to classify xrRNAs and to find new examples associated with both non-coding and coding RNAs. These successes now define several new questions. First, xrRNAs are often found in multiple copies ‘in tandem’ where their function is coupled in some way, but the structural basis of this coupling, and the effects of breaking the coupling on both sfRNA formation and viral infection kinetics, are unknown. Second, although we have a good understanding of several classes of xrRNAs, we have yet to solve the structure of an xrRNA from a tick-borne flavivirus, which appear to have interesting and unique properties. Third, although we have found many new examples of xrRNAs, it appears there are many more that are undiscovered, and we also do not understand how the various classes of xrRNA relate evolutionarily. How do these structures diversify and evolve in 3-D given the tight constraints on their folding? Here, we propose to answer these questions in three aims, employing a strategy that combines biochemistry, x- ray crystallography, cryo-EM, virology, and in vitro selections coupled with computational tools. The research described here will contribute significant basic knowledge regarding an important molecular process of broad applicability to viral disease, a necessary step between the discovery of a mechanism and the targeting of it for therapeutic intervention.