Mechanisms of viral RNA maturation by co-opting cellular exonucleases

项目摘要 黄病毒是单链正义RNA病毒，包括危险的人类病原体，如 登革热、西尼罗河病毒、黄热病、寨卡病毒和其他许多疾病。在感染过程中，这些病毒产生一系列非- 编码RNA称为“亚基因组黄病毒RNA”（sfRNA），与细胞蛋白质相互作用， 细胞环境，包括抑制抗病毒反应。sfRNA与细胞病变直接相关， 和致病性结果，不能产生sfRNA的病毒被减弱，促使人们努力 了解xrRNA产生的机制。当细胞5 'à3'核糖核酸外切酶（在 特别是，Xrn 1）原酶降解病毒基因组RNA，但随后在特异性结构的RNA处停止 病毒3' UTR中的元件称为核糖核酸外切酶抗性RNA（xrRNA）。通过求解 通过X射线晶体学并结合生物化学，生物物理学和病毒学，我们 显示xrRNA折叠成独特的环状拓扑结构，该拓扑结构产生了核糖核酸外切酶的机械阻断， 不能通过。此外，我们利用我们的发现对xrRNA进行分类，并找到新的例子。 与非编码和编码RNA相关。这些成功现在界定了几个新的问题。第一、 xrRNA通常以“串联”的多个拷贝存在，其中它们的功能以某种方式偶联，但是 这种偶联的结构基础，以及破坏偶联对sfRNA形成和病毒感染的影响。 感染动力学尚不清楚。其次，虽然我们对几类xrRNA有很好的了解， 我们还没有解决来自蜱传黄病毒的xrRNA的结构，它似乎具有有趣的， 独特的属性。第三，虽然我们已经发现了许多新的xrRNA的例子， 更多的是未被发现的，我们也不知道各种类型的xrRNA是如何联系在一起的。 从进化的角度来看这些结构如何在3D中多样化和进化，因为它们的折叠受到严格限制？ 在这里，我们建议回答这些问题的三个目标，采用的战略，结合生物化学，x- 射线晶体学、冷冻EM、病毒学和与计算工具相结合的体外选择。研究 这里所描述的将有助于重要的基础知识，关于一个重要的分子过程的广泛 适用于病毒性疾病，这是发现机制和靶向它之间的必要步骤， 治疗干预

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.