Mechanisms of viral RNA maturation by co-opting cellular exonucleases

项目摘要 作为专性细胞寄生虫，病毒采用多种策略来利用宿主细胞的机制，利用它来产生 成功感染所需的分子这些策略中的许多涉及形成特定结构的病毒RNA 能够与细胞成分相互作用并操纵细胞成分。一个重要的例子是蚊子传播的黄病毒， 其选择细胞核糖核酸外切酶并使用其产生致病重要的非编码RNA。具体而言是 5 '端3'核糖核酸外切酶Xrn1被募集到基因组RNA中，并被原核生物降解，但随后在特定位置停止 在基因组中。这种程序化的“核糖核酸外切酶抗性”依赖于特定的三维RNA结构， 嵌入在黄病毒RNA中蚊媒黄病毒的核糖核酸外切酶抗性RNA（xrRNA）是 这个过程的原型，我们通过研究它们学到了很多东西。然而，现在很明显，联合国的战略， 选择和利用细胞核糖核酸外切酶并不限于这些病毒，而是可能广泛存在。证据表明 不同的病毒使用不同类型的抗核糖核酸外切酶的RNA元件作为加工长前体的手段， RNA转化为更短的具有生物活性的RNA。然而，尽管这些新的核糖核酸外切酶的重要性正在显现， 尽管我们对耐药RNA的结构和它们的作用机制几乎一无所知。在燃烧的 基本问题：所有这些假定的Xrn1抗性元件都使用类似的机制吗？尽管没有明显的 序列相似性，它们都是折叠的RNA吗？它们都是由RNA结构驱动的吗？还是有些需要结合蛋白质？是 这些不同RNA的折叠相似，或者自然进化出许多方式来实现阻断肿瘤进展的目标。 核糖核酸外切酶由于缺乏基本信息，我们对不同病毒中这些过程的理解受到阻碍 关于各种xrRNA结构。因此，本提案的重点是通过研究几个 未被探索的xrRNA的例子。我们的目标是深入了解核糖核酸外切酶抗性的广度和多样性， 现象，发现核糖核酸外切酶抗性的基本原则，可能适用于更大的病毒 世界，并开发新技术，使我们能够找到或预测其他病毒和环境中的核糖核酸外切酶结构。 我们提出了三个目标：（1）确定的基本序列，结构决定因素，和机械特性， 通过一组不同的黄病毒RNA的核糖核酸外切酶抗性。(2)定义RNA的序列和结构， 香石竹病毒和裂谷热病毒，赋予核糖核酸外切酶抗性，和（3）开发一种合成的扩增的 Xrn1抗性RNA的遗传学，并使用它来计算搜索其他病毒中未识别的抗性RNA。 我们的方法是结合联合收割机生化分析，这是我们实验室独有的，包括一套全面的工具 为了探索这些RNA，结构生物学，包括X射线晶体学，以及体外选择， 计算工具。这里描述的研究将有助于重要的基础知识， 广泛适用于病毒性疾病的分子过程，是发现机制和治疗之间的必要步骤。 将其作为治疗干预的目标。

Project Summary As obligate cellular parasites, viruses employ a variety of strategies to co-opt the host cell’s machinery, using it to generate the molecules needed for successful infection. Many of these strategies involve viral RNA that forms specific structures able to interact with and manipulate cellular components. An important example is found in the mosquito-borne flaviviruses, which co-opt a cellular exoribonuclease and use it to generate pathogenically-important non-coding RNAs. Specifically, the 5’3’ exoribonuclease Xrn1 is recruited to the genomic RNA, processively degrades it, but then halts at specific locations in the genome. This programmed “exoribonuclease resistance” depends on specific three-dimensional RNA structures that are embedded in the flaviviral RNA. The exoribonuclease-resistant RNAs (xrRNAs) of the mosquito-borne flaviviruses are the prototypes of this process and we have learned much by studying them. However, it is now clear that the strategy of co- opting and exploiting cellular exoribonucleases is not limited to these viruses, but may be widespread. Evidence suggests that diverse viruses use different types of exoribonuclease-resistant RNA elements as a means to process long precursor RNAs into shorter, biologically active RNAs. However, despite the emerging importance of these novel exoribonuclease- resistant RNA structures and the mechanisms they perform, we know almost nothing about them. Among the burning fundamental questions: Do all of these putative Xrn1-resistant elements use a similar mechanism? Despite no obvious sequence similarity, are they all folded RNAs? Are they all RNA structure-driven, or do some require bound proteins? Are the folds of these different RNAs similar, or has nature evolved many ways to achieve the goal of blocking progression of an exoribonuclease? Our understanding of these processes in diverse viruses is hampered by a lack of basic information about various xrRNA structures. The focus of this proposal is therefore to drive the field forward by studying several unexplored examples of xrRNAs. We aim to gain insight into the breadth and diversity of the exoribonuclease resistance phenomenon, to discover fundamental principles of exoribonuclease resistance that may be applicable across the larger viral world, and to develop new technology to enable us to find or predict exoribonuclease structures in other viruses and contexts. We propose three aims: (1) Determine the essential sequences, structural determinants, and mechanistic characteristics of exoribonuclease resistance by a diverse set of flaviviral RNAs. (2) Define sequences and structures of RNAs from the Dianthoviruses and Rift Valley Fever Virus that confer exoribonuclease resistance, and (3) Develop a synthetic expanded phylogeny of Xrn1-resistant RNAs and use this to computationally search for unidentified resistant RNAs in other viruses. Our approach is to combine biochemical assays that are unique to our lab and that comprise a comprehensive set of tools for exploring these RNAs, structural biology to include x-ray crystallography, 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.