Mechanisms of viral RNA maturation by co-opting cellular exonucleases

项目摘要 作为专性的细胞寄生虫，病毒采用各种策略来利用宿主细胞的机器，利用它来产生 成功感染所需的分子。这些策略中的许多都涉及形成特定结构的病毒RNA 能够与蜂窝组件交互并操纵这些组件。一个重要的例子是蚊媒黄病毒， 它选择了一种细胞外切核糖核酸酶，并用它来产生具有致病意义的非编码RNA。具体地说， 5‘3’外切核糖核酸酶Xrn1被招募到基因组rna中，不断地降解它，但随后在特定位置停止 在基因组中。这种程序化的“外切核糖核酸酶抗性”依赖于特定的三维RNA结构 都嵌在黄病毒的RNA中。蚊媒黄病毒的抗外切核糖核酸酶RNA(XrRNAs)是 这一过程的原型，我们通过研究它们学到了很多。然而，现在很明显，联合- 选择和利用细胞外切核糖核酸酶并不局限于这些病毒，而是可能广泛存在。有证据表明 不同的病毒使用不同类型的抗外切核糖核酸酶的RNA元件作为一种手段来处理长前体 RNA转化为更短的、具有生物活性的RNA。然而，尽管这些新的外切核糖核酸酶的重要性正在显现- 对于抗性RNA结构及其执行机制，我们几乎一无所知。在燃烧的人群中 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？尽管没有明显的证据 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，还是有些需要结合蛋白？是 这些不同RNA的折叠相似，或者说大自然进化了许多方式来实现阻止 外切核糖核酸酶？由于缺乏基本信息，我们对不同病毒的这些过程的理解受到阻碍 关于各种xrRNA结构。因此，这项建议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外切核酸酶耐药性的广度和多样性。 现象，以发现可能适用于较大病毒的外切核糖核酸酶抗性的基本原理 并开发新技术，使我们能够在其他病毒和环境中发现或预测外切核糖核酸酶结构。 我们提出了三个目标：(1)确定基本序列、结构决定因素和机械特征 一组不同的黄病毒RNA对外切核糖核酸酶的抗性。(2)定义RNA的序列和结构 具有外切核糖核酸酶抗性的斑点病毒和裂谷热病毒，以及(3)开发一种合成的扩展的 Xrn1抗性RNA的系统发育，并利用这一点在计算机上搜索其他病毒中未识别的抗性RNA。 我们的方法是结合我们实验室独有的生化分析，并包括一套全面的工具 为了探索这些RNA，结构生物学包括x射线结晶学，以及与 计算工具。这里描述的研究将有助于对一个重要的 广泛适用于病毒疾病的分子过程，是从发现机制到 以此为靶点进行治疗干预。

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.