Mechanisms of viral RNA maturation by co-opting cellular exonucleases

通过选择细胞核酸外切酶使病毒 RNA 成熟的机制

基本信息

批准号：
9372352
负责人：
Jeffrey S Kieft
金额：
$ 38.23万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-06-26 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9372352
关键词：
Address Affect Agriculture Basic Science Binding Proteins Biochemical Biochemistry Biological Biological Assay Biophysics Cells Characteristics Computational Biology Coupled Culicidae Darkness Databases Dianthoviruses Dimensions Disease Distant Elements Environment Enzymes Exonuclease Exoribonucleases Flavivirus Genome Goals Health Human In Vitro Infection Knowledge Location Methods Molecular Nature Parasites Pathogenicity Phylogenetic Analysis Phylogeny Precursor RNA Process Proteins RNA RNA Decay RNA Folding RNA Precursors RNA Sequences Recruitment Activity Research Resistance Rift Valley fever virus Role Structure Testing Therapeutic Intervention Untranslated RNA Validation Viral Viral Genome Virus Virus Diseases X-Ray Crystallography combat computerized tools follow-up genomic RNA innovation insight new technology novel novel therapeutics poly A specific exoribonuclease protein folding prototype structural biology three dimensional structure tool viral RNA

项目摘要

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.

项目概要作为专性细胞寄生虫，病毒采用多种策略来选择宿主细胞的机器，利用它来产生许多这些策略都涉及形成特定结构的病毒RNA。能够与细胞成分相互作用并对其进行操纵，一个重要的例子是在蚊媒黄病毒中发现的，它选择细胞外核糖核酸酶并用它来产生致病性重要的非编码RNA。 5'3' 核糖核酸外切酶 Xrn1 被招募到基因组 RNA 中，逐渐降解它，但随后在特定位置停止这种程序化的“外切核糖核酸酶抗性”取决于特定的三维 RNA 结构。蚊媒黄病毒的抗核糖核酸酶 RNA (xrRNA) 嵌入黄病毒 RNA 中。这个过程的原型，我们通过研究它们学到了很多东西。但是，现在很明显，合作的策略。有证据表明，选择和利用细胞外核糖核酸酶不仅限于这些病毒，而且可能广泛存在。不同的病毒使用不同类型的外切核糖核酸酶抗性 RNA 元件作为处理长前体的手段然而，尽管这些新型核糖核酸外切酶的重要性日益凸显，抗性RNA结构及其发挥的机制，我们对它们几乎一无所知。基本问题：尽管没有明显的机制，但所有这些假定的 Xrn1 抗性元件是否都使用类似的机制？序列相似性，它们都是折叠的RNA吗？它们都是RNA结构驱动的吗？还是有些需要结合蛋白质？这些不同RNA的折叠相似，或者已经自然进化出多种方式来达到阻断进展的目的缺乏基本信息阻碍了我们对各种病毒中这些过程的理解因此，该提案的重点是通过研究几种 xrRNA 结构来推动该领域的发展。我们的目标是深入了解外切核糖核酸酶抗性的广度和多样性。现象，发现外核糖核酸酶抗性的基本原理，这些原理可能适用于更大的病毒世界，并开发新技术，使我们能够发现或预测其他病毒和环境中的外切核糖核酸酶结构。我们提出三个目标：（1）确定基本序列、结构决定因素和机制特征多种黄病毒 RNA 对核糖核酸外切酶的抗性 (2) 定义来自黄病毒 RNA 的序列和结构。赋予外核糖核酸酶抗性的石竹病毒和裂谷热病毒，以及 (3) 开发合成的扩展病毒 Xrn1 抗性 RNA 的系统发育，并使用它来通过计算搜索其他病毒中未识别的抗性 RNA。我们的方法是将我们实验室独有的生化检测结合起来，并包含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）和体外选择结合这里描述的研究将贡献有关重要的基础知识。广泛适用于病毒性疾病的分子过程，是机制发现和研究之间的必要步骤将其作为治疗干预的目标。