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

• 批准号: 10153681
• 负责人: Jeffrey S Kieft
• 金额: $ 38.09万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 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/10153681
• 关键词: 3-Dimensional; Address; Affect; Agriculture; Basic Science; Binding Proteins; Biochemical; Biochemistry; Biological; Biological Assay; Biophysics; Cells; Characteristics; Computational Biology; Coupled; Databases; Dianthoviruses; 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; Process; Proteins; RNA; RNA Decay; RNA Folding; RNA Precursors; RNA Sequences; Research; Resistance; Rift Valley fever virus; Role; Structure; Testing; Therapeutic Intervention; Untranslated RNA; Validation; Viral; Viral Genome; Virus; Virus Diseases; X-Ray Crystallography; computerized tools; follow-up; genomic RNA; innovation; insight; mosquito-borne; new technology; novel; novel therapeutics; pathogenic virus; poly A specific exoribonuclease; protein folding; prototype; recruit; structural biology; three dimensional structure; tool; viral RNA; 3-Dimensional; Address; Affect; Agriculture; Basic Science; Binding Proteins; Biochemical; Biochemistry; Biological; Biological Assay; Biophysics; Cells; Characteristics; Computational Biology; Coupled; Databases; Dianthoviruses; 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; Process; Proteins; RNA; RNA Decay; RNA Folding; RNA Precursors; RNA Sequences; Research; Resistance; Rift Valley fever virus; Role; Structure; Testing; Therapeutic Intervention; Untranslated RNA; Validation; Viral; Viral Genome; Virus; Virus Diseases; X-Ray Crystallography; computerized tools; follow-up; genomic RNA; innovation; insight; mosquito-borne; new technology; novel; novel therapeutics; pathogenic virus; poly A specific exoribonuclease; protein folding; prototype; recruit; structural biology; three dimensional structure; tool; viral RNA

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.

项目摘要 作为强制性的细胞寄生虫，病毒员工使用了各种选择宿主细胞机械的策略，并使用它来生成 成功感染所需的分子。这些策略中有许多涉及形成特定结构的病毒RNA 可以与细胞成分相互作用并操纵细胞成分。在蚊子传播的黄病毒中找到一个重要的例子， 它选择了细胞驱虫夹，并使用它来产生病原体至关重要的非编码RNA。具体来说， 5'3'驱核核酸酶XRN1被募集到基因组RNA，然后降解，但在特定位置停止 在基因组中。该编程的“驱虫核酸酶抗性”取决于特定的三维RNA结构 嵌入黄素RNA中。蚊子传播黄素的耐核糖核酸酶耐药性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。但是，现在很明显，共同的战略 选择和利用细胞驱虫核酸酶不限于这些病毒，但可能是广泛的。有证据表明 潜水病毒使用不同类型的驱虫酶耐药的RNA元素作为处理长前体的一种手段 RNA变短，生物活性RNA。但是，这些新颖的驱虫蛋白酶的重要性是 抗性RNA结构及其执行的机制，我们对它们几乎一无所知。在燃烧中 基本问题：所有这些推定的XRN1抗性元素是否使用类似的机制？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RNA结构驱动的，还是有些需要结合的蛋白质？是 这些不同的RNA的折叠相似，或者具有多种方法来实现阻止进展的目标 驱虫核酸酶？缺乏基本信息，我们对潜水病毒中这些过程的理解受到了阻碍 关于各种XRRNA结构。因此，该提案的重点是通过研究几个 XRRNA的未开发示例。我们旨在深入了解驱虫酶抵抗的广度和多样性 现象，发现可能适用于较大病毒的驱虫核酸酶阻力的基本原理 世界，并开发新技术，使我们能够在其他病毒和环境中找到或预测驱虫酶结构。 我们提出了三个目标：（1）确定基本序列，结构决定者和机械特征 多种黄素RNA的驱虫核酸酶耐药性。 （2）从 Dianthoviruses和Rift Valley Fever病毒，会议驱虫核酸酶阻力，并且（3）发展合成的扩展 XRN1耐药性RNA的系统发育，并使用它来计算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测定法，并完成一套全面的工具 用于探索这些RNA，结构生物学，包括X射线晶体学，并在体外选择与 计算工具。这里描述的研究将为重要的基本知识提供重要的基础知识 广泛适用于病毒疾病的分子过程，在发现机制和 靶向治疗干预。