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：
10463469
负责人：
Jeffrey S Kieft
金额：
$ 45.31万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-01 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10463469
关键词：
3&apos Untranslated Regions 3-Dimensional 5&apos Untranslated Regions Address Antiviral Response Architecture Attenuated Biochemistry Bioinformatics Biophysics Cells Characteristics Code Complex Coupled Coupling Cryoelectron Microscopy Cultured Cells Dangerousness Data Dengue Directed Molecular Evolution Disease Elements Environment Enzymes Exonuclease Exoribonucleases Flavivirus Foundations Health In Vitro Individual Infection Japanese Encephalitis Kinetics Knowledge Laboratories Link Location Mechanics Modeling Molecular Outcome Pathogenicity Pattern Powassan virus Process Production Property Proteins RNA RNA Decay RNA Folding RNA Viruses Regulation Research Resistance Resolution Structure Surface Therapeutic Intervention Tick-Borne Encephalitis Untranslated RNA Variant Viral Viral Genome Virus Virus Diseases West Nile virus Work X-Ray Crystallography Yellow Fever ZIKA arthropod-borne base computerized tools genomic RNA human pathogen insight mosquito-borne poly A specific exoribonuclease structural biology success three dimensional structure tick-borne tick-borne flavivirus viral RNA viral genomics virology

项目摘要

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.

项目概要黄病毒是单链正义 RNA 病毒，包括危险的人类病原体，例如登革热、西尼罗河病毒、黄热病、寨卡病毒和许多其他病毒在感染过程中会产生一系列非病毒。编码被称为“亚基因组黄病毒RNA”（sfRNA）的RNA，它们与细胞蛋白质相互作用以操纵细胞环境，包括抑制抗病毒反应，与细胞病变直接相关。和致病结果，并且不能产生 sfRNA 的病毒被减弱，从而激励人们努力了解细胞 5'à3' 核糖核酸酶（在细胞中）产生 xrRNA 的机制。特别是 Xrn1) 逐渐降解病毒基因组 RNA，但随后停止于特定结构的 RNA 病毒 3' UTR 中的元件称为外核糖核酸酶抗性 RNA (xrRNA)。通过 X 射线晶体学分析多种 xrRNA，并将其与生物化学、生物物理学和病毒学相结合，我们表明 xrRNA 折叠成独特的环状拓扑结构，从而产生机械阻断外切核糖核酸酶此外，我们利用我们的发现对 xrRNA 进行分类并寻找新的例子。这些成功现在定义了几个新问题。 xrRNA 通常以“串联”的多个拷贝形式存在，它们的功能以某种方式耦合，但是这种偶联的结构基础，以及打破偶联对 sfRNA 形成和病毒的影响其次，尽管我们对几类 xrRNA 有很好的了解，我们尚未解析蜱传黄病毒的 xrRNA 结构，该结构似乎具有有趣且第三，虽然我们发现了许多 xrRNA 的新例子，但看起来还有很多。还有更多未被发现的，我们也不了解不同类别的 xrRNA 之间的关系考虑到折叠的严格限制，这些结构如何在 3D 中多样化和进化？在这里，我们建议通过三个目标来回答这些问题，采用结合生物化学、x- 射线晶体学、冷冻电镜、病毒学和体外选择与计算工具相结合。这里描述的将贡献关于广泛的重要分子过程的重要基础知识对病毒性疾病的适用性，是发现机制和靶向治疗之间的必要步骤治疗干预。