Rotavirus Genome Replication and Virion Assembly

轮状病毒基因组复制和病毒粒子组装

基本信息

批准号：
10576929
负责人：
Deborah F Kelly
金额：
$ 45.82万
依托单位：
WAKE FOREST UNIVERSITY
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：
2022-01-14 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10576929
关键词：
2019-nCoV 3-Dimensional Antiviral Agents Armadillo Repeat Binding Biochemical Biological Assay Biological Models Cells Child Complex Cryoelectron Microscopy Data Defect Diarrhea Disease Double Stranded RNA Virus Double-Stranded RNA Drug Design Ebola virus Economic Burden Engineering Enzymes Family Foundations Genetic Techniques Genetic Transcription Genome Genomic Segment Ice Image In Situ In Vitro Infection Influenza A virus Knowledge Lesion Life Liquid substance Mammalian Cell Maps Medical Microfluidics Mission Molecular Conformation N-terminal Pathogenesis Performance Plasmids Proteins Publishing RNA RNA Viruses RNA chemical synthesis RNA-Directed RNA Polymerase Recombinants Regulation Reoviridae Research Resolution Rotavirus Site Societies Structure System Techniques Testing Transcriptase United States National Institutes of Health Viral Virion Virus Virus Replication Virus-like particle Work flexibility human disease human pathogen in vitro activity insight mutant particle prevent public health relevance rational design reconstruction replicase reverse genetics scaffold structural biology three dimensional structure viral RNA

项目摘要

PROJECT SUMMARY/ABSTRACT (DESCRIPTION): RNA viruses can be devastating human pathogens that impart large medical and economic burdens to society (e.g., SARS-CoV-2, influenza A virus, Ebola virus, rotavirus, etc.). While these viruses can differ quite dramatically in their pathogenesis, they share a common replication feature—they must synthesize new RNA molecules from RNA templates. Because host cell enzymes lack this activity, RNA viruses encode a specialized RNA-dependent RNA polymerase (RdRp). Viral RdRps are structurally- and functionally-conserved among diverse RNA viral families, and they directly catalyze all stages of viral transcription and genome replication. However, these enzymes rarely function alone in the context of infected cells. Instead, the viral RdRps are tightly regulated by other proteins in multi-subunit transcriptase/replicase complexes so as to maximize the type and timing of viral RNA synthesis. The overall objective of this application is to gain mechanistic insight into RdRp regulation for rotavirus—an 11-segmented, double-stranded (ds) RNA virus that causes life-threatening diarrhea in young children. The rotavirus VP1 RdRp functions only when bound beneath the icosahedral VP2 core shell layer of intact, or partially intact, particles. Engagement of VP1 by VP2 during early particle assembly activates the RdRp so that it functions as a replicase, converting packaged, single-stranded positive sense (+) RNA templates into dsRNA genome segments. Early assembly intermediates then morph into double-layered particles, wherein the VP2-bound, VP1 RdRp switches to a transcriptase activity, synthesizing +RNAs using dsRNA templates. Still, major gaps in knowledge remain about the structure of the VP1 RdRp as a replicase during dsRNA synthesis and its regulation by the VP2 core shell protein. Here, well-established in vitro biochemical and genetic techniques are combined with state-of-the-art structural approaches to close these gaps in knowledge and inform a deep understanding of rotavirus RdRp regulation. AIM 1 will elucidate VP2 core shell determinants critical for VP1 replicase activity, and AIM 2 will determine the first-ever in situ 3D atomic structures of VP1 as a replicase in both ice (using cryo-EM) and liquid (using a microfluidics system). The work outlined in this application is significant, as it is expected to reveal detailed structure-function information about the rotavirus RdRp that could be applied to rational antiviral drug design.

项目摘要/摘要（描述）： RNA 病毒可能是毁灭性的人类病原体，给社会带来巨大的医疗和经济负担。（例如 SARS-CoV-2、甲型流感病毒、埃博拉病毒、轮状病毒等）。在其发病机制中，它们具有一个共同的复制特征——它们必须合成新的RNA 来自 RNA 模板的分子。由于宿主细胞酶缺乏这种活性，RNA 病毒编码一种特殊的 RNA 依赖性 RNA 聚合酶 (RdRp) 在结构和功能上都是保守的。不同的RNA病毒家族，它们直接催化病毒转录和基因组复制的所有阶段。然而，这些酶很少在受感染的细胞中单独发挥作用，相反，病毒 RdRps 紧密结合在一起。受多亚基转录酶/复制酶复合物中其他蛋白质的调节，以最大化类型和复制酶病毒 RNA 合成的时间。该应用程序的总体目标是深入了解 RdRp 的机制轮状病毒的监管——一种 11 段双链 (ds) RNA 病毒，可导致危及生命的腹泻轮状病毒 VP1 RdRp 仅当结合在二十面体 VP2 核壳下方时才发挥作用。完整或部分完整的颗粒层。在早期颗粒组装过程中，VP1 与 VP2 的接合被激活 RdRp，使其充当复制酶，转换包装的单链正义 (+) RNA 模板进入 dsRNA 基因组片段，然后演变成双层。颗粒，因此 VP2 结合的 VP1 RdRp 切换到转录酶活性，使用合成 +RNA 尽管如此，关于 VP1 RdRp 作为复制酶的结构的知识仍然存在重大差距。 dsRNA 合成过程中及其受 VP2 核壳蛋白的调节，在体外已得到证实。生化和遗传技术与最先进的结构方法相结合来缩小这些差距深入了解轮状病毒 RdRp 调控的知识和信息将阐明 VP2 核心壳。对于 VP1 复制酶活性至关重要的决定因素，AIM 2 将确定首个原位 3D 原子结构 VP1 作为冰（使用冷冻电镜）和液体（使用微流体系统）中的复制酶。该应用意义重大，因为它有望揭示轮状病毒的详细结构功能信息 RdRp 可应用于合理的抗病毒药物设计。