Genome Structure, Transcription and Packaging of dsRNA Viruses

dsRNA 病毒的基因组结构、转录和包装

• 批准号: 10820018
• 负责人: Z Hong ZHOU
• 金额: $ 3.18万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-16 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10820018
• 关键词: 2019-nCoV; Academia; Animals; Antiviral Response; Antiviral Therapy; Aquareoviruses; Area; Award; Bacteria; Baculovirus Expression System; Basic Science; Binding Proteins; Bluetongue virus; Capsid; Capsid Proteins; Cells; Cessation of life; Child; Chimeric Proteins; Classification; Clinical Trials; Complement; Cryoelectron Microscopy; Cues; Cytoplasmic Polyhedrosis Viruses; Detection; Development; Diarrhea; Double Stranded RNA Virus; Double-Stranded RNA; Drug Delivery Systems; Economics; Electrons; Engineering; Event; Family; Fellowship; Flu virus; Funding; Future; Gastroenteritis; Genetic Transcription; Genome; Goals; HIV; Health Sciences; Human; Insecta; Intestines; Investigation; Ions; Iris; Life; Livestock; Modeling; Multienzyme Complexes; Mutagenesis; Oncolytic; Outcome; Pathogenicity; Pathway interactions; Phase; Plants; Positioning Attribute; Postdoctoral Fellow; Principal Investigator; Proteins; Public Health; RNA; RNA Caps; Recombinants; Reoviridae; Reovirus; Reovirus 3; Reovirus Type 1; Research; Research Personnel; Resolution; Respiratory Tract Infections; Rhesus; Rotavirus; Rotavirus Vaccines; Science; Structure; Study models; Surface; Technology; Testing; Transcriptional Activation; Vaccines; Variant; Viral Genome; Viral Proteins; Virion; Virus; Virus Assembly; Virus-like particle; Visualization; Work; antiviral drug development; career; electron tomography; enteric infection; fungus; genomic RNA; human pathogen; immunogenic; improved; insight; mRNA capping; member; nanocarrier; nanoparticle delivery; parent grant; particle; pathogen; pre-doctoral; receptor binding; self assembly; social; structural biology; vaccine delivery; vaccine development

PROJECT SUMMARY/ABSTRACT Double-stranded RNA (dsRNA) viruses comprise a large group of non-enveloped viruses characterized by their ability to transcribe their RNA within an intact capsid (i.e., endogenous RNA transcription), thus evading cellular antiviral responses to dsRNA. Among them, members of the Reoviridae family of dsRNA viruses are of significance in both public health and basic science, exemplified respectively by the gastroenteritis-causing rotavirus which is responsible for approximately half a million child deaths annually worldwide and the insect- killing cytoplasmic polyhedrosis virus (CPV) which was used historically as a model in the discovery of RNA capping. We have studied non-enveloped dsRNA viruses with single-layered (CPV), double-layered [mammalian reovirus (MRV) and aquareovirus (ARV)], and triple-layered [rhesus rotavirus (RRV), Bluetongue virus (BTV)] capsid. These viruses could also be classified based on the presence (such as CPV and reoviruses) or absence (such as BTV and RRV) of an mRNA-capping turret on the icosahedral vertices of their innermost shell. Results from the prior funding cycles have uncovered that BTV and CPV both use surface trimers bearing similarities to fusion proteins of enveloped viruses (e.g., flu, AIDS and COVID-19 viruses) for cell entry. We have also captured the asymmetrically attached transcriptional enzyme complex (TEC) at the quiescent, initiation and transcribing stages of CPV, BTV and RRV; and identified both conserved and diverse features among their structures and organizations of TEC and RNA capping. Our studies showed that, upon cell entry, these viruses sense different environmental cues for internal transcription activation; and in the case of CPV, sensing of SAM and ATP by the RNA-capping turret triggers a cascade of events: opening of the turret iris, detachment of the trimeric spike, and initiation of endogenous transcription. The need to conserve endogenous RNA transcription and the structural diversities uncovered in our prior studies have led to our overall hypothesis: genomes of dsRNA viruses have diverged substantially to allow incorporation of RNA segments encoding the distinct proteins required to interact with different host cells, giving rise to different genome and TEC organizations and variations to both RNA unwinding during transcription and RNA capping during release. The goal of this renewal application is to test this hypothesis with state-of-the-art cryogenic electron microscopy (cryoEM) and tomography (cryoET) by determining representative dsRNA viruses’ genome organizations during quiescence, unwinding and capping during transcription, and genome packing during assembly. We will model the genomes inside CPV, BTV, as well as dsRNA viruses with one and two dsRNA segments for comparison (Aim 1). Capping and cap-snatching during RNA transcription will then be investigated (Aim 2). Finally, we will visualize how different genomic RNA and capsid proteins assemble to form infectious virion particles (Aim 3). As demonstrated in our prior work, these studies will be complemented by structure-based mutagenesis for functional verification.

项目概要/摘要 双链 RNA (dsRNA) 病毒由一大类无包膜病毒组成，其特征是 它们在完整衣壳内转录 RNA 的能力（即内源 RNA 转录），从而逃避 细胞对 dsRNA 的抗病毒反应。其中，dsRNA病毒呼肠孤病毒科的成员有 对公共卫生和基础科学都具有重要意义，分别以引起胃肠炎的例子为例 轮状病毒每年导致全世界约 50 万儿童死亡，而昆虫 杀死细胞质多角体病毒 (CPV)，该病毒历史上曾被用作发现 RNA 的模型 封顶。我们研究了单层（CPV）、双层的无包膜 dsRNA 病毒 [哺乳动物呼肠孤病毒 (MRV) 和水呼肠孤病毒 (ARV)]，以及三层 [恒河猴轮状病毒 (RRV)、蓝舌病 病毒(BTV)]衣壳。这些病毒还可以根据是否存在进行分类（例如 CPV 和 呼肠孤病毒）或在其二十面体顶点上缺少 mRNA 帽转塔（例如 BTV 和 RRV） 最里面的壳。先前融资周期的结果表明，BTV 和 CPV 均使用表面 三聚体与包膜病毒（例如流感、艾滋病和 COVID-19 病毒）的融合蛋白具有相似性 细胞进入。我们还捕获了不对称附着的转录酶复合物（TEC） CPV、BTV 和 RRV 的静止期、起始期和转录期；并确定了保守性和多样性 它们的 TEC 和 RNA 封端的结构和组织具有特征。我们的研究表明，基于 进入细胞后，这些病毒会感知不同的环境线索以进行内部转录激活；在这种情况下 CPV 中，RNA 封盖转塔对 SAM 和 ATP 的感应会触发一系列事件：转塔打开 虹膜、三聚体刺突的分离以及内源转录的启动。 保护内源 RNA 转录的需要以及我们之前发现的结构多样性 研究得出了我们的总体假设：dsRNA 病毒的基因组已发生显着差异，从而允许 掺入编码与不同宿主细胞相互作用所需的不同蛋白质的RNA片段， 产生不同的基因组和 TEC 组织以及 RNA 解旋过程中的变化 释放过程中的转录和RNA加帽。此更新应用程序的目标是检验这一假设 使用最先进的低温电子显微镜 (cryoEM) 和断层扫描 (cryoET) 确定 代表性 dsRNA 病毒在静止、解旋和加帽过程中的基因组组织 转录和组装过程中的基因组包装。我们将对 CPV、BTV 以及 具有一个和两个 dsRNA 片段的 dsRNA 病毒用于比较（目标 1）。期间封盖和抢盖 然后将研究 RNA 转录（目标 2）。最后，我们将可视化基因组 RNA 和 衣壳蛋白组装形成感染性病毒颗粒（目标 3）。正如我们之前的工作所证明的，这些 研究将得到基于结构的突变的补充，以进行功能验证。