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.

项目摘要/摘要 双链RNA（DSRNA）病毒包括一大批以特征的非发育病毒 它们在完整的capsid中转录RNA的能力（即内源性RNA转录），从而逃避 细胞对DSRNA的抗病毒反应。其中，dsRNA病毒家族的Reoviridae家族的成员是 在公共卫生和基础科学中的重要性，分别是由胃炎引起的 轮状病毒，每年在全球范围内约有50万儿童死亡以及绝缘材料 - 杀死细胞质多层病毒（CPV），该病毒历史上用作发现RNA的模型 上限。我们已经使用单层（CPV），双层研究了非开发的DSRNA病毒 [哺乳动物依然病毒（MRV）和Aquareovirus（ARV）]和三层层[Rhesus rotavirus（RRV），蓝牙 病毒（BTV）]衣壳。这些病毒也可以根据存在分类（例如CPV和 静脉病毒）或缺席（例如BTV和RRV）在其二十面体顶点的mRNA盖炮塔 最内向的壳。先前的资金周期的结果发现了BTV和CPV都使用表面 与包络病毒的融合蛋白相似的三聚体（例如流感，艾滋病和covid-19病毒） 细胞输入。我们还捕获了不对称连接的转录酶复合物（TEC） CPV，BTV和RRV的静止，主动性和转录阶段；并确定了保守和潜水员 TEC和RNA封盖的结构和组织之间的特征。我们的研究表明，在 细胞进入，这些病毒感知到内部转录激活的不同环境提示。在情况下 CPV，RNA限制炮塔对SAM和ATP的敏感性触发了一系列事件：炮塔的开放 虹膜，三聚体尖峰的脱离以及内源性转录的主动性。 需要保护内源性RNA转录和我们先前发现的结构多样性的需求 研究已经提出了我们的总体假设：DSRNA病毒的基因组大大分歧以允许 掺入RNA片段，编码与不同宿主细胞相互作用所需的不同蛋白 引起不同的基因组和TEC组织，并在RNA中脱颖而出。 发行过程中的转录和RNA封盖。此更新应用的目的是检验该假设 通过确定最先进的低温电子显微镜（冷冻）和层析成像（冷冻） 在静止期间，在 在组装过程中的转录和基因组包装。我们将对CPV，BTV以及 具有一个和两个dsRNA段的DSRNA病毒进行比较（AI​​M 1）。封盖和盖帽捕捉 然后将研究RNA转录（AIM 2）。最后，我们将可视化不同的基因组RNA和 衣壳蛋白组装以形成感染性病毒粒子颗粒（AIM 3）。正如我们先前的工作中所证明的那样，这些 研究将通过基于结构的诱变来完成功能验证。