Genome structure, transcription and packaging of dsRNA viruses

双链RNA病毒的基因组结构、转录和包装

基本信息

批准号：
10554343
负责人：
Z Hong ZHOU
金额：
$ 45.62万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
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/10554343
关键词：
2019-nCoV Animals Antiviral Response Aquareoviruses Bacteria Basic Science Biochemical Biological Models Bluetongue virus Capsid Capsid Proteins Cells Cessation of life Child Chimeric Proteins Classification Communities Complement Complex Cryoelectron Microscopy Cues Cytoplasm Cytoplasmic Polyhedrosis Viruses Detection Double Stranded RNA Virus Double-Stranded RNA Drug Delivery Systems Economics Electrons Engineering Environment Enzymes Event Family Flu virus Funding Gastroenteritis General Population Genetic Transcription Genome Goals Grant HIV Health Sciences Human In Situ Infectious bursal disease virus Insect Viruses Insecta Invaded Ions Iris Lead Life Cycle Stages Link Literature Livestock Membrane Proteins Messenger RNA Modeling Molecular Biology Molecular Conformation Multienzyme Complexes Mutagenesis N-terminal Pattern Phase Plants Polymerase Proteins Public Health Publishing RNA RNA Caps RNA replication RNA-Directed RNA Polymerase Recording of previous events Reoviridae Reovirus Rhesus Role Rotavirus Structure Surface System Techniques Technology Testing Transcriptional Activation Trichomonas vaginalis Vaccines Variant Viral Viral Genome Virion Virus Virus Assembly Visualization Work antiviral drug development comparative electron tomography endosome membrane fungus genomic RNA insight interest mRNA capping member nanometer resolution novel nucleoside triphosphatase particle pathogen reconstruction replicator social vaccine development virus core

项目摘要

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.

双链RNA（DSRNA）病毒组成了一大批非发育病毒，其特征是它们在完整的capsID（即内源性RNA转录）中转录其RNA的能力，从而避免了细胞对DSRNA的抗病毒反应。其中，DSRNA病毒家族的成员在公共卫生和基础科学中都具有重要意义，分别由引起胃炎的轮状病毒造成的胃炎的轮状病毒分别示例，每年大约每年大约有半百万个儿童死亡，以及绝缘效果 - 杀死细胞质的多毛病毒病毒病毒（CPV），这是一个模型，该模型是模型的。我们已经研究了单层（CPV），双层[哺乳动物[MRV）和Aquareovirus（ARV）]和三层式[恒河虫（RRV），蓝牙病毒（BTV）capsid capsid研究了非发育的DSRNA病毒。病毒也可以根据其最内膜壳的二十面体顶点上的mRNA贴塔炮塔的存在（例如CPV和依伏病毒）或不存在（例如BTV和RRV）进行分类。先前的资金周期的结果发现，BTV和CPV都使用与信封病毒的融合蛋白相似的表面三聚体（例如流感，艾滋病和Covid-19病毒）进行细胞进入。我们还捕获了在CPV，BTV和RRV的静止，起始和转录阶段，在静止，起始和转录阶段捕获了不对称的转录酶复合物（TEC）；并确定了TEC和RNA封盖的结构和组织之间的保守和潜水员的特征。我们的研究表明，在细胞进入后，这些病毒感知到内部转录激活的不同环境线索。在CPV的情况下，RNA贴塔炮塔对SAM和ATP的敏感性触发了一系列事件：炮塔虹膜的打开，三聚体峰值的脱离以及内源性转录的倡议。在我们先前的研究中保存内源性RNA转录和发现的结构多样性的需求导致了我们的总体假设：DSRNA病毒的基因组已经大大差异，以允许编写编码与不同宿主相互作用所需的RNA片段，从而使不同的宿主相互作用，从而使不同的基因组和TEC的释放在RNA的转录过程中产生，并在RNA不感染了RNA期间和变异过程中均可构图和变异。这种更新应用的目的是通过确定具有代表性的DSRNA病毒的基因组组织在静止，转录过程中的放松和封盖以及组装过程中的基因组包装的过程中确定代表性的DSRNA病毒组织的基因组组织来检验这一假设。我们将模拟CPV，BTV内的基因组以及一个DSRNA病毒，其中一个和两个DSRNA片段进行比较（AIM 1）。然后，将研究RNA转录过程中的封盖和盖帽捕捉（AIM 2）。最后，我们将可视化不同的基因组RNA和衣壳蛋白如何组装以形成传染性病毒颗粒（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进行功能验证完成。