Genome structure, transcription and packaging of dsRNA viruses

双链RNA病毒的基因组结构、转录和包装

• 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
• 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

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) 病毒由一大类无包膜病毒组成，其特征是能够在完整的衣壳内转录其 RNA（即内源 RNA 转录），从而逃避细胞对 dsRNA 的抗病毒反应。其中，dsRNA病毒呼肠孤病毒科成员在公共卫生和基础科学方面都具有重要意义，例如引起胃肠炎的轮状病毒（每年导致全世界约50万儿童死亡）和杀虫细胞质多角体病毒（CPV），该病毒历史上被用作发现RNA加帽的模型。我们研究了具有单层（CPV）、双层[哺乳动物呼肠孤病毒（MRV）和水生病毒（ARV）]和三层[恒河猴轮状病毒（RRV）、蓝舌病毒（BTV）]衣壳的无包膜dsRNA病毒。这些病毒还可以根据其最内壳的二十面体顶点上是否存在 mRNA 帽转塔（例如 CPV 和呼肠孤病毒）或不存在（例如 BTV 和 RRV）来分类。先前资助周期的结果表明，BTV 和 CPV 均使用与包膜病毒（例如流感、艾滋病和 COVID-19 病毒）融合蛋白相似的表面三聚体进入细胞。我们还捕获了 CPV、BTV 和 RRV 的静止、起始和转录阶段的不对称附着转录酶复合物 (TEC)；并鉴定了它们的 TEC 和 RNA 封端结构和组织中保守且多样化的特征。我们的研究表明，进入细胞后，这些病毒会感知到内部转录激活的不同环境线索；就 CPV 而言，RNA 加帽转塔对 SAM 和 ATP 的感知会触发一系列事件：转塔虹膜打开、三聚体刺突分离以及内源转录启动。保护内源RNA转录的需要和我们之前研究中发现的结构多样性导致了我们的总体假设：dsRNA病毒的基因组已经发生了很大的分歧，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，从而产生不同的基因组和TEC组织以及转录过程中RNA解旋和释放过程中RNA加帽的变化。这一更新应用的目标是通过确定代表性 dsRNA 病毒在静止期间、转录期间解旋和加帽期间以及组装期间基因组包装期间的基因组组织，使用最先进的低温电子显微镜 (cryoEM) 和断层扫描 (cryoET) 来测试这一假设。我们将使用一个和两个 dsRNA 片段对 CPV、BTV 以及 dsRNA 病毒内部的基因组进行建模以进行比较（目标 1）。然后将研究 RNA 转录过程中的加帽和帽抢夺（目标 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。