Genome Structure, Transcription and Packaging of dsRNA Viruses

项目摘要/摘要 双链RNA(DsRNA)病毒由一大群无包膜病毒组成，其特征是 他们能够在完整的衣壳内转录他们的RNA(即内源RNA转录)，从而避免 细胞对dsRNA的抗病毒反应。其中呼肠孤病毒科dsRNA病毒家族成员包括 在公共卫生和基础科学方面的意义，分别以引起胃肠炎为例 轮状病毒每年导致全球约50万儿童死亡，这种昆虫- 杀死细胞质多角体病毒(CPV)，它在历史上被用作发现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)和水生呼肠孤病毒(ARV)]，以及三层[恒河猴轮状病毒(RRV)，蓝舌 病毒(BTV)]衣壳。这些病毒也可以根据存在的情况进行分类(如CPV和 呼肠孤病毒)或(如BTV和RRV)在其二十面体顶点上没有mRNA封顶的转塔 最里面的壳。前几个资金周期的结果发现，BTV和CPV都使用Surface 与包膜病毒(如流感、艾滋病和新冠肺炎病毒)的融合蛋白具有相似性的三聚体 单元格输入。我们还捕获了不对称连接的转录酶复合体(TEC) CPV、BTV和RRV的静止期、起始期和转录阶段；并鉴定了保守和多样性 它们的TEC和RNA封端的结构和组织特征。我们的研究表明，在 细胞进入，这些病毒感知不同的环境提示进行内部转录激活；在这种情况下 对于CPV，RNA覆盖的炮塔检测到SAM和ATP会触发一系列事件：炮塔的打开 虹膜、三聚体棘突的分离和内源转录的启动。 保存内源RNA转录和我们以前发现的结构多样性的需要 研究已经导致了我们的总体假设：dsRNA病毒的基因组已经发生了实质性的分化，使得 结合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， 导致不同的基因组和TEC组织以及在 转录和RNA在释放过程中的封顶。此续订应用程序的目标是验证这一假设 利用最先进的低温电子显微镜(CryoEM)和断层扫描(CryoET)通过确定 典型dsRNA病毒在静止、解旋和封顶过程中的基因组组织 转录，以及组装过程中的基因组组装。我们将对CPV、BTV以及 DsRNA病毒与一个和两个dsRNA片段进行比较(目标1)。封盖和抢盖过程中 然后将研究RNA转录(目标2)。最后，我们将可视化不同的基因组RNA和 衣壳蛋白聚集形成具有感染性的病毒粒子(目标3)。正如我们之前的工作所展示的，这些 研究将得到基于结构的突变的补充，以进行功能验证。

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.