Genome structure, transcription and packaging of dsRNA viruses

双链RNA （dsRNA）病毒包括一大群非包膜病毒，其特点是能够在完整的衣壳内转录其RNA（即内源性RNA转录），从而逃避细胞对dsRNA的抗病毒反应。其中呼肠病毒科的dsRNA病毒在公共卫生和基础科学中都具有重要意义，分别是引起肠胃炎的轮状病毒，它每年造成全世界大约50万儿童死亡，以及杀死昆虫的细胞质多角体病毒（CPV），它在历史上被用作发现RNA盖层的模型。我们研究了具有单层（CPV）、双层[哺乳动物呼肠孤病毒（MRV）和水病毒（ARV）]和三层[恒河轮状病毒（RRV）、蓝舌病病毒（BTV）]衣壳的非包膜dsRNA病毒。这些病毒也可以根据在其最内层壳的二十面体顶点上是否存在（如CPV和呼肠孤病毒）或是否存在（如BTV和RRV） mRNA-capping转塔来分类。先前资助周期的结果表明，BTV和CPV都使用与包膜病毒（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。我们还在CPV、BTV和RRV的静止、起始和转录阶段捕获了不对称附着的转录酶复合物（TEC）；并鉴定出它们的TEC和RNA capping结构和组织的保守性和多样性特征。我们的研究表明，在进入细胞后，这些病毒感知不同的环境信号进行内部转录激活；在CPV的情况下，rna盖顶转塔对SAM和ATP的感知触发了一系列事件：转塔虹膜的打开，三聚体穗的脱离，以及内源性转录的启动。保存内源性RNA转录的需要和我们先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化，从而允许编码与不同宿主细胞相互作用所需的不同蛋白质的RNA片段的结合，从而导致不同的基因组和TEC组织以及转录过程中的RNA解绕和释放过程中的RNA capping的变化。这项更新申请的目的是通过测定代表性dsRNA病毒在静止、转录过程中解绕和盖帽以及组装过程中基因组包装期间的基因组组织，用最先进的低温电子显微镜（cryoEM）和断层扫描（cryoET）来验证这一假设。我们将建立CPV、BTV以及dsRNA病毒的基因组模型，用一个和两个dsRNA片段进行比较（目的1）。然后将研究RNA转录过程中的Capping和cap-snatching （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形成感染性病毒粒子（目标3）。正如我们之前的工作所证明的那样，这些研究将通过基于结构的诱变来补充功能验证。

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.