Genome structure, transcription and packaging of dsRNA viruses

双链RNA（dsRNA）病毒包括一大组无包膜病毒，其特征在于它们能够在完整衣壳内转录其RNA（即，内源RNA转录），从而逃避对dsRNA的细胞抗病毒应答。其中，dsRNA病毒的呼肠孤病毒科（Reoviridae）家族的成员在公共卫生和基础科学中均具有重要意义，分别以每年造成全球约50万儿童死亡的引起胃肠炎的轮状病毒和历史上用作发现RNA加帽模型的杀昆虫细胞质多角体病毒（CPV）为例。我们已经研究了具有单层（CPV）、双层[哺乳动物呼肠孤病毒（MRV）和水生呼肠孤病毒（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。这些病毒也可以根据其最内层外壳的二十面体顶点上的mRNA加帽转塔的存在（如CPV和呼肠孤病毒）或不存在（如BTV和RRV）进行分类。来自先前资助周期的结果已经发现，BTV和CPV都使用与包膜病毒的融合蛋白具有相似性的表面三聚体（例如，流感、艾滋病和COVID-19病毒）用于细胞进入。我们还捕获了CPV，BTV和RRV在静止，起始和转录阶段的不对称连接的转录酶复合物（TEC）;并确定了TEC和RNA加帽的结构和组织中的保守和多样性特征。我们的研究表明，在进入细胞后，这些病毒感知不同的内部转录激活的环境线索;在CPV的情况下，RNA加帽转塔对SAM和ATP的感知触发了一系列事件：转塔虹膜的打开，三聚体刺突的脱离和内源性转录的启动。需要保存内源性RNA转录和在我们先前的研究中发现的结构差异导致了我们的总体假设：dsRNA病毒的基因组已经显著分化，以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组织以及转录期间RNA解旋和释放期间RNA加帽的变化。该更新申请的目标是通过确定静止期间的代表性dsRNA病毒的基因组组织、转录期间的解旋和加帽以及组装期间的基因组包装，用最先进的低温电子显微镜（cryoEM）和断层扫描（cryoET）来测试该假设。我们将用一个和两个dsRNA片段对CPV、BTV以及dsRNA病毒内的基因组进行建模以进行比较（目的1）。然后将研究RNA转录过程中的加帽和帽抢夺（目的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（目的3）。正如我们先前的工作所证明的，这些研究将通过基于结构的诱变进行功能验证。

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.