Genome structure, transcription and packaging of dsRNA viruses

双链RNA(DsRNA)病毒由一大群无包膜的病毒组成，其特征是能够在完整的衣壳内转录RNA(即内源性RNA转录)，从而逃避细胞对dsRNA的抗病毒反应。其中，呼肠孤病毒科dsRNA病毒家族的成员在公共卫生和基础科学方面都具有重要意义，分别以引起胃肠炎的轮状病毒和杀虫细胞质多角体病毒(CPV)为例，前者每年导致全球约50万儿童死亡，后者历史上被用作发现RNA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)和水禽病毒(ARV)]和三层[恒河猴轮状病毒(RRV)、蓝舌病毒(BTV)]衣壳的非包膜dsRNA病毒。这些病毒也可以根据其最内壳的二十面体顶点上是否存在(如CPV和RRV)或不存在(如BTV和RRV)进行分类。先前资助周期的结果发现，牛细小病毒和柯萨奇病毒都使用与被包裹病毒(例如流感、艾滋病和新冠肺炎病毒)的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在静止期、起始期和转录阶段的不对称连接转录酶复合体(TEC)，并鉴定了它们的结构和组织之间的保守和多样性特征。我们的研究表明，在进入细胞时，这些病毒感知不同的环境信号进行内部转录激活；对于CPV，RNA覆盖的炮塔感知SAM和ATP会触发一系列事件：炮塔虹膜打开，三聚体棘突脱离，以及内源转录的启动。保存内源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歧，允许整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，以及在转录过程中RNA解开和释放过程中的RNA封顶的变异。这一更新应用的目标是通过确定典型dsRNA病毒在静止过程中的基因组组织、转录过程中的解卷和封顶以及组装过程中的基因组组装，使用最先进的低温电子显微镜(CryoEM)和断层扫描(CryoET)来验证这一假说。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(目标1)。然后将研究RNA转录过程中的封顶和抢帽(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成具有感染性的病毒粒子(目标3)。正如我们以前的工作所表明的那样，这些研究将得到基于结构的突变的补充，以进行功能验证。

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.