Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands.
Section Electron Microscopy, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands.
PLoS Biol. 2020 Jun 8;18(6):e3000715. doi: 10.1371/journal.pbio.3000715. eCollection 2020 Jun.
Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection.
动物源冠状病毒（CoV）感染，例如导致当前严重急性呼吸系统综合征-CoV 2（SARS-CoV-2）大流行的病毒，引起了严重的国际公共卫生关注。在受感染的细胞中，CoV RNA 合成机制与经过修饰的内质网膜结合，内质网膜转化为病毒复制细胞器（RO）。尽管双层膜囊泡（DMVs）似乎是泛 CoV RO 元件，但迄今为止的研究描述了多种额外的 CoV 诱导的膜结构。尽管有很多推测，但仍不清楚哪种 RO 元件（多个）容纳病毒 RNA 合成。在这里，我们提供了 CoV RO 的详细 2D 和 3D 分析，并表明不同的 CoV 基本上会引起相同的膜修饰，包括以前被认为仅限于γ和δ CoV 感染并被提议作为复制部位的小开口双层膜球体（DMS）。用新合成的病毒 RNA 进行代谢标记，然后进行定量电子显微镜（EM）放射自显影，结果显示β CoV 中东呼吸综合征-CoV（MERS-CoV）和 SARS-CoV 以及γ-CoV 传染性支气管炎病毒感染的细胞中存在大量与 DMVs 相关的病毒 RNA 合成。无法将 RNA 合成与 DMS 或任何其他细胞或病毒诱导的结构联系起来。我们的结果提供了一个统一的 CoV RO 模型，并明确将 DMVs 确立为病毒 RNA 合成的中心枢纽，以及 CoV 感染的潜在药物靶标。
