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.

人畜共患冠状病毒 (CoV) 感染，例如导致当前严重急性呼吸综合征-冠状病毒 2 (SARS-CoV-2) 大流行的感染，引起了严重的国际公共卫生问题。在受感染的细胞中，冠状病毒RNA合成机制与修饰的内质网膜相关联，内质网膜被转化为病毒复制细胞器（RO）。尽管双膜囊泡 (DMV) 似乎是泛冠状病毒 RO 元件，但迄今为止的研究描述了各种额外的冠状病毒诱导的膜结构。尽管有很多猜测，但仍不清楚哪些 RO 元件能够调节病毒 RNA 的合成。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 本质上诱导相同的膜修饰，包括小的开放双膜球体（DMS），以前被认为仅限于 γ-和 δ-CoV 感染，并提出：复制位点。对新合成的病毒 RNA 进行代谢标记，然后进行定量电子显微镜 (EM) 放射自显影，结果显示感染 β-CoV 中东呼吸综合征冠状病毒 (MERS-CoV) 和 SARS-CoV 以及 γ 病毒的细胞中与 DMV 相关的大量病毒 RNA 合成-CoV 传染性支气管炎病毒。 RNA 合成不能与 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 CoV RO 的统一模型，并明确将 DMV 确立为病毒 RNA 合成的中心枢纽和 CoV 感染的潜在药物靶点。