A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis

TLDR

Zoonotic coronaviruses, including SARS‑CoV‑2, hijack modified endoplasmic reticulum membranes to form replication organelles, but it remains unclear which of the diverse membrane structures—such as double‑membrane vesicles and other CoV‑induced elements—serve as the sites of viral RNA synthesis. The study aims to identify the specific replication organelle structures that support coronavirus RNA synthesis. The authors used metabolic labeling of nascent viral RNA combined with quantitative electron microscopy autoradiography to map RNA synthesis to double‑membrane vesicles in cells infected with MERS‑CoV, SARS‑CoV, and infectious bronchitis virus. The analyses demonstrate that coronavirus replication organelles across different strains share similar membrane modifications, and that RNA synthesis is confined to double‑membrane vesicles rather than to spherules or other structures, establishing DMVs as the central hub for viral RNA synthesis and a potential drug target.

Abstract

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.