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

Eric J Snijder,Frank F G A Faas,Abraham J Koster,Ronald W A L Limpens,Montserrat Bárcena,Helena J Maier,Jessika C Zevenhoven-Dobbe,Anja W M De Jong,Adriaan H De Wilde,Andrea Cimarelli

doi:10.1371/journal.pbio.3000715

Eric J Snijder, Frank F G A Faas + Show 8 more

Open Access

https://doi.org/10.1371/journal.pbio.3000715

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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.

Highlights

We used a radiolabeled nucleoside ([3H]uridine) and applied the classic and highly sensitive technique of electron microscopy (EM) autoradiography [32,33] in combination with advanced quantitative analysis tools. This approach revealed that doublemembrane vesicle (DMV) are the primary site of CoV positive-stranded RNA (RNA) synthesis, with neither double-membrane spherule (DMS) nor convoluted membranes (CM) nor zippered endoplasmic reticulum (ER) being labeled to a significant extent
After somewhat disparate observations [15,20,21,26,27,47], the unifying model that emerges from our study is that of a
CoV replication organelle (RO) comprising 3 basic types of double-membrane structural elements: (1) CM or zippered ER, which would represent branched or unbranched configurations of paired-ER membranes; (2) small DMSs that appear to arise from CM or zippered ER; and (3) DMVs

Summary

Introduction

The RNA synthesis of all positive-stranded RNA (+RNA) viruses of eukaryotes occurs in the cytoplasm of the host cell, in conjunction with modified endomembranes that are often. The morphological and functional characterization of ROs of the second, vesiculotubular type is lagging behind Such structures, which always include double-membrane vesicles (DMVs), commonly derive from membranes of the secretory pathway and have been found in cells infected with, e.g., picornaviruses [10,11], noroviruses [12], hepatitis C virus (HCV) [13], and different nidoviruses, including the arterivirus and coronavirus (CoV) families [14,15,16,17,18]. We used a radiolabeled nucleoside ([3H]uridine) and applied the classic and highly sensitive technique of EM autoradiography [32,33] in combination with advanced quantitative analysis tools This approach revealed that DMVs are the primary site of CoV RNA synthesis, with neither DMSs nor CM nor zippered ER being labeled to a significant extent. It returns DMVs to center stage as the hub of CoV RNA synthesis and a potential antiviral drug target

Results

Discussion

Materials and methods