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

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.

Inhibiting coronavirus replication in cultured cells by chemical ER stress

“…Similarly, IRE1α is suggested to mediate protective and adaptive responses suitable to alleviate ER stress, e.g. by balancing lipid bilayer stress, an aberrant perturbation of ER membrane structures, which may be expected to occur upon DMV formation in CoV-infected cells (Chen & Brandizzi, 2013;Halbleib et al, 2017;Snijder et al, 2020). Accordingly, high levels of BiP, HERPUD1 and IRE1α may increase in general the resilience of cells when infected by diverse pathogens.…”

“…In common with other cellular stress conditions, including infections by diverse pathogens, CoVs are known to activate the NF-κB, JNK and p38 MAPK pathways and to reprogram host cell transcriptomes (Liao et al, 2011;Mizutani et al, 2005;Poppe et al, 2017). In addition, they induce the formation of replicative organelles (ROs), an intracellular network of double-membrane vesicles (DMV) that harbors the viral replication/transcription complexes (RTC) and shields these complexes from recognition by cellular defense mechanisms (Snijder et al, 2020). The combination of these and other events leads to cell damage and cell death upon virus budding and release within a few days (de Wilde et al, 2018).…”

Structures, conformations and distributions of SARS-CoV-2 spike protein trimers on intact virions

“…During infection, S protein production takes place in an abnormal cellular environment. Coronaviruses extensively remodel the internal membrane organisation of the cell, generating viral replication organelles in which replication takes place (15)(16)(17). The S protein, together with the other membrane protein M, and E, are inserted into membranes of the endoplasmic reticulum (ER), and traffic to the ER Golgi intermediate compartment (ERGIC).…”

“…The S protein, together with the other membrane protein M, and E, are inserted into membranes of the endoplasmic reticulum (ER), and traffic to the ER Golgi intermediate compartment (ERGIC). The encapsidated genome buds into the ERGIC to form virions which are then trafficked to the plasma membrane and released (15)(16)(17).…”

How Signaling Games Explain Mimicry at Many Levels: From Viral Epidemiology to Human Sociology

“…There follows a sample of some of the mimicry strategies that SARS-CoV-2 utilizes. i) Replication organelles, inside which the virus replicates [44], are a form of camouflage. ii) The addition of a cap-like structure onto the 5'end of viral mRNA by SARS-CoV-2 nsp16 [45], produces virus deceiver mRNAs, as discussed in Section 3.1.…”