Expression and Cleavage of Middle East Respiratory Syndrome Coronavirus nsp3-4 Polyprotein Induce the Formation of Double-Membrane Vesicles That Mimic Those Associated with Coronaviral RNA Replication

Abstract

Betacoronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), are important pathogens causing potentially lethal infections in humans and animals. Coronavirus RNA synthesis is thought to be associated with replication organelles (ROs) consisting of modified endoplasmic reticulum (ER) membranes. These are transformed into double-membrane vesicles (DMVs) containing viral double-stranded RNA and into other membranous elements such as convoluted membranes, together forming a reticulovesicular network. Previous evidence suggested that the nonstructural proteins (nsp’s) 3, 4, and 6 of the severe acute respiratory syndrome coronavirus (SARS-CoV), which contain transmembrane domains, would all be required for DMV formation. We have now expressed MERS-CoV replicase self-cleaving polyprotein fragments encompassing nsp3-4 or nsp3-6, as well as coexpressed nsp3 and nsp4 of either MERS-CoV or SARS-CoV, to characterize the membrane structures induced. Using electron tomography, we demonstrate that for both MERS-CoV and SARS-CoV coexpression of nsp3 and nsp4 is required and sufficient to induce DMVs. Coexpression of MERS-CoV nsp3 and nsp4 either as individual proteins or as a self-cleaving nsp3-4 precursor resulted in very similar DMVs, and in both setups we observed proliferation of zippered ER that appeared to wrap into nascent DMVs. Moreover, when inactivating nsp3-4 polyprotein cleavage by mutagenesis, we established that cleavage of the nsp3/nsp4 junction is essential for MERS-CoV DMV formation. Addition of the third MERS-CoV transmembrane protein, nsp6, did not noticeably affect DMV formation. These findings provide important insight into the biogenesis of coronavirus DMVs, establish strong similarities with other nidoviruses (specifically, the arteriviruses), and highlight possible general principles in viral DMV formation.

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

“…However, given the prevalence of DMSs and CM across different CoV genera, it is tempting to speculate that these structures must play a role in virus replication. It has been proposed that CM could be a form of cubic membranes [25, 56], which are membrane aggregates resulting from ER protein overexpression [57]. CM, which abundantly label for viral nsps [15, 20] and proliferate late in infection [15, 20, 21], could thus be a by-product of viral protein overexpression.…”

Post-Translational Modifications of Coronavirus Proteins: Roles and Function

“…In fact, co-expression of SARS-CoV nsp3, nsp4 and nsp6 induced DMV formation in the transfected cells [143]. A more recent study showed that, for both MERS-CoV and SARS-CoV, co-expression of nsp3 and nsp4 was already sufficient to induce DMV formation [144]. On the other hand, overexpression of coronavirus nsp6 induced the formation of autophagosomes [145], but at the same time restricted its expansion [146].…”

Innate Immune Evasion by Human Respiratory RNA Viruses

“…Also, the negative-stranded RSV genome and its replication enzymes are found associated with cytosolic occluded structures, in that case named inclusion bodies [38,39]. Expression of a selection of specific hydrophobic viral proteins can usually mimic the formation of these structures, for example, nsp3 and nsp4 of CoVs [40], the N and P proteins of RSV [41], and 2B,2C and 3A proteins of enterovirus (polio; [42]). All these structures, while divers in morphology and contents, seem to concentrate the viral replication machinery, intermediates and products inside membrane-bound vesicles or invaginations, seemingly unreachable for the innate immune sensors of the cytosol.…”