Conserved structural features of RNA export pores spanning the double membrane of arterivirus and coronavirus replication organelles

Corona- and arteriviruses are distantly related positive-stranded RNA virus families within the order Nidovirales. Both transform intracellular membranes into double-membrane vesicles (DMVs) that serve as replication organelles. Newly made viral RNA presumably exits DMVs through double-membrane-spanning molecular pores formed by coronavirus nsp3-nsp4 and arterivirus nsp2-nsp3. Although sub-nm resolution structural information is available for the coronavirus DMV pore, only low-resolution data exist for the pore of the prototypic arterivirus equine arteritis virus (EAV). Here, we modeled arterivirus nsp2 and nsp3 to define their domain architecture, membrane topology, and potential organization into a DMV pore containing 12 copies of each protein. Despite the much smaller dimensions of the arterivirus pore, our analysis suggests striking conservation of features also found in coronaviruses, including conserved positively-charged residues lining analogous pore channel constrictions implicated in RNA export. We investigated their importance in arteriviruses using EAV reverse genetics and nsp2-nsp3 expression systems. While none of these mutations impaired DMV formation, charge-neutralizing substitutions were lethal, whereas conservative substitutions were partially tolerated. We also identified an additional conserved positively-charged residue in the nsp2 C-terminal domain, likely facing the outer DMV membrane, that is essential for DMV formation. Together, these findings reveal nidovirus-wide conservation of important DMV pore features despite the large evolutionary distance between arteri- and coronaviruses.