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. ImportanceFor eukaryotic positive-strand RNA viruses, efficient replication depends on the spatial organization of viral RNA synthesis within replication organelles in the infected cells cytoplasm. These virus-induced, membrane-bound compartments are thought to provide an optimized environment for viral RNA synthesis. In corona- and arteriviruses, distantly related families within the nidovirus order, unusual double-membrane vesicles (DMVs) have been identified as the primary site of viral RNA synthesis. A hallmark of these structures is a double-membrane-spanning pore complex that connects their interior to the cytosol, thereby presumably enabling export of newly synthesized viral RNA. Here, we analyze the arterivirus DMV pore complex and identify several conserved structural features, including positively-charged residues in the pore-forming proteins nsp2 and nsp3, which are essential for viral replication, likely due to their role in RNA export. Together, our findings underscore the functional conservation and fundamental importance of DMV pore complexes across evolutionary distant nidovirus families.