Architecture of torovirus replicative organelles

Plus-stranded RNA viruses replicate in the cytosol of infected cells, in membrane-bound replication complexes. We previously identified double membrane vesicles (DMVs) in the cytoplasm of cells infected with Berne virus (BEV), the prototype member of the Torovirus genus (Nidovirales Order). Our previous analysis by transmission electron microscopy suggested that the DMVs form a reticulovesicular network (RVN) analogous those described for the related severe acute respiratory syndrome coronavirus (SARS-CoV-1). Here, we used serial sectioning and electron tomography to characterize the architecture of torovirus replication organelles, and to learn about their biogenesis and dynamics during the infection. The formation of a RVN in BEV infected cells was confirmed, where the outer membranes of the DMVs are interconnected with each other and with the ER. Paired or zippered ER membranes connected with the DMVs were also observed, and likely represent early structures that evolve to give rise to DMVs. Also, paired membranes forming small spherule-like invaginations were observed at late time post-infection. Although resembling in size, the tomographic analysis show that these structures are clearly different from the true spherules described previously for coronaviruses. Hence, BEV shows important similarities, but also some differences, in the architecture of the replication organelles with other nidoviruses.This work was supported by grants AGL2017-87464-C2-1-P to DR and SAF2017-84565-R to JJF funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”, by the European Union. GAP was supported by a FPU fellowship from the Spanish Ministry of Education, Culture and Sport. We acknowledge access to the cryoEM CNB-CSIC facility in the context of the CRIOMECORR project (ESFRI-2019-01-CSIC-16).Peer reviewe