title: Ultrastructural characterization of membranous torovirus replication factories.
abstract: Plus-stranded RNA viruses replicate in the cytosol of infected cells, in membrane-bound replication complexes containing the replicase proteins, the viral RNA and host proteins. The formation of the replication and transcription complexes (RTCs) through the rearrangement of cellular membranes is currently being actively studied for viruses belonging to different viral families. In this work, we identified double-membrane vesicles (DMVs) in the cytoplasm of cells infected with the equine torovirus Berne virus (BEV), the prototype member of the Torovirus genus (Coronaviridae family, Nidovirales order). Using confocal microscopy and transmission electron microscopy, we observed a close relationship between the RTCs and the DMVs of BEV. The examination of BEV-infected cells revealed that the replicase proteins colocalize with each other and with newly synthesized RNA and are associated to the membrane rearrangement induced by BEV. However, the double-stranded RNA, an intermediate of viral replication, is exclusively limited to the interior of DMVs. Our results with BEV resemble those obtained with other related viruses in the Nidovirales order, thus providing new evidence to support the idea that nidoviruses share a common replicative structure based on the DMV arranged clusters.
authors.affiliation: Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, CSIC, C/Darwin 3, 28049, Madrid, Spain.
authors.forename: Ginés
authors.initials: G
authors.lastname: Ávila-Pérez
authors.affiliation: Electron Microscopy Facility, Centro de Biología Molecular Severo Ochoa, CSIC, C/Nicolás Cabrera 1, 28049, Madrid, Spain.
authors.forename: María Teresa
authors.initials: MT
authors.lastname: Rejas
authors.affiliation: Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, CSIC, C/Darwin 3, 28049, Madrid, Spain.
authors.forename: Dolores
authors.identifier: http://orcid.org/0000-0001-6365-4501
authors.initials: D
authors.lastname: Rodríguez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure / Q000821:virology; D022162:Cytoplasmic Vesicles / Q000378:metabolism / Q000648:ultrastructure* / Q000821:virology; D020405:Dermis; D004847:Epithelial Cells / Q000378:metabolism / Q000648:ultrastructure / Q000821:virology; D005347:Fibroblasts / Q000378:metabolism / Q000648:ultrastructure / Q000821:virology; D015967:Gene Expression Regulation, Viral; D006736:Horses; D054884:Host-Pathogen Interactions; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure* / Q000821:virology; D046529:Microscopy, Electron, Transmission; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D017003:Torovirus / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
chemical_list: D012330:RNA, Double-Stranded; D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase