title: Biogenesis and architecture of arterivirus replication organelles.
abstract: All eukaryotic positive-stranded RNA (+RNA) viruses appropriate host cell membranes and transform them into replication organelles, specialized micro-environments that are thought to support viral RNA synthesis. Arteriviruses (order Nidovirales) belong to the subset of +RNA viruses that induce double-membrane vesicles (DMVs), similar to the structures induced by e.g. coronaviruses, picornaviruses and hepatitis C virus. In the last years, electron tomography has revealed substantial differences between the structures induced by these different virus groups. Arterivirus-induced DMVs appear to be closed compartments that are continuous with endoplasmic reticulum membranes, thus forming an extensive reticulovesicular network (RVN) of intriguing complexity. This RVN is remarkably similar to that described for the distantly related coronaviruses (also order Nidovirales) and sets them apart from other DMV-inducing viruses analysed to date. We review here the current knowledge and open questions on arterivirus replication organelles and discuss them in the light of the latest studies on other DMV-inducing viruses, particularly coronaviruses. Using the equine arteritis virus (EAV) model system and electron tomography, we present new data regarding the biogenesis of arterivirus-induced DMVs and uncover numerous putative intermediates in DMV formation. We generated cell lines that can be induced to express specific EAV replicase proteins and showed that DMVs induced by the transmembrane proteins nsp2 and nsp3 form an RVN and are comparable in topology and architecture to those formed during viral infection. Co-expression of the third EAV transmembrane protein (nsp5), expressed as part of a self-cleaving polypeptide that mimics viral polyprotein processing in infected cells, led to the formation of DMVs whose size was more homogenous and closer to what is observed upon EAV infection, suggesting a regulatory role for nsp5 in modulating membrane curvature and DMV formation.
authors.affiliation: Electron Microscopy Section, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Barbara
authors.initials: B
authors.lastname: van der Hoeven
authors.affiliation: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Diede
authors.initials: D
authors.lastname: Oudshoorn
authors.affiliation: Electron Microscopy Section, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Abraham J
authors.initials: AJ
authors.lastname: Koster
authors.affiliation: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Snijder
authors.affiliation: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands. Electronic address: m.kikkert@lumc.nl.
authors.forename: Marjolein
authors.initials: M
authors.lastname: Kikkert
authors.affiliation: Electron Microscopy Section, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands. Electronic address: m.barcena@lumc.nl.
authors.forename: Montserrat
authors.initials: M
authors.lastname: Bárcena
mesh_terms: D000818:Animals; D018018:Arterivirus / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D018174:Arterivirus Infections / Q000662:veterinary / Q000821:virology; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure* / Q000821:virology; D017934:Coronavirus / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D055032:Electron Microscope Tomography; D004721:Endoplasmic Reticulum / Q000648:ultrastructure* / Q000821:virology; D015870:Gene Expression; D054884:Host-Pathogen Interactions; D015388:Organelles / Q000648:ultrastructure* / Q000821:virology*; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D017361:Viral Nonstructural Proteins
keywords: Correlative light-electron microscopy; Membrane remodelling; Nidoviruses; Replication complex; Replication structures; Three-dimensional electron microscopy