The replication of all positive-stranded RNA viruses of eukaryotes is thought to take place at cytoplasmic membranous replication organelles. One of the most prominent types of viral ROs induced by... Show moreThe replication of all positive-stranded RNA viruses of eukaryotes is thought to take place at cytoplasmic membranous replication organelles. One of the most prominent types of viral ROs induced by a number of these viruses, including coronaviruses and arteriviruses, are double-membrane vesicles (DMVs) that contain viral double-stranded RNA. This thesis discusses the formation of these replication organelles by arteri- and coronaviruses and provides new insights in the viral proteins involved. The ultrastructure of the replication organelles was unravelled using both electron microscopy and tomography. Furthermore, this research described in this thesis also shows that the innate immune system in infected cells can prevent the formation of the membrane structures. Show less
Oudshoorn, D.; Hoeven, B. van der; Limpens, R.W.A.L.; Beugeling, C.; Snijder, E.J.; Barcena, M.; Kikkert, M. 2016
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... Show moreAll 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. (C) 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license. Show less
