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.
Highlights
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Using immuno electron microscopy (IEM) to detect BrUTP incorporated into viral RNA, the interior of these spherules was shown to contain newly-synthesized viral RNA that is thought to be exported to the cytosol through a neck-like channel of ∼10 nm in diameter, which could be clearly visualized in the 3D reconstruction
Our results show that the expression of nsp2-3 suffices to reproduce the doublemembrane architecture, topology and connectivity of the doublemembrane vesicles (DMVs) found in infected cells, and constitutes a useful system to study these features of the equine arteritis virus (EAV)-induced reticulovesicular network (RVN)
Summary
The arterivirus prototype EAV was discovered in 1953 (Bryans et al, 1957), the family Arteriviridae was not established until 1996 (Cavanagh, 1997). When the TM1-TM2-Mpro-TM3 regions of the arterivirus and coronavirus pp1a polyproteins are compared (Fig. 1), their general organization is strikingly similar, despite the 2.5-fold overall pp1a size difference between the two families In both virus groups, the junction between the nsps containing TM1 and TM2 is cleaved by a PLP located in the TM1-containing nsp (PLP2 in nsp for arteriviruses and PLPro in nsp for coronaviruses) (Harcourt et al, 2004; Kanjanahaluethai and Baker, 2000; Snijder et al, 1995; Ziebuhr et al, 2001). The fact that the general arrangement of hydrophobic domains in pp1a is conserved between arteriviruses and coronaviruses probably points to a common, and important, function in virus replication
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