The Origin, Dynamic Morphology, and PI4P-Independent Formation of Encephalomyocarditis Virus Replication Organelles

Abstract

Picornaviruses induce dramatic rearrangements of endomembranes in the cells that they infect to produce dedicated platforms for viral replication. These structures, termed replication organelles (ROs), have been well characterized for the Enterovirus genus of the Picornaviridae. However, it is unknown whether the diverse RO morphologies associated with enterovirus infection are conserved among other picornaviruses. Here, we use serial electron tomography at different stages of infection to assess the three-dimensional architecture of ROs induced by encephalomyocarditis virus (EMCV), a member of the Cardiovirus genus of the family of picornaviruses that is distantly related. Ultrastructural analyses revealed connections between early single-membrane EMCV ROs and the endoplasmic reticulum (ER), establishing the ER as a likely donor organelle for their formation. These early single-membrane ROs appear to transform into double-membrane vesicles (DMVs) as infection progresses. Both single- and double-membrane structures were found to support viral RNA synthesis, and progeny viruses accumulated in close proximity, suggesting a spatial association between RNA synthesis and virus assembly. Further, we explored the role of phosphatidylinositol 4-phosphate (PI4P), a critical host factor for both enterovirus and cardiovirus replication that has been recently found to expedite enterovirus RO formation rather than being strictly required. By exploiting an EMCV escape mutant, we found that low-PI4P conditions could also be overcome for the formation of cardiovirus ROs. Collectively, our data show that despite differences in the membrane source, there are striking similarities in the biogenesis, morphology, and transformation of cardiovirus and enterovirus ROs, which may well extend to other picornaviruses.

A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis

“…Our results add to studies that, in the last years and after much speculation, have started to provide experimental evidence that the DMVs induced by +RNA viruses are active sites of vRNA synthesis [11, 58–60]. However, it is not clear that DMVs always play the primary role in virus replication that we demonstrate here for CoV.…”

“…However, it is not clear that DMVs always play the primary role in virus replication that we demonstrate here for CoV. For picornaviruses, for example, virus-induced single membrane structures, which are DMV precursors and also active sites of vRNA synthesis, could well be more relevant as they predominate at the peak of vRNA replication [10, 11, 60]. By clearly pointing to DMVs as the key sites for CoV replication, our results also bring back to centre stage some of the challenges that CoV-induced DMVs pose, which extend to the distantly-related arterivirus family, and probably also to other members of the order Nidovirales .…”

“…By clearly pointing to DMVs as the key sites for CoV replication, our results also bring back to centre stage some of the challenges that CoV-induced DMVs pose, which extend to the distantly-related arterivirus family, and probably also to other members of the order Nidovirales . In contrast with the DMVs induced by other +RNA viruses [10, 11, 13, 60], the DMVs in nidovirus-infected cells appear to lack membrane openings that would connect their inner compartment with the cytosol to allow import of precursors and export of genomic and subgenomic viral mRNAs [15, 16, 29]. This topological conundrum, however, starts with the assumption that vRNA synthesis takes place inside the DMVs, yet the evidence so far is insufficient to ascertain this point.…”

The viral replication organelles within cells studied by electron microscopy

“…Since then, many studies have demonstrated the power of these methods. Structures harboring the VRCs such as single-membrane spherules and double membrane vesicles have been studied in detail within the replication organelles of West Nile Virus (Gillespie et al, 2010), HCV (Romero-Brey et al, 2012), coxsackievirus B3 (Limpens et al, 2011), Bunyamwera virus (Fontana et al, 2008), Rubella virus (Fontana et al, 2010), Tombusvirus (Fernández de Castro et al, 2017a), a cardiovirus (Melia et al, 2018) and the human reovirus (Fernández de Castro et al, 2014a;Tenorio et al, 2018). The reovirus replication neoorganelle visualized in 3D with two different methods is shown in Fig.…”

High‐pressure freezing followed by freeze substitution of a complex and variable density miniorgan: the wool follicle

“…Lack of cell membrane visibility was proclaimed either due to extraction of lipids or to the failure of the bilayer membrane to bind electron-dense fixatives or stains. Addition of water to the substitution medium was shown to considerably improve membrane visibility (Limpens et al, 2011;Melia et al, 2018). The FS media used in this study contained more water and although was shown to clearly preserve the Golgi bodies, it is not clear why the cell membrane complexes are not visible in the follicles.…”