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Abstract
ABSTRACTPicornaviruses 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.
Highlights
Picornaviruses induce dramatic rearrangements of endomembranes in the cells that they infect to produce dedicated platforms for viral replication
Putative transition states between singlemembrane tubules and double-membrane vesicles (DMVs) were found that mirror the transformation intermediates observed for enterovirus replication organelles (ROs) [21]. This chronological progression of RO morphology contextualizes the previous observations of single- or double-membrane encephalomyocarditis virus (EMCV) ROs and represents a striking parallel between cardiovirus and enterovirus DMV formation that may represent a conserved mechanism among picornaviruses. Given this cohesion in RO morphology between enteroviruses and EMCV, we investigated whether phosphatidylinositol 4-kinase type III alpha (PI4KA) plays a role in the formation of EMCV ROs, as PI4KB does for enterovirus ROs [20]
Samples for immunofluorescence and electron microscopy (EM) analyses were fixed between 3 and 9 hpi. While data from these chemically fixed EM samples were assessed to quantify the emergence of ROs, representative images are presented from a subsequent experiment using an improved sample preparation method that more faithfully preserves RO morphology
Summary
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. It is unknown whether the diverse RO morphologies associated with enterovirus infection are conserved among other picornaviruses. IMPORTANCE Like all positive-sense RNA viruses, picornaviruses induce the rearrangement of host cell membranes to form unique structures, or replication organelles (ROs), that support viral RNA synthesis. We show that PI4P, a critical lipid for cardiovirus and enterovirus replication, is not strictly required for the formation of cardiovirus ROs, as functional ROs with typical morphologies are formed under phosphatidylinositol 4-kinase type III alpha (PI4KA) inhibition in cells infected with an escape mutant. As with cardioviruses and enteroviruses, PI4P is required by the more distantly related hepatitis C virus (HCV), which depends upon PI4KA for its replication [19]
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