Picornavirus security proteins promote the release of extracellular vesicle enclosed viruses via the modulation of host kinases.

The discovery that extracellular vesicles (EVs) serve as carriers of virus particles calls for a reevaluation of the release strategies of non-enveloped viruses. Little is currently known about the molecular mechanisms that determine the release and composition of EVs produced by virus-infected cells, as well as conservation of these mechanisms among viruses. We previously described an important role for the Leader protein of the picornavirus encephalomyocarditis virus (EMCV) in the induction of virus-carrying EV subsets with distinct molecular and physical properties. EMCV L acts as a 'viral security protein' by suppressing host antiviral stress and type-I interferon (IFN) responses. Here, we tested the ability of functionally related picornavirus proteins of Theilers murine encephalitis virus (TMEV L), Saffold virus (SAFV L), and coxsackievirus B3 (CVB3 2Apro), to rescue EV and EV-enclosed virus release when introduced in Leader-deficient EMCV. We show that all viral security proteins tested were able to promote virus packaging in EVs, but that only the expression of EMCV L and CVB3 2Apro increased overall EV production. We provide evidence that one of the main antiviral pathways counteracted by this class of picornaviral proteins, i.e. the inhibition of PKR-mediated stress responses, affected EV and EV-enclosed virus release during infection. Moreover, we show that the enhanced capacity of the viral proteins EMCV L and CVB3 2Apro to promote EV-enclosed virus release is linked to their ability to simultaneously promote the activation of the stress kinase P38 MAPK. Taken together, we demonstrate that cellular stress pathways involving the kinases PKR and P38 are modulated by the activity of non-structural viral proteins to increase the release EV-enclosed viruses during picornavirus infections. These data shed new light on the molecular regulation of EV production in response to virus infection.

Modulation of nucleotide metabolism by picornaviruses.

Viruses actively reprogram the metabolism of the host to ensure the availability of sufficient building blocks for virus replication and spreading. However, relatively little is known about how picornaviruses-a large family of small, non-enveloped positive-strand RNA viruses-modulate cellular metabolism for their own benefit. Here, we studied the modulation of host metabolism by coxsackievirus B3 (CVB3), a member of the enterovirus genus, and encephalomyocarditis virus (EMCV), a member of the cardiovirus genus, using steady-state as well as 13C-glucose tracing metabolomics. We demonstrate that both CVB3 and EMCV increase the levels of pyrimidine and purine metabolites and provide evidence that this increase is mediated through degradation of nucleic acids and nucleotide recycling, rather than upregulation of de novo synthesis. Finally, by integrating our metabolomics data with a previously acquired phosphoproteomics dataset of CVB3-infected cells, we identify alterations in phosphorylation status of key enzymes involved in nucleotide metabolism, providing insight into the regulation of nucleotide metabolism during infection.

Comprehensive Elucidation of the Role of L and 2A Security Proteins on Cell Death during EMCV Infection.

The EMCV L and 2A proteins are virulence factors that counteract host cell defense mechanisms. Both L and 2A exhibit antiapoptotic properties, but the available data were obtained in different cell lines and under incomparable conditions. This study is aimed at checking the role of these proteins in the choice of cell death type in three different cell lines using three mutants of EMCV lacking functional L, 2A, and both proteins together. We have found that both L and 2A are non-essential for viral replication in HeLa, BHK, and RD cell lines, as evidenced by the viability of the virus in the absence of both functional proteins. L-deficient infection led to the apoptotic death of HeLa and RD cells, and the necrotic death of BHK cells. 2A-deficient infection induced apoptosis in BHK and RD cells. Infection of HeLa cells with the 2A-deficient mutant was finalized with exclusive caspase-dependent death with membrane permeabilization, morphologically similar to pyroptosis. We also demonstrated that inactivation of both proteins, along with caspase inhibition, delayed cell death progression. The results obtained demonstrate that proteins L and 2A play a critical role in choosing the path of cell death during infection, but the result of their influence depends on the properties of the host cells.

Antiviral responses are shaped by heterogeneity in viral replication dynamics.

Antiviral signalling, which can be activated in host cells upon virus infection, restricts virus replication and communicates infection status to neighbouring cells. The antiviral response is heterogeneous, both quantitatively (efficiency of response activation) and qualitatively (transcribed antiviral gene set). To investigate the basis of this heterogeneity, we combined Virus Infection Real-time IMaging (VIRIM), a live-cell single-molecule imaging method, with real-time readouts of the dsRNA sensing pathway to analyse the response of human cells to encephalomyocarditis virus (EMCV) infection. We find that cell-to-cell heterogeneity in viral replication rates early in infection affect the efficiency of antiviral response activation, with lower replication rates leading to more antiviral response activation. Furthermore, we show that qualitatively distinct antiviral responses can be linked to the strength of the antiviral signalling pathway. Our analyses identify variation in early viral replication rates as an important parameter contributing to heterogeneity in antiviral response activation.

Encephalomyocarditis Virus 2A Protein Inhibited Apoptosis by Interaction with Annexin A2 through JNK/c-Jun Pathway.

Encephalomyocarditis virus can cause myocarditis and encephalitis in pigs and other mammals, thus posing a potential threat to public health safety. The 2A protein is an important virulence factor of EMCV. Previous studies have shown that the 2A protein may be related to the inhibition of apoptosis by virus, but its specific molecular mechanism is not clear. In this study, the 2A protein was expressed in Escherichia coli in order to find interacting cell proteins. A pull down assay, coupled with mass spectrometry, revealed that the 2A protein possibly interacted with annexin A2. Co-immunoprecipitation assays and confocal imaging analysis further demonstrated that the 2A protein interacted with annexin A2 in cells. In reducing the expression of annexin A2 by siRNA, the ability of the 2A protein to inhibit apoptosis was weakened and the proliferation of EMCV was slowed down. These results suggest that annexin A2 is closely related to the inhibition of apoptosis by 2A. Furthermore, both RT-PCR and western blot results showed that the 2A protein requires annexin A2 interaction to inhibit apoptosis via JNK/c-Jun pathway. Taken together, our data indicate that the 2A protein inhibits apoptosis by interacting with annexin A2 via the JNK/c-Jun pathway. These findings provide insight into the molecular pathogenesis underlying EMCV infection.

DDX56 antagonizes IFN-ß production to enhance EMCV replication by inhibiting IRF3 nuclear translocation.

DEAD (Asp-Glu-Ala-Asp)-box RNA helicases (DDX) play important roles in viral infection, either as cytosolic viral nucleic acids sensors or as essential host factors for viral replication. In this study, we identified DDX56 as a positive regulator for encephalomyocarditis virus (EMCV) replication. EMCV infection promotes DDX56 expression via its viral proteins, VP3 and 3C. We showed that DDX56 overexpression promotes EMCV replication whereas its loss dampened EMCV replication. Consequently, knockdown of DDX56 increases type I interferon (IFN) expression during EMCV infection. We also showed that DDX56 interrupts IFN regulatory factor 3 (IRF3) phosphorylation and its nucleus translocation by directly targeting KPNA3 and KPNA4 in an EMCV-triggered MDA5 signaling activation cascade leading to the blockade of IFN-ß production. Overall, we showed that DDX56 is a novel negative regulator of EMCV-mediated IFN-ß responses and that DDX56 plays a critical role in EMCV replication. These findings reveal a novel strategy for EMCV to utilize a host factor to evade the host innate immune response and provide us new insight into the function of DDX56.

Caveolin-1 is involved in encephalomyocarditis virus replication in BHK-21 cells.

BACKGROUND: Encephalomyocarditis virus, member of Cardiovirus genus within Picornaviridae family, is an important pathogen that infects different domestic and wild animals. However, the molecular mechanism of its entry remains unclear. In this study, we investigated the mechanism of EMCV infectivity in relation to endocytic pathway using BHK-21 cells. METHODS: The function of numerous cellular key factors implicated in the various endocytic mechanisms were systematically explored using chemical inhibitors. Furthermore, RNA interference (RNAi) as well as the overexpression of dominant protein combined to virus infectivity assays, and confocal microscopy was used to examine EMCV infection in details. RESULTS: The results indicated that the EMCV entry into BHK-21 cells depends on caveolin, dynamin, and actin but not clathrin nor macropinocytosis pathways. The effects of overexpression and knockdown of caveolin-1, one components of the caveolae, was examined on EMCV infection. The results showed that EMCV infection was positive correlation with caveolin-1 expression. Confocal microscopy analysis and internalization assay showed that caveolin-1 is required at the early stage of EMCV infection. CONCLUSIONS: Caveolin-1, dynamin, and actin-dependent endocytosis pathways are necessary for EMCV infection in vitro.

Encephalomyocarditis Virus Abrogates the Interferon Beta Signaling Pathway via Its Structural Protein VP2.

Type I interferon (IFN)-mediated antiviral responses are critical for modulating host-virus responses, and indeed, viruses have evolved strategies to antagonize this pathway. Encephalomyocarditis virus (EMCV) is an important zoonotic pathogen, which causes myocarditis, encephalitis, neurological disease, reproductive disorders, and diabetes in pigs. This study aims to understand how EMCV interacts with the IFN pathway. EMCV circumvents the type I IFN response by expressing proteins that antagonize cellular innate immunity. Here, we show that EMCV VP2 is a negative regulator of the IFN-ß pathway. This occurs via the degradation of the MDA5-mediated cytoplasmic double-stranded RNA (dsRNA) antiviral sensing RIG-I-like receptor (RLR) pathway. We show that structural protein VP2 of EMCV interacts with MDA5, MAVS, and TBK1 through its C terminus. In addition, we found that EMCV VP2 could significantly degrade RLRs by the proteasomal and lysosomal pathways. For the first time, EMCV VP2 was shown to play an important role in EMCV evasion of the type I IFN signaling pathway. This study expands our understanding that EMCV utilizes its capsid protein VP2 to evade the host antiviral response.IMPORTANCE Encephalomyocarditis virus is an important pathogen that can cause encephalitis, myocarditis, neurological diseases, and reproductive disorders. It also causes huge economic losses for the swine industry worldwide. Innate immunity plays an important role in defending the host from pathogen infection. Understanding pathogen microorganisms evading the host immune system is of great importance. Currently, whether EMCV evades cytosolic RNA sensing and signaling is still poorly understood. In the present study, we found that viral protein VP2 antagonized the RLR signaling pathway by degrading MDA5, MAVS, and TBK1 protein expression to facilitate viral replication in HEK293 cells. The findings in this study identify a new mechanism for EMCV evading the host's innate immune response, which provide new insights into the virus-host interaction and help develop new antiviral approaches against EMCV.

NUDT21 Links Mitochondrial IPS-1 to RLR-Containing Stress Granules and Activates Host Antiviral Defense.

Viral RNA in the cytoplasm of mammalian host cells is recognized by retinoic acid-inducible protein-I-like receptors (RLRs), which localize to cytoplasmic stress granules (SGs). Activated RLRs associate with the mitochondrial adaptor protein IPS-1, which activates antiviral host defense mechanisms, including type I IFN induction. It has remained unclear, however, how RLRs in SGs and IPS-1 in the mitochondrial outer membrane associate physically and engage in information transfer. In this study, we show that NUDT21, an RNA-binding protein that regulates alternative transcript polyadenylation, physically associates with IPS-1 and mediates its localization to SGs in response to transfection with polyinosinic-polycytidylic acid [poly(I:C)], a mimic of viral dsRNA. We found that despite its well-established function in the nucleus, a fraction of NUDT21 localizes to mitochondria in resting cells and becomes localized to SGs in response to poly(I:C) transfection. NUDT21 was also found to be required for efficient type I IFN induction in response to viral infection in both human HeLa cells and mouse macrophage cell line RAW264.7 cells. Our results together indicate that NUDT21 links RLRs in SGs to mitochondrial IPS-1 and thereby activates host defense responses to viral infection.

Inhibition of oxidative metabolism by nitric oxide restricts EMCV replication selectively in pancreatic beta-cells.

Environmental factors, such as viral infection, are proposed to play a role in the initiation of autoimmune diabetes. In response to encephalomyocarditis virus (EMCV) infection, resident islet macrophages release the pro-inflammatory cytokine IL-1ß, to levels that are sufficient to stimulate inducible nitric oxide synthase (iNOS) expression and production of micromolar levels of the free radical nitric oxide in neighboring ß-cells. We have recently shown that nitric oxide inhibits EMCV replication and EMCV-mediated ß-cell lysis and that this protection is associated with an inhibition of mitochondrial oxidative metabolism. Here we show that the protective actions of nitric oxide against EMCV infection are selective for ß-cells and associated with the metabolic coupling of glycolysis and mitochondrial oxidation that is necessary for insulin secretion. Inhibitors of mitochondrial respiration attenuate EMCV replication in ß-cells, and this inhibition is associated with a decrease in ATP levels. In mouse embryonic fibroblasts (MEFs), inhibition of mitochondrial metabolism does not modify EMCV replication or decrease ATP levels. Like most cell types, MEFs have the capacity to uncouple the glycolytic utilization of glucose from mitochondrial respiration, allowing for the maintenance of ATP levels under conditions of impaired mitochondrial respiration. It is only when MEFs are forced to use mitochondrial oxidative metabolism for ATP generation that mitochondrial inhibitors attenuate viral replication. In a ß-cell selective manner, these findings indicate that nitric oxide targets the same metabolic pathways necessary for glucose stimulated insulin secretion for protection from viral lysis.

Inhibition of mitochondrial oxidative metabolism attenuates EMCV replication and protects ß-cells from virally mediated lysis.

Viral infection is one environmental factor that may contribute to the initiation of pancreatic ß-cell destruction during the development of autoimmune diabetes. Picornaviruses, such as encephalomyocarditis virus (EMCV), induce a pro-inflammatory response in islets leading to local production of cytokines, such as IL-1, by resident islet leukocytes. Furthermore, IL-1 is known to stimulate ß-cell expression of iNOS and production of the free radical nitric oxide. The purpose of this study was to determine whether nitric oxide contributes to the ß-cell response to viral infection. We show that nitric oxide protects ß-cells against virally mediated lysis by limiting EMCV replication. This protection requires low micromolar, or iNOS-derived, levels of nitric oxide. At these concentrations nitric oxide inhibits the Krebs enzyme aconitase and complex IV of the electron transport chain. Like nitric oxide, pharmacological inhibition of mitochondrial oxidative metabolism attenuates EMCV-mediated ß-cell lysis by inhibiting viral replication. These findings provide novel evidence that cytokine signaling in ß-cells functions to limit viral replication and subsequent ß-cell lysis by attenuating mitochondrial oxidative metabolism in a nitric oxide-dependent manner.

Cholesterol 25-hydroxylase inhibits encephalomyocarditis virus replication through enzyme activity-dependent and independent mechanisms.

Cholesterol-25-hydroxylase (CH25 H) is a reticulum-associated membrane protein induced by an important interferon-stimulating gene (ISG) and can significantly inhibit some virus replication. But the effect of CH25H on encephalomyocarditis virus (EMCV) is still not clear. In this study, we found that EMCV infection increases significantly the endogenous CH25H expression in BHK-21 and N2a cells. CH25H and cholesterol catalytic oxidation product 25-hydroxycholesterol (25HC) obviously inhibits EMCV infection by inhibiting the viral penetration. But the CH25H mutant lacking hydroxylase activity repairs the ability to inhibit the viral replication. Meanwhile, ß-cyclodextrin crystalline as a cholesterol inhibitor significantly decreases the viral replication. In addition, CH25H can selectively interact and degrade the viral RNA-Dependent RNA Polymerase-3D protein by independent on the association of proteasome, lysosome and caspase manner. It provides new insights into the interplay mechanisms between CH25H and non-enveloped single-stranded positive RNA viruses.

Inhibition of encephalomyocarditis virus replication by shRNA targeting 1C and 2A genes in vitro and in vivo.

Encephalomyocarditis virus (EMCV) infects many mammalian species, causing myocarditis, encephalitis and reproductive disorders. The small interference RNA (siRNA) targeting to the virus has not been understood completely. Here, two out of six interference sequences were screened to inhibit significantly EMCV replication by using recombinant plasmids expressing small hairpin RNA (shRNA) targeting to the viral 1C or 2A genes in BHK-21 cells. And two recombinant adenoviruses expressing the shRNAs were constructed and named as rAd-1C-1 and rAd-2A-3. They inhibit EMCV replication in BHK-21 cells in protein levels, as well as the virus yields by approximately 1000 times. Furthermore, they provide high protective efficacy against the challenge with virulent EMCV NJ08 strain in mice. And the EMCV loads in the live mice in rAd-1C-1 and rAd-2A-3 groups decrease by more than 90 % compared with those in the dead mice in the challenge control groups at the same times. It indicates that the adenoviruses medicated shRNA targeting to 1C and 2A genes might provide a potential strategy for combating EMCV infection.

ZFYVE1 negatively regulates MDA5- but not RIG-I-mediated innate antiviral response.

The retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I and melanoma differentiation-associated gene 5 (MDA5), sense cytoplasmic viral RNA and initiate innate antiviral responses. How RIG-I and MDA5 are differentially regulated remains enigmatic. In this study, we identified the guanylate-binding protein (GBP) and zinc-finger FYVE domain-containing protein ZFYVE1 as a negative regulator of MDA5- but not RIG-I-mediated innate antiviral responses. ZFYVE1-deficiency promoted MDA5- but not RIG-I-mediated transcription of downstream antiviral genes. Comparing to wild-type mice, Zfyve1-/- mice were significantly protected from lethality induced by encephalomyocarditis virus (EMCV) that is sensed by MDA5, whereas Zfyve1-/- and Zfyve1+/+ mice were comparable to death induced by vesicular stomatitis virus (VSV) that is sensed by RIG-I. Mechanistically, ZFYVE1 interacted with MDA5 but not RIG-I. ZFYVE1 bound to viral RNA and decreased the ligand binding and oligomerization of MDA5. These findings suggest that ZFYVE1 acts as a specific negative regulator of MDA5-mediated innate immune responses by inhibiting its ligand binding and oligomerization.

Identification of the Cell-Surface Protease ADAM9 as an Entry Factor for Encephalomyocarditis Virus.

Encephalomyocarditis virus (EMCV) is an animal pathogen and an important model organism, whose receptor requirements are poorly understood. Here, we employed a genome-wide haploid genetic screen to identify novel EMCV host factors. In addition to the previously described picornavirus receptors sialic acid and glycosaminoglycans, this screen unveiled important new host factors for EMCV. These factors include components of the fibroblast growth factor (FGF) signaling pathway, such as the potential receptors FGFR1 and ADAM9, a cell-surface metalloproteinase. By employing various knockout cells, we confirmed the importance of the identified host factors for EMCV infection. The largest reduction in infection efficiency was observed in cells lacking ADAM9. Pharmacological inhibition of the metalloproteinase activity of ADAM9 did not affect virus infection. Moreover, reconstitution of inactive ADAM9 in knockout cells restored susceptibility to EMCV, pointing to a proteinase-independent role of ADAM9 in mediating EMCV infection. Using neutralization assays with ADAM9-specific antiserum and soluble receptor proteins, we provided evidence for a role of ADAM9 in EMCV entry. Finally, binding assays showed that ADAM9 facilitates attachment of EMCV to the cell surface. Together, our findings reveal a role for ADAM9 as a novel receptor or cofactor for EMCV.IMPORTANCE EMCV is an animal pathogen that causes acute viral infections, usually myocarditis or encephalitis. It is thought to circulate mainly among rodents, from which it is occasionally transmitted to other animal species, including humans. EMCV causes fatal outbreaks of myocarditis and encephalitis in pig farms and zoos, making it an important veterinary pathogen. Although EMCV has been widely used as a model to study mechanisms of viral disease in mice, little is known about its entry mechanism. Here, we employ a haploid genetic screen for EMCV host factors and identify an essential role for ADAM9 in EMCV entry.

SIDT1 Localizes to Endolysosomes and Mediates Double-Stranded RNA Transport into the Cytoplasm.

dsRNA is a common by-product of viral replication and acts as a potent trigger of antiviral immunity. SIDT1 and SIDT2 are closely related members of the SID-1 transmembrane family. SIDT2 functions as a dsRNA transporter and is required to traffic internalized dsRNA from endocytic compartments into the cytosol for innate immune activation, but the role of SIDT1 in dsRNA transport and in the innate immune response to viral infection is unclear. In this study, we show that Sidt1 expression is upregulated in response to dsRNA and type I IFN exposure and that SIDT1 interacts with SIDT2. Moreover, similar to SIDT2, SIDT1 localizes to the endolysosomal compartment, interacts with the long dsRNA analog poly(I:C), and, when overexpressed, enhances endosomal escape of poly(I:C) in vitro. To elucidate the role of SIDT1 in vivo, we generated SIDT1-deficient mice. Similar to Sidt2-/- mice, SIDT1-deficient mice produced significantly less type I IFN following infection with HSV type 1. In contrast to Sidt2-/- mice, however, SIDT1-deficient animals showed no impairment in survival postinfection with either HSV type 1 or encephalomyocarditis virus. Consistent with this, we observed that, unlike SIDT2, tissue expression of SIDT1 was relatively restricted, suggesting that, whereas SIDT1 can transport extracellular dsRNA into the cytoplasm following endocytosis in vitro, the transport activity of SIDT2 is likely to be functionally dominant in vivo.

RNA viruses promote activation of the NLRP3 inflammasome through cytopathogenic effect-induced potassium efflux.

Early detection of viruses by the innate immune system is crucial for host defense. The NLRP3 inflammasome, through activation of caspase-1, promotes the maturation of IL-1ß and IL-18, which are critical for antiviral immunity and inflammatory response. However, the mechanism by which viruses activate this inflammasome is still debated. Here, we report that the replication of cytopathogenic RNA viruses such as vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) induced a lytic cell death leading to potassium efflux, the common trigger of NLRP3 inflammasome activation. This lytic cell death was not prevented by a chemical or genetic inhibition of apoptosis, pyroptosis, or necroptosis but required the viral replication. Hence, the viruses that stimulated type I IFNs production after their sensing did not activate NLRP3 inflammasome due to an inhibition of their replication. In contrast, NLRP3 inflammasome activation induced by RNA virus infection was stimulated in IFNAR-deficient or MAVS-deficient cells consequently to an increased viral replication and ensuing lytic cell death. Therefore, in a context of inefficient IFN response, viral replication-induced lytic cell death activates of the NLRP3 inflammasome to fight against infection.

Exploitation of nuclear protein SFPQ by the encephalomyocarditis virus to facilitate its replication.

The encephalomyocarditis virus (EMCV) is a single-stranded RNA virus that induces sudden death, diabetes, myocarditis and nervous disorders in non-human primates. The rapid development of xenografts such as heart transplantation from pig to human raises the issue of EMCV safety in human cells. SFPQ, a proline and glutamine rich splicing factor that participates in diverse molecular functions including paraspeckle formation, microRNA synthesis and transcription regulation, is known to regulate host innate immune response to viruses. However, the role of SFPQ in EMCV infection remains unclear. Here we reported that the SFPQ was essential for EMCV replication. Depletion of SFPQ impaired EMCV production, while forced expression of SFPQ promoted viral replication. Mechanistically, loss of SFPQ affected the transcription profile of host mitochondria pathway related genes. In addition, cellular SFPQ was exploited by EMCV and accumulated in cytoplasm and it interacted with eukaryotic initiation factors and ribosomal proteins to facilitate internal ribosome entry site (IRES)-dependent translation of EMCV protein. Altogether, our work discovered host SFPQ as a new target to inhibit EMCV replication and infection.

The ubiquitin-protein ligase E6AP/UBE3A supports early encephalomyocarditis virus replication.

Many viruses make use of, and even direct, the ubiquitin-proteasome system to facilitate the generation of a cellular environment favorable for virus replication, while host cells use selected protein ubiquitylation pathways for antiviral defense. Relatively little information has been acquired, however, regarding the extent to which protein ubiquitylation determines the replication success of picornaviruses. Here we report that the ubiquitin-protein ligase E6AP/UBE3A, recently shown to be a participant in encephalomyocarditis virus (EMCV) 3C protease concentration regulation, also facilitates the early stages of EMCV replication, probably by a mechanism that does not involve 3C protease ubiquitylation. Using stably transfected E6AP knockdown cells, we found that reduced E6AP concentration extends the time required for infected cells to undergo the morphological changes caused by virally induced pathogenesis and to begin the production of infectious virions. This lag in virion production is accompanied by a corresponding delay in the appearance of detectable levels of viral proteins and RNA. We also found, by using both immunofluorescence microscopy and cell fractionation, that E6AP is partially redistributed from the nucleus to the cytoplasm in EMCV-infected cells, thereby increasing its availability to participate in cytoplasmic virus replication processes.

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

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.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. Here, we investigate the architecture and biogenesis of cardiovirus ROs and compare them with those induced by enteroviruses, members of the well-characterized picornavirus genus Enterovirus The origins and dynamic morphologies of cardiovirus ROs are revealed using electron tomography, which points to the endoplasmic reticulum as the donor organelle usurped to produce single-membrane tubules and vesicles that transform into double-membrane vesicles. 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. Our data show that the transformation from single-membrane structures to double-membrane vesicles is a conserved feature of cardiovirus and enterovirus infections that likely extends to other picornavirus genera.