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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., Competing Interests: The authors have declared that no competing interest exists., (Copyright: © 2024 Defourny et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection., (© 2021. The Author(s).)

Viral infections of the central nervous system cause acute or delayed neuropathology and clinical consequences ranging from asymptomatic courses to chronic, debilitating diseases. The outcome of viral encephalitis is partially determined by genetically programed immune response patterns of the host. Experimental infection of mice with Theiler's murine encephalomyelitis virus (TMEV) causes diverse neurologic diseases, including TMEV-induced demyelinating disease (TMEV-IDD), depending on the used mouse strain. The aim of the present study was to compare initial transcriptomic changes occurring in the brain of TMEV-infected SJL (TMEV-IDD susceptible) and C57BL/6 (TMEV-IDD resistant) mice. Animals were infected with TMEV and sacrificed 4, 7, or 14 days post infection. RNA was isolated from brain tissue and analyzed by whole-transcriptome sequencing. Selected differences were confirmed on a protein level by immunohistochemistry. In mock-infected SJL and C57BL/6 mice, >200 differentially expressed genes (DEGs) were detected. Following TMEV-infection, the number of DEGs increased to >700. Infected C57BL/6 mice showed a higher expression of transcripts related to antigen presentation via major histocompatibility complex (MHC) I, innate antiviral immune responses and cytotoxicity, compared with infected SJL animals. Expression of many of those genes was weaker or delayed in SJL mice, associated with a failure of viral clearance in this mouse strain. SJL mice showed prolonged elevation of MHC II and chemotactic genes compared with C57BL/6 mice, which presumably facilitates the induction of chronic demyelinating disease. In addition, elevated expression of several genes associated with immunomodulatory or -suppressive functions was observed in SJL mice. The exploratory study confirms previous observations in the model and provides an extensive list of new immunologic parameters potentially contributing to different outcomes of viral encephalitis in two mouse strains., (© 2021 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology.)

Several virus-induced models were used to study the underlying mechanisms of multiple sclerosis (MS). The infection of susceptible mice with Theiler's murine encephalomyelitis virus (TMEV) establishes persistent viral infections and induces chronic inflammatory demyelinating disease. In this review, the innate and adaptive immune responses to TMEV are discussed to better understand the pathogenic mechanisms of viral infections. Professional (dendritic cells (DCs), macrophages, and B cells) and non-professional (microglia, astrocytes, and oligodendrocytes) antigen-presenting cells (APCs) are the major cell populations permissive to viral infection and involved in cytokine production. The levels of viral loads and cytokine production in the APCs correspond to the degrees of susceptibility of the mice to the TMEV-induced demyelinating diseases. TMEV infection leads to the activation of cytokine production via TLRs and MDA-5 coupled with NF-κB activation, which is required for TMEV replication. These activation signals further amplify the cytokine production and viral loads, promote the differentiation of pathogenic Th17 responses, and prevent cellular apoptosis, enabling viral persistence. Among the many chemokines and cytokines induced after viral infection, IFN α/β plays an essential role in the downstream expression of costimulatory molecules in APCs. The excessive levels of cytokine production after viral infection facilitate the pathogenesis of TMEV-induced demyelinating disease. In particular, IL-6 and IL-1β play critical roles in the development of pathogenic Th17 responses to viral antigens and autoantigens. These cytokines, together with TLR2, may preferentially generate deficient FoxP3 + CD25 - regulatory cells converting to Th17. These cytokines also inhibit the apoptosis of TMEV-infected cells and cytolytic function of CD8 + T lymphocytes (CTLs) and prolong the survival of B cells reactive to viral and self-antigens, which preferentially stimulate Th17 responses.

Eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), better known as PKR, plays a key role in the response to viral infections and cellular homeostasis by regulating mRNA translation. Upon binding dsRNA, PKR is activated through homodimerization and subsequent autophosphorylation on residues Thr446 and Thr451. In this study, we identified a novel PKR phosphorylation site, Ser6, located 3 amino acids upstream of the first double-stranded RNA binding motif (DRBM1). Another Ser residue occurs in PKR at position 97, the very same position relative to the DRBM2. Ser or Thr residues also occur 3 amino acids upstream DRBMs of other proteins such as ADAR1 or DICER. Phosphoinhibiting mutations (Ser-to-Ala) introduced at Ser6 and Ser97 spontaneously activated PKR. In contrast, phosphomimetic mutations (Ser-to-Asp) inhibited PKR activation following either poly (I:C) transfection or virus infection. These mutations moderately affected dsRNA binding or dimerization, suggesting a model where negative charges occurring at position 6 and 97 tighten the interaction of DRBMs with the kinase domain, thus keeping PKR in an inactive closed conformation even in the presence of dsRNA. This study provides new insights on PKR regulation mechanisms and identifies Ser6 and Ser97 as potential targets to modulate PKR activity for therapeutic purposes.

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., (Copyright © 2020 by The American Association of Immunologists, Inc.)

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-1β, 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 β-cells. We have recently shown that nitric oxide inhibits EMCV replication and EMCV-mediated β-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 β-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 β-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 β-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., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Stafford et al.)

Background: Experimental autoimmune encephalitis (EAE) and virally induced demyelinating disease are two major experimental model systems used to study human multiple sclerosis. Although endothelin-1 level elevation was previously observed in the CNS of mice with EAE and viral demyelinating disease, the potential role of endothelin-1 in the development of these demyelinating diseases is unknown., Methods and Results: In this study, the involvement of endothelin-1 in the development and progression of demyelinating diseases was investigated using these two experimental models. Administration of endothelin-1 significantly promoted the progression of both experimental diseases accompanied with elevated inflammatory T cell responses. In contrast, administration of specific endothelin-1 inhibitors (BQ610 and BQ788) significantly inhibited progression of these diseases accompanied with reduced T cell responses to the respective antigens., Conclusions: These results strongly suggest that the level of endothelin-1 plays an important role in the pathogenesis of immune-mediated CNS demyelinating diseases by promoting immune responses.

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., (Copyright © 2019 Baggen et al.)

Galectin-3 is a β-galactoside-binding lectin which is important in cell proliferation and apoptotic regulation. Recently, serum galectin-3 has been shown to have prognostic value as a biomarker in heart failure. Encephalomyocarditis virus (EMCV) can cause severe myocarditis, congestive heart failure and dilated cardiomyopathy as well as encephalitis in various animals including mice. The pathophysiological role of galectin-3 in acute myocarditis following viral infection is not fully understood. The goal of this study is to determine the cardiac localization and the time-course of galectin-3 expression in heart failure after viral inoculation with EMCV. At 12, 24, 48, 96 hours, 7 and 10 days after intraperitoneal EMCV inoculation, animals were examined histologically and analyzed for the expression of galectin-3 and Iba1. Galectin-3 was up-regulated in degenerated fibrotic lesions of cardiac tissues 96 hours after viral inoculation and were followed by myocardial fibrosis. At the same time, Iba1 positive macrophages were observed within the inflammatory sites. A time-course correlation between the number of galectin-3 positive cells and the cardiac area of degenerated fibrotic lesions was detected-serum galectin-3 increased at 96 hours and correlated well with the number of cardiac galectin-3 positive cells. Our results indicate that galectin-3 expression may be a useful biomarker of cardiac fibrotic degeneration in acute myocarditis following viral infection. In addition, measuring serum galectin-3 levels might be an early diagnostic method for detecting cardiac degeneration in acute myocarditis., Competing Interests: The authors have declared that no competing interests exist.

Multiple sclerosis (MS) is a high prevalence degenerative disease characterized at the cellular level by glial and neuronal cell death. The causes of cell death during the disease course are not fully understood. In this work we demonstrate that in a MS model induced by Theiler's murine encephalomyelitis virus (TMEV) infection, the inward rectifier (Kir) 4.1 potassium channel subunit is overexpressed in astrocytes. In voltage clamp experiments the inward current density from TMEV-infected astrocytes was significantly larger than in mock-infected ones. The cRNA hybridization analysis from mock- and TMEV-infected cells showed an upregulation of a potassium transport channel coding sequence. We validated this mRNA increase by RT-PCR and quantitative PCR using Kir 4.1 specific primers. Western blotting experiments confirmed the upregulation of Kir 4.1, and alignment between sequences provided the demonstration that the over-expressed gene encodes for a Kir family member. Flow cytometry showed that the Kir 4.1 protein is located mainly in the cell membrane in mock and TMEV-infected astrocytes. Our results demonstrate an increase in K + inward current in TMEV-infected glial cells, this increment may reduce the neuronal depolarization, contributing to cell resilience mechanisms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cardioviruses cause diseases in many animals including, in rare cases, humans. Although they share common features with all picornaviruses, cardioviruses have unique properties that distinguish them from other family members, including enteroviruses. One feature shared by all picornaviruses is the covalent attachment of VPg to the 5' end of genomic RNA via a phosphotyrosyl linkage. For enteroviruses, this linkage is cleaved by a host cell protein, TDP2. Since TDP2 is divergently required during enterovirus infections, we determined if TDP2 is necessary during infection by the prototype cardiovirus, EMCV. We found that EMCV yields are reduced in the absence of TDP2. We observed a decrease in viral protein accumulation and viral RNA replication in the absence of TDP2. In contrast to enterovirus infections, we found that TDP2 is modified at peak times of EMCV infection. This finding suggests a unique mechanism for cardioviruses to regulate TDP2 activity during infection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Viral encephalitis is a dangerous compromise between the need to robustly clear pathogen from the brain and the need to protect neurons from bystander injury. Theiler's murine encephalomyelitis virus (TMEV) infection of C57Bl/6 mice is a model of viral encephalitis in which the compromise results in hippocampal damage and permanent neurological sequelae. We previously identified brain-infiltrating inflammatory monocytes as the primary driver of this hippocampal pathology, but the mechanisms involved in recruiting these cells to the brain were unclear., Methods: Chemokine expression levels in the hippocampus were assessed by microarray, ELISA, RT-PCR, and immunofluorescence. Monocyte infiltration during acute TMEV infection was measured by flow cytometry. CCL2 levels were manipulated by immunodepletion and by specific removal from neurons in mice generated by crossing a line expressing the Cre recombinase behind the synapsin promoter to animals with floxed CCL2., Results: Inoculation of the brain with TMEV induced hippocampal production of the proinflammatory chemokine CCL2 that peaked at 6 h postinfection, whereas inoculation with UV-inactivated TMEV did not elicit this response. Immunofluorescence revealed that hippocampal neurons expressed high levels of CCL2 at this timepoint. Genetic deletion of CCR2 and systemic immunodepletion of CCL2 abrogated or blunted the infiltration of inflammatory monocytes into the brain during acute infection. Specific genetic deletion of CCL2 from neurons reduced serum and hippocampal CCL2 levels and inhibited inflammatory monocyte infiltration into the brain., Conclusions: We conclude that intracranial inoculation with infectious TMEV rapidly induces the expression of CCL2 in neurons, and this cellular source is necessary for CCR2-dependent infiltration of inflammatory monocytes into the brain during the most acute stage of encephalitis. These findings highlight a unique role for neuronal production of chemokines in the initiation of leukocytic infiltration into the infected central nervous system.

Seizure disorders are often associated with infectious etiologies. Infection, via the intracerebral (i.c.) route, of C57BL/6J mice with the Daniels (DA) strain of Theiler's murine encephalomyelitis virus (TMEV) results in approximately 50% of the mice developing acute behavioral seizures. TMEV-DA is the wild-type strain of the virus that replicates within the parenchyma of the brain. A variant of TMEV-DA, TMEV-H101, does not replicate within the parenchyma of the brain. However, infection with TMEV-H101 via the i.c. route still results in approximately 40% of the mice developing acute behavioral seizures. Infiltrating macrophages producing interleukin-6 (IL-6) have been implicated in the induction of acute seizures following TMEV-DA infection. We examined macrophage infiltration and microglial activation within the brain and cytokine levels in the periphery in mice infected with TMEV-DA or TMEV-H101 and assessed the effects of the addition of recombinant IL-6 to the periphery in wild-type and IL-6 knockout mice infected with TMEV-DA. We found that pathologic levels of IL-6 in the periphery may play a role in the development of seizures when viral replication within the brain is limited. Examination of the role played by the peripheral immune system in the development of seizures/epilepsy in the TMEV-induced seizure model, the first viral infection driven model for epilepsy, could lead to the elucidation of novel therapeutics.

Viral infections can give rise to secondary bacterial infections. In the present study, we examined the role of invariant natural killer T (iNKT) cells in lipopolysaccharide (LPS)-induced lethal shock during encephalomyocarditis virus (EMCV) infection. Wild-type (WT) mice and Jα18 gene knockout (Jα18 KO) mice were inoculated with EMCV, 5days prior to challenging with LPS. The survival rate of Jα18 KO mice subjected to EMCV and LPS was significantly higher than that of WT mice. TNF-α and nitric oxide (NO) production were increased in WT mice, than that in Jα18 KO mice, after the administration of EMCV and LPS. EMCV infection increased the number of iNKT cells and IFN-γ production by iNKT cells in WT mice. Moreover, EMCV infection enhanced the expression of Toll-like receptor 4 (TLR4) in the lung and spleen. IFN-γ also increased the expression of TLR4 in splenocytes. These findings indicated that EMCV infection activated iNKT cells, and IFN-γ secreted from the iNKT cells up-regulated the expression of TLR4 in various tissues. As a result, EMCV-infected mice were susceptible to LPS and easily developed the lethal shock. In conclusion, iNKT cells were involved in the development of LPS-induced lethal shock during EMCV infection., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

LGP2 and MDA5 cooperate to detect viral RNA in the cytoplasm of Picornavirus-infected cells and activate innate immune responses. To further define regulatory components of RNA recognition by LGP2/MDA5, a yeast two-hybrid screen was used to identify LGP2-interacting proteins. The screening has identified the TAR-RNA binding protein (TRBP), which is known to be an essential factor for RNA interference (RNAi). Immuno-precipitation experiments demonstrated that TRBP interacted specifically with LGP2 but not with related RIG-I-like receptors, RIG-I or MDA5. siRNA knockdown experiments indicate that TRBP is important for Cardiovirus-triggered interferon responses, but TRBP is not involved in Sendai virus-triggered interferon response that is mediated mainly by RIG-I. To support functional interaction with LGP2, overexpressed TRBP increased Cardiovirus-triggered interferon promoter activity only when LGP2 and MDA5 are co-expressed but not MDA5 alone. Together, our findings illustrate a possible connection between an RNAi-regulatory factor and antiviral RNA recognition that is specifically required for a branch of the virus induced innate immune response., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The VP1 subunit of the picornavirus capsid is the major antigenic determinant and mediates host cell attachment and virus entry. To investigate the localisation of Theiler's murine encephalomyelitis virus (TMEV) VP1 during infection, a bioinformatics approach was used to predict a surface-exposed, linear epitope region of the protein for subsequent expression and purification. This region, comprising the N-terminal 112 amino acids of the protein, was then used for rabbit immunisation, and the resultant polyclonal antibodies were able to recognise full length VP1 in infected cell lysates by Western blot. Following optimisation, the antibodies were used to investigate the localisation of VP1 in relation to Hsp90 in infected cells by indirect immunofluorescence and confocal microscopy. At 5h post infection, VP1 was distributed diffusely in the cytoplasm with strong perinuclear staining but was absent from the nucleus of all cells analysed. Dual-label immunofluorescence using anti-TMEV VP1 and anti-Hsp90 antibodies indicated that the distribution of both proteins colocalised in the cytoplasm and perinuclear region of infected cells. This is the first report describing the localisation of TMEV VP1 in infected cells, and the antibodies produced provide a valuable tool for investigating the poorly understood mechanisms underlying the early steps of picornavirus assembly., (Copyright © 2016 Elsevier B.V. All rights reserved.)

4.5SH and 4.5SI RNA are two abundant small non-coding RNAs specific for several related rodent families including Muridae. These RNAs have a number of common characteristics such as the short length (about 100nt), transcription by RNA polymerase III, and origin from Short Interspersed Elements (SINEs). However, their stabilities in cells substantially differ: the half-life of 4.5SH RNA is about 20min, while that of 4.5SI RNA is 22h. Here we studied the influence of cell stress such as heat shock or viral infection on these two RNAs. We found that the level of 4.5SI RNA did not change in stressed cells; whereas heat shock increased the abundance of 4.5SH RNA 3.2-10.5 times in different cell lines; and viral infection, 5 times. Due to the significant difference in the turnover rates of these two RNAs, a similar activation of their transcription by heat shock increases the level of the short-lived 4.5SH RNA and has minor effect on the level of the long-lived 4.5SI RNA. In addition, the accumulation of 4.5SH RNA results not only from the induction of its transcription but also from a substantial retardation of its decay. To our knowledge, it is the first example of a short-lived non-coding RNA whose elongated lifetime contributes significantly to its accumulation in stressed cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Neurologic complications associated with viral encephalitis, including seizures and cognitive impairment, are a global health issue, especially in children. We previously showed that hippocampal injury during acute picornavirus infection in mice is associated with calpain activation and is the result of neuronal death triggered by brain-infiltrating inflammatory monocytes. We therefore hypothesized that treatment with a calpain inhibitor would protect neurons from immune-mediated bystander injury. C57BL/6J mice infected with the Daniel's strain of Theiler's murine encephalomyelitis virus were treated with the FDA-approved drug ritonavir using a dosing regimen that resulted in plasma concentrations within the therapeutic range for calpain inhibition. Ritonavir treatment significantly reduced calpain activity in the hippocampus, protected hippocampal neurons from death, preserved cognitive performance, and suppressed seizure escalation, even when therapy was initiated 36 hours after disease onset. Calpain inhibition by ritonavir may be a powerful tool for preserving neurons and cognitive function and preventing neural circuit dysregulation in humans with neuroinflammatory disorders.

The present study was carried out to clarify the mechanisms of EMC virus-induced sialodacryoadenitis in mice during the acute phase infection focusing on the activation of type I interferon (IFN) signaling in the parotid and exorbital lachrymal glands. In the parotid gland, a few apoptotic acinar cells were detected at 2days post inoculation (DPI). The ratio of apoptotic acinar cells increased at 3 and 4DPI. On the other hand, in the exorbital lachrymal gland, apoptosis of acinar cells and infiltration of inflammatory cells mainly composed of mononuclear cells started at 3DPI, and prominent acinar cell damage developed at 4DPI. Viral RNA was detected at 3 and 4DPI in both glands and the expression level was higher in the exorbital lachrymal gland than in the parotid gland. The up-regulation of IFN-stimulated genes (ISGs), such as Irf7, Pkr and Oas, was quickly induced at 2DPI in the parotid gland, and this probably contributed to suppress viral replication and to eliminate affected cells by apoptosis. In the exorbital lachrymal gland, the expression levels of ISGs mRNAs were not elevated at 2DPI, suggesting no induction of an effective anti-viral response such as apoptosis at this time point. In the exorbital lachrymal gland, the mRNA expression of IFN beta and IFN alpha (type I IFNs) was weak- to strong-positive at 1DPI, and became negative at 2DPI. The weak- to strong-positive expression of IFNs at 1DPI is likely related to the abrupt viral replication and pathological changes in the exorbital lachrymal gland through activating the negative feedback regulation that depressed the IFN signaling cascade at 2DPI. In conclusion, the present study showed the changes in factors involved in the activation of type I IFN signaling cascade in the parotid and exorbital lachrymal glands and their differences between the two glands during the acute phase of EMC virus infection in mice., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Microglia and macrophages play a central role for demyelination in Theiler's murine encephalomyelitis (TME) virus infection, a commonly used infectious model for chronic-progressive multiple sclerosis. In order to determine the dynamic changes of microglia/macrophage polarization in TME, the spinal cord of Swiss Jim Lambert (SJL) mice was investigated by gene expression profiling and immunofluorescence. Virus persistence and demyelinating leukomyelitis were confirmed by immunohistochemistry and histology. Electron microscopy revealed continuous myelin loss together with abortive myelin repair during the late chronic infection phase indicative of incomplete remyelination. A total of 59 genes out of 151 M1- and M2-related genes were differentially expressed in TME virus-infected mice over the study period. The onset of virus-induced demyelination was associated with a dominating M1 polarization, while mounting M2 polarization of macrophages/microglia together with sustained prominent M1-related gene expression was present during the chronic-progressive phase. Molecular results were confirmed by immunofluorescence, showing an increased spinal cord accumulation of CD16/32(+) M1-, arginase-1(+) M2- and Ym1(+) M2-type cells associated with progressive demyelination. The present study provides a comprehensive database of M1-/M2-related gene expression involved in the initiation and progression of demyelination supporting the hypothesis that perpetuating interaction between virus and macrophages/microglia induces a vicious circle with persistent inflammation and impaired myelin repair in TME., (© 2014 International Society of Neuropathology.)

Cardioviruses, including encephalomyocarditis virus (EMCV) and the human Saffold virus, are small non-enveloped viruses belonging to the Picornaviridae, a large family of positive-sense RNA [(+)RNA] viruses. All (+)RNA viruses remodel intracellular membranes into unique structures for viral genome replication. Accumulating evidence suggests that picornaviruses from different genera use different strategies to generate viral replication organelles (ROs). For instance, enteroviruses (e.g. poliovirus, coxsackievirus, rhinovirus) rely on the Golgi-localized phosphatidylinositol 4-kinase III beta (PI4KB), while cardioviruses replicate independently of the kinase. By which mechanisms cardioviruses develop their ROs is currently unknown. Here we show that cardioviruses manipulate another PI4K, namely the ER-localized phosphatidylinositol 4-kinase III alpha (PI4KA), to generate PI4P-enriched ROs. By siRNA-mediated knockdown and pharmacological inhibition, we demonstrate that PI4KA is an essential host factor for EMCV genome replication. We reveal that the EMCV nonstructural protein 3A interacts with and is responsible for PI4KA recruitment to viral ROs. The ensuing phosphatidylinositol 4-phosphate (PI4P) proved important for the recruitment of oxysterol-binding protein (OSBP), which delivers cholesterol to EMCV ROs in a PI4P-dependent manner. PI4P lipids and cholesterol are shown to be required for the global organization of the ROs and for viral genome replication. Consistently, inhibition of OSBP expression or function efficiently blocked EMCV RNA replication. In conclusion, we describe for the first time a cellular pathway involved in the biogenesis of cardiovirus ROs. Remarkably, the same pathway was reported to promote formation of the replication sites of hepatitis C virus, a member of the Flaviviridae family, but not other picornaviruses or flaviviruses. Thus, our results highlight the convergent recruitment by distantly related (+)RNA viruses of a host lipid-modifying pathway underlying formation of viral replication sites.

Changes in the expression of xCT, the specific subunit of system xc(-) or the cystine/glutamate antiporter, have been associated with several neurological disorders and system xc(-) was recently proposed as a potential target for the development of new treatment strategies for multiple sclerosis (MS). In this study we used Theiler's murine encephalomyelitis virus (TMEV) infection, both in vitro and in vivo, as a model to further evaluate the involvement of system xc(-) in MS. Protein levels of xCT, as well as activity of system xc(-) were unaffected in RAW264.7 macrophages after infection with the demyelinating DA strain of TMEV. Also, protein expression of xCT remained stable in spinal cord and brain of FVB mice 1-2 and 6 weeks after intracranial injection of the DA strain of TMEV. These results demonstrate that TMEV infection of macrophages or FVB mice has no effect on system xc(-) and as such cannot be used as a model to study the involvement of system xc(-) in MS., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Galectin-3 is a β-galactosidase-binding lectin which is important in cell proliferation and apoptotic regulation. Encephalomyocarditis virus (EMCV), which includes the Enterovirus genus, can cause not only acute myocarditis but also neuronal degeneration of central nervous system in various animals including mice. The pathophysiological role of galectin-3 in central nervous system following acute viral infection is not fully understood. The goal of this study is to determine the localization and time-course of galectin-3 expression after acute viral inoculation with EMCV. Galectin-3 is up-regulated in degenerated lesions of brain area including cerebellum, hippocampus, thalamus and cerebral hemisphere, 96 h after EMCV inoculation. At the same time, Iba-1 positive microglia was morphologically activated within and around the focus of infection. Interestingly, in cerebellum, the microlesions containing a few galectin-3 cells were detected in the immediate-early phase of infection, as early as 48 h after EMCV inoculation. Thus, our results indicate that galectin-3 expression may be a key mediator between viral infection and neuronal degeneration in central nervous system including cerebellum. Furthermore, detection of galectin-3 might be an early diagnostic method for neuronal degeneration after virus infection., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Viral double-stranded RNA (dsRNA) is the most important viral structure recognized by cytosolic pattern-recognition receptors of the innate immune system, and its recognition results in the activation of signaling cascades that stimulate the production of antiviral cytokines and apoptosis of infected cells. 14-3-3 proteins are ubiquitously expressed regulatory molecules that participate in a variety of cellular processes, and 14-3-3 protein-mediated signaling pathways are activated by cytoplasmic dsRNA in human keratinocytes. However, the functional role of 14-3-3 protein-mediated interactions during viral dsRNA stimulation has remained uncharacterized. Here, we used functional proteomics to identify proteins whose phosphorylation and interaction with 14-3-3 is modulated by dsRNA and to characterize the signaling pathways activated during cytosolic dsRNA-induced innate immune response in human HaCaT keratinocytes. Phosphoproteome analysis showed that several MAPK- and immune-response-related signaling pathways were activated after dsRNA stimulation. Interactome analysis identified RelA-associated inhibitor, high-mobility group proteins, and several proteins associated with host responses to viral infection as novel 14-3-3 target proteins. Functional studies showed that RelA-associated inhibitor regulated dsRNA-induced apoptosis and TNF production. Integrated network analyses of proteomic data revealed that sirtuin1 was a central molecule regulated by 14-3-3s during dsRNA stimulation. Further experiments showed that sirtuin 1 negatively regulated dsRNA-induced NFκB transcriptional activity, suppressed expression of antiviral cytokines, and protected cells from apoptosis in dsRNA-stimulated and encephalomyocarditis-virus-infected keratinocytes. In conclusion, our data highlight the importance of 14-3-3 proteins in antiviral responses and identify RelA-associated inhibitor and sirtuin 1 as novel regulators of antiviral innate immune responses., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Theiler's murine encephalomyelitis virus (TMEV) induces demyelination in susceptible strains of mice (SJL/J) through an immunopathological process that is mediated by CD4(+) Th1 T cell. These T cells are chemoattracted to the central nervous system by chemokines. Hence, in this study, we focused on the production of the chemokine "interferon-gamma-inducible protein 10 kDa," or IP-10/CXCL10, by cultured SJL/J mouse astrocytes infected with the BeAn strain of TMEV and its capacity to attract activated T cells. The analysis of the whole murine genome by DNA hybridization with cRNAs from mock- and TMEV-infected cultures revealed the upregulation of six sequences that potentially encode for CXCL10. This increased CXCL10 expression was validated by PCR and qPCR. The presence of this chemokine was further demonstrated by enzyme-linked immunoassay (ELISA). Significantly, astrocytes from BALB/c mice, a strain resistant to demyelination, did not produce CXCL10. The secreted CXCL10 was biologically active, inducing chemoattraction of activated lymphocytes. The inflammatory cytokines, IL-1α, IFN-γ, and TNF-α, were strong inducers of CXCL10 in astrocytes. Serum from TMEV-infected SJL/J but not BALB/c mice contains CXCL10, the levels of which peak at the onset of the clinical disease. Finally, this in vitro inflammation model was fully inhibited by 17β-estradiol and four selective estrogen receptor modulators, as demonstrated by ELISA and qPCR.

Unlabelled: Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are essential intracellular detectors of viral RNA. They contribute to the type I interferon (IFN) response that is crucial for host defense against viral infections. Given the potent antiviral and proinflammatory activities elicited by the type I IFNs, induction of the type I IFN response is tightly regulated. Members of the tripartite motif (TRIM) family of proteins have recently emerged as key regulators of antiviral immunity. We show that TRIM13, an E3 ubiquitin ligase, is expressed in immune cells and is upregulated in bone marrow-derived macrophages upon stimulation with inducers of type I IFN. TRIM13 interacts with MDA5 and negatively regulates MDA5-mediated type I IFN production in vitro, acting upstream of IFN regulatory factor 3. We generated Trim13(-/-) mice and show that upon lethal challenge with encephalomyocarditis virus (EMCV), which is sensed by MDA5, Trim13(-/-) mice produce increased amounts of type I IFNs and survive longer than wild-type mice. Trim13(-/-) murine embryonic fibroblasts (MEFs) challenged with EMCV or poly(I · C) also show a significant increase in beta IFN (IFN-β) levels, but, in contrast, IFN-β responses to the RIG-I-detected Sendai virus were diminished, suggesting that TRIM13 may play a role in positively regulating RIG-I function. Together, these results demonstrate that TRIM13 regulates the type I IFN response through inhibition of MDA5 activity and that it functions nonredundantly to modulate MDA5 during EMCV infection., Importance: The type I interferon (IFN) response is crucial for host defense against viral infections, and proper regulation of this pathway contributes to maintaining immune homeostasis. Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are intracellular detectors of viral RNA that induce the type I IFN response. In this study, we show that expression of the gene tripartite motif 13 (Trim13) is upregulated in response to inducers of type I IFN and that TRIM13 interacts with both MDA5 and RIG-I in vitro. Through the use of multiple in vitro and in vivo model systems, we show that TRIM13 is a negative regulator of MDA5-mediated type I IFN production and may also impact RIG-I-mediated type I IFN production by enhancing RIG-I activity. This places TRIM13 at a key junction within the viral response pathway and identifies it as one of the few known modulators of MDA5 activity., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Deletion of cellular isoform of prion protein (PrP(C)) increases neuronal predisposition to damage by modulating apoptosis and the negative consequences of oxidative stress. In vivo studies have demonstrated that PrP(C)-deficient mice are more prone to seizure, depression, and induction of epilepsy and experience extensive cerebral damage following ischemic challenge or viral infection. In addition, adenovirus-mediated overexpression of PrP(C) reduces brain damage in rat models of cerebral ischemia. In experimental autoimmune encephalomyelitis, PrP(C)-deficient mice reportedly have a more aggressive disease onset and less clinical improvement during the chronic phase than wild-type mice mice. In mice given oral dextran sulfate, PrP(C) has a potential protective role against inflammatory bowel disease. PrP(C)-deficient mice demonstrate significantly greater increases in blood glucose concentrations after intraperitoneal injection of glucose than wild-type mice. Further in vivo challenges to PrP gene-deficient models and conditional knockout models with siRNA and in vivo administration of PrP-ligating agents may assist in refining knowledge of the lymphoid function of PrP(C) and predicting the effects of anti-PrP treatment on the immune system. Together, these findings indicate that PrP(C) may have multiple neuroprotective and anti-inflammatory roles, which explains why this protein is so widely expressed., (© 2014 The Societies and Wiley Publishing Asia Pty Ltd.)

Indoleamine 2,3-dioxygenase1 (IDO1) is the rate-limiting enzyme in the kynurenine pathway that converts l-tryptophan to l-kynurenine. Encephalomyocarditis virus (EMCV) can cause acute myocarditis in various animals including mice. Previously, IDO1 has been reported to have an important immunomodulatory function in immune-related diseases. However, the pathophysiological roles of IDO1 following acute viral infection of central nervous system are not fully understood. We observed that acute EMCV infection leads to a highly reproducible neuronal degeneration in mouse cerebellum. The goal of this study is to determine tissue/cell-specific and time-dependent expressions of IDO1 during acute EMCV infection in mouse cerebellum. IDO1 was up-regulated in microglia, which was recognized to be activated morphologically and positive for ionized calcium-binding adapter molecule 1 (Iba-1), a protein expressed in microglia, within EMCV-induced cerebellar lesions showing neuronal degeneration although the very weak expression of IDO1 is detected only in cytoplasm of Purkinje cells. No GFAP immunostaining was observed in EMCV-induced cerebellar lesions although many reactive astrocytes surrounding the lesions showed strongly positive immunostaining for GFAP 10 days after the viral inoculation. Thus, IDO1 expression may affect EMCV-induced neuronal degeneration in cerebellum., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The Theiler's murine encephalomyelitis virus (TMEV) leader (L) protein zinc-finger domain was mutated to study its role in cell death in infection of the murine macrophage cell line M1-D, revealing that an intact zinc-finger domain is required for full apoptotic activity. A functional L zinc-finger domain was also required for activation of p38 MAPK that results in phosphorylation and activation of p53, and in turn, alteration of the conformation of the anti-apoptotic proteins Puma and Mcl-1, leading to the release of pro-apoptotic Bax and apoptosis through the intrinsic pathway. TMEV infection also inhibits host protein synthesis, a stress shown by others to induce apoptosis. Since inhibition of host protein synthesis follows rather than precedes activation of MKK3/6 and p38, it seems less likely that it triggers apoptosis in infected cells. Finally, we showed that the levels of reactive oxygen species following infection were consistent with apoptotic rather than necrotic cell death. Thus, these experiments support an important role for the TMEV L protein zinc-finger domain in apoptosis in an infected murine macrophage line., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Viral myocarditis is among the most common causes of heart failure in children and young adults. The RNA helicase melanoma differentiation-associated gene-5 (MDA5) is critical for host antiviral responses against members of the Picornaviridae family, such as encephalomyocarditis virus (EMCV). MDA5-knockout mice are highly susceptible to EMCV infection and develop significant myocardial injury and left ventricular dysfunction. However, the mechanisms by which MDA5 signaling within cardiac myocytes contributes to the host response against viral infection have not been defined., Methods and Results: We generated cardiac-specific MDA5 transgenic (alpha-myosin heavy chain [αMHC]-MDA5) mice. These mice showed increased baseline cardiac expression of antiviral cytokines and increased cellular infiltration but no alterations in cardiac function and structure. αMHC-MDA5 mice were less susceptible to EMCV infection and had a significantly lower cardiac viral load compared with littermate control mice. The severity of myocarditis, prevalence of cardiac myocyte apoptosis, and cleavage of caspase 3 after EMCV infection were attenuated in αMHC-MDA5 mice. Furthermore, αMHC-MDA5 mice were protected against EMCV-induced myocardial dysfunction., Conclusions: Our data suggest that myocardial MDA5 may be a key molecule in protecting the heart from direct viral injury and myocardial dysfunction.

Theiler's virus is a neurotropic picornavirus responsible for chronic infections of the central nervous system. The establishment of a persistent infection and the subsequent demyelinating disease triggered by the virus depend on the expression of L*, a viral accessory protein encoded by an alternative open reading frame of the virus. We discovered that L* potently inhibits the interferon-inducible OAS/RNase L pathway. The antagonism of RNase L by L* was particularly prominent in macrophages where baseline oligoadenylate synthetase (OAS) and RNase L expression levels are elevated, but was detectable in fibroblasts after IFN pretreatment. L* mutations significantly affected Theiler's virus replication in primary macrophages derived from wild-type but not from RNase L-deficient mice. L* counteracted the OAS/RNase L pathway through direct interaction with the ankyrin domain of RNase L, resulting in the inhibition of this enzyme. Interestingly, RNase L inhibition was species-specific as Theiler's virus L* protein blocked murine RNase L but not human RNase L or RNase L of other mammals or birds. Direct RNase L inhibition by L* and species specificity were confirmed in an in vitro assay performed with purified proteins. These results demonstrate a novel viral mechanism to elude the antiviral OAS/RNase L pathway. By targeting the effector enzyme of this antiviral pathway, L* potently inhibits RNase L, underscoring the importance of this enzyme in innate immunity against Theiler's virus.

Autophagy is a tightly regulated mechanism that mediates sequestration, degradation, and recycling of cellular proteins, organelles, and pathogens. Several proteins associated with autophagy regulate host responses to viral infections. Ribonuclease L (RNase L) is activated during viral infections and cleaves cellular and viral single-stranded RNAs, including rRNAs in ribosomes. Here we demonstrate that direct activation of RNase L coordinates the activation of c-Jun N-terminal kinase (JNK) and double-stranded RNA-dependent protein kinase (PKR) to induce autophagy with hallmarks as accumulation of autophagic vacuoles, p62(SQSTM1) degradation and conversion of Microtubule-associated Protein Light Chain 3-I (LC3-I) to LC3-II. Accordingly, treatment of cells with pharmacological inhibitors of JNK or PKR and mouse embryonic fibroblasts (MEFs) lacking JNK1/2 or PKR showed reduced autophagy levels. Furthermore, RNase L-induced JNK activity promoted Bcl-2 phosphorylation, disrupted the Beclin1-Bcl-2 complex and stimulated autophagy. Viral infection with Encephalomyocarditis virus (EMCV) or Sendai virus led to higher levels of autophagy in wild-type (WT) MEFs compared with RNase L knock out (KO) MEFs. Inhibition of RNase L-induced autophagy using Bafilomycin A1 or 3-methyladenine suppressed viral growth in initial stages; in later stages autophagy promoted viral replication dampening the antiviral effect. Induction of autophagy by activated RNase L is independent of the paracrine effects of interferon (IFN). Our findings suggest a novel role of RNase L in inducing autophagy affecting the outcomes of viral pathogenesis.

The present in vitro study aimed to define the involvement of astrocytes and microglia in the initial inflammatory response of Theiler's murine encephalomyelitis (TME), a virus-induced mouse model of multiple sclerosis, and whether intralesional microglia exert pro- (M1) or anti-inflammatory (M2) effects following TME virus (TMEV) infection. Therefore astrocytes and microglia were purified from neonatal murine brains and inoculated either with TMEV or mock-solution. Gene expression of IL-1, IL-2, IL-10, IL-12, TNF, TNF receptors (TNFR1, TNFR2), TGFβ1, IFNγ and transcription factors NF-κB (p50, p65) and AP-1 (c-jun, c-fos) were quantified using RT-qPCR at 6, 48, and 240h post infection (hpi). In addition, IL-1, IL-10, IL-12, TNF and TGFβ1 mRNA transcripts were investigated at 168 hpi in TMEV- and mock-infected SJL/J mice. Overall in vitro astrocytes showed a significant higher amount of viral RNA compared to microglia. In addition, TMEV-infected astrocytes showed higher numbers of IL-1, IL-12 and TNF transcripts at 48 hpi. In microglia high IL-10 and low IL-12 mRNA levels were detected at 48 hpi, while the opposite was the case at 240 hpi. In addition, TNF mRNA was increased in microglia at 240 hpi. In addition, the observed up-regulation of IL-1, IL-12 and IL-10 in the early phase of TME in vivo substantiates the relevance of these cytokines during the disease induction. Summarized data indicate that TMEV infection of microglia induces a switch from the anti-inflammatory (M2) during the early phase to the pro-inflammatory (M1) phenotype in the later phase of the infection. The simultaneous expression of TNF and its receptors by both cell types might generate autocrine feedback loops possibly associated with pro-inflammatory actions of astrocytes via TNFR1., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This paper reports the upregulation of the gene coding for the apoptosis regulator family member "surviving" in SJL/J mouse brain astrocyte cultures infected with the BeAn strain of TMEV. cRNA from mock- and TMEV-infected SJL/J astrocytes was hybridised to an Affymetrix whole murine genome DNA microarray. Analysis revealed the upregulation of two sequences coding for the survivin protein in infected cells; this was confirmed by RT-PCR and qPCR. Western blotting showed an increase in the synthesis of survivin and caspase-3 after infection. Unexpectedly, no enzymatic activity was detected in BeAn-infected cell lysates in caspase-3-specific colorimetric assays. Cross-linking experiments showed survivin and caspase-3 to exist as a complex containing one molecule of caspase-3 (17 kDa) and one of either 16 kDa or 14 kDa survivin. The neutralization of caspase-3 by survivin-containing lysates was demonstrated using recombinant caspase-3. Brains from TMEV-infected mice, but not from naïve mice, contained survivin mRNA during the acute phase of encephalitis. The present results suggest that astrocytes infected by the BeAn strain do not undergo apoptosis due to the production of survivin.

Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease characterized by myelin and axonal pathology. In a viral model of MS, we tested whether axonopathy initiation and development are based on an impaired transport of neurofilaments. Spinal cords of Theiler's murine encephalomyelitis virus (TMEV)-infected and mock-infected mice and TMEV infected neuroblastoma N1E-115 cells were analyzed by microarray analysis, light microscopy and electron and laser confocal microscopy. In vivo axonal accumulation of non-phosphorylated neurofilaments after TMEV infection revealed a temporal development caused by the impairments of the axonal traffic consisting of the downregulation of kinesin family member 5A, dynein cytoplasmic heavy chain 1, tau-1 and β-tubulin III expression. In addition, alterations of the protein metabolism were also noticed. In vitro, the TMEV-infected N1E-115 cells developed tandem-repeated swellings similar to in vivo alterations. Furthermore, the hypothesis of an underlying axonal self-destruction program involving nicotinamide adenine dinucleotide depletion was supported by molecular findings. The obtained data indicate that neurofilament accumulation in TME is mainly the result of dysregulation of their axonal transport machinery and impairment of neurofilament phosphorylation and protein metabolism. The present findings allow a more precise understanding of the complex interactions responsible for initiation and development of axonopathies in inflammatory degenerative diseases., (© 2011 The Authors; Brain Pathology © 2011 International Society of Neuropathology.)

Theiler's murine encephalomyelitis virus (TMEV) results in a persistent central nervous system infection (CNS) and immune-mediated demyelination in mice. TMEV largely persists in macrophages (Ms) in the CNS, and infected Ms in vitro undergo apoptosis, whereas the infection of other rodent cells produces necrosis. We have found that necrosis is the dominant form of cell death in BeAn virus-infected BHK-21 cells but that ~20% of cells undergo apoptosis. Mcl-1 was highly expressed in BHK-21 cells, and protein levels decreased upon infection, consistent with onset of apoptosis. In infected BHK-21 cells in which Mcl-1 expression was knocked down using silencing RNAs there was a 3-fold increase in apoptotic cell death compared to parental cells. The apoptotic program switched on by BeAn virus is similar to that in mouse Ms, with hallmarks of activation of the intrinsic apoptotic pathway in a tumor suppressor protein p53-dependent manner. Infection of stable Mcl-1-knockdown cells led to restricted virus titers and increased physical to infectious particle (PFU) ratios, with additional data suggesting that a late step in the viral life cycle after viral RNA replication, protein synthesis, and polyprotein processing is affected by apoptosis. Together, these results indicate that Mcl-1 acts as a critical prosurvival factor that protects against apoptosis and allows high yields of infectious virus in BHK-21 cells.

Nod-like receptors (NLRs) comprise a large family of intracellular pattern- recognition receptors. Members of the NLR family assemble into large multiprotein complexes, termed the inflammasomes. The NLR family, pyrin domain-containing 3 (NLRP3) is triggered by a diverse set of molecules and signals, and forms the NLRP3 inflammasome. Recent studies have indicated that both DNA and RNA viruses stimulate the NLRP3 inflammasome, leading to the secretion of interleukin 1 beta (IL-1β) and IL-18 following the activation of caspase-1. We previously demonstrated that the proton-selective ion channel M2 protein of influenza virus activates the NLRP3 inflammasome. However, the precise mechanism by which NLRP3 recognizes viral infections remains to be defined. Here, we demonstrate that encephalomyocarditis virus (EMCV), a positive strand RNA virus of the family Picornaviridae, activates the NLRP3 inflammasome in mouse dendritic cells and macrophages. Although transfection with RNA from EMCV virions or EMCV-infected cells induced robust expression of type I interferons in macrophages, it failed to stimulate secretion of IL-1β. Instead, the EMCV viroporin 2B was sufficient to cause inflammasome activation in lipopolysaccharide-primed macrophages. While cells untransfected or transfected with the gene encoding the EMCV non-structural protein 2A or 2C expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells transfected with the gene encoding the EMCV 2B or influenza virus M2 protein. 2B proteins of other picornaviruses, poliovirus and enterovirus 71, also caused the NLRP3 redistribution. Elevation of the intracellular Ca(2+) level, but not mitochondrial reactive oxygen species and lysosomal cathepsin B, was important in EMCV-induced NLRP3 inflammasome activation. Chelation of extracellular Ca(2+) did not reduce virus-induced IL-1β secretion. These results indicate that EMCV activates the NLRP3 inflammasome by stimulating Ca(2+) flux from intracellular storages to the cytosol, and highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation.

Secondary bacterial infection in humans is one of the pathological conditions requiring clinical attention. In this study, we examined the effect of lipopolysaccharide (LPS) on encephalomyocarditis virus (EMCV) infected mice. All mice inoculated with EMCV at 5 days before LPS challenge died within 24 h. LPS-induced TNF-α mRNA expression was significantly increased in the brain and heart at 5 days after EMCV infection. CD11b(+)/TLR4(+) cell population in the heart was remarkably elevated at 5 days after EMCV infection, and sorted CD11b(+) cells at 5 days after EMCV infection produced a large amount of TNF-α on LPS stimulation in vivo and in vitro. In conclusion, we found that the infiltration of CD11b(+) cells into infected organs is involved in the subsequent LPS-induced lethal shock in viral encephalomyocarditis. This new experimental model can help define the mechanism by which secondary bacterial infection causes a lethal shock in viral encephalomyocarditis.

The single-stranded RNA encephalomyocarditis virus (EMCV) can replicate in the central nervous system (CNS) and lead to prominent brain lesions in the stratum pyramidale hippocampus and the stratum granulosum cerebelli. Activated microglia cells infected by EMCV produce a massive burst of reactive oxygen species (ROS) via NADPH oxidase 2 (NOX2) activation, leading to neuronal death. Balancing this effect is mechanisms by which ROS are eliminated from the CNS. Cellular prion protein (PrP(C)) plays an important antioxidant role and contributes to cellular defense against EMCV infection. This review introduces recent knowledge on brain injury induced by EMCV infection via ROS generation as well as the involvement of various mediators and regulators in the pathogenesis.

Background: We have previously shown that toll-like receptor 3 (TLR3)-mediated signaling plays an important role in the induction of innate cytokine responses to Theiler's murine encephalomyelitis virus (TMEV) infection. In addition, cytokine levels produced after TMEV infection are significantly higher in the glial cells of susceptible SJL mice compared to those of resistant C57BL/6 mice. However, it is not known whether TLR3-mediated signaling plays a protective or pathogenic role in the development of demyelinating disease., Methods: SJL/J and B6;129S-Tlr3tm1Flv/J (TLR3KO-B6) mice, and TLR3KO-SJL mice that TLR3KO-B6 mice were backcrossed to SJL/J mice for 6 generations were infected with Theiler's murine encephalomyelitis virus (2 × 105 PFU) with or without treatment with 50 μg of poly IC. Cytokine production and immune responses in the CNS and periphery of infected mice were analyzed., Results: We investigated the role of TLR3-mediated signaling in the protection and pathogenesis of TMEV-induced demyelinating disease. TLR3KO-B6 mice did not develop demyelinating disease although they displayed elevated viral loads in the CNS. However, TLR3KO-SJL mice displayed increased viral loads and cellular infiltration in the CNS, accompanied by exacerbated development of demyelinating disease, compared to the normal littermate mice. Late, but not early, anti-viral CD4+ and CD8+ T cell responses in the CNS were compromised in TLR3KO-SJL mice. However, activation of TLR3 with poly IC prior to viral infection also exacerbated disease development, whereas such activation after viral infection restrained disease development. Activation of TLR3 signaling prior to viral infection hindered the induction of protective IFN-γ-producing CD4+ and CD8+ T cell populations. In contrast, activation of these signals after viral infection improved the induction of IFN-γ-producing CD4+ and CD8+ T cells. In addition, poly IC-pretreated mice displayed elevated PDL-1 and regulatory FoxP3+ CD4+ T cells in the CNS, while poly IC-post-treated mice expressed reduced levels of PDL-1 and FoxP3+ CD4+ T cells., Conclusions: These results suggest that TLR3-mediated signaling during viral infection protects against demyelinating disease by reducing the viral load and modulating immune responses. In contrast, premature activation of TLR3 signal transduction prior to viral infection leads to pathogenesis via over-activation of the pathogenic immune response.

Theiler's murine encephalomyelitis virus (TMEV) is a positive-sense RNA virus belonging to the Cardiovirus genus in the family Picornaviridae. In addition to other host cellular factors and pathways, picornaviruses utilise heat shock proteins (Hsps) to facilitate their propagation in cells. This study investigated the localisation of Hsps 70 and 90 in TMEV-infected BHK-21 cells by indirect immunofluorescence and confocal microscopy. The effect of Hsp90 inhibitors novobiocin (Nov) and geldanamycin (GA) on the development of cytopathic effect (CPE) induced by infection was also examined. Hsp90 staining was uniformly distributed in the cytoplasm of uninfected cells but was found concentrated in the perinuclear region during late infection where it overlapped with the signal for non-structural protein 2C within the viral replication complex. Hsp70 redistributed into the vicinity of the viral replication complex during late infection, but its distribution did not overlap with that of 2C. Inhibition of Hsp90 by GA and Nov had a negative effect on virus growth over a 48-h period as indicated by no observable CPE in treated compared to untreated cells. 2C was detected by Western analysis of GA-treated infected cell lysates at doses between 0.01 and 0.125 μM, suggesting that processing of viral precursors was not affected in the presence of this drug. In contrast, 2C was absent in cell lysates of Nov-treated cells at doses above 10 μM, although CPE was evident 48 hpi. This is the first study describing the dynamic behaviour of Hsps 70 and 90 in TMEV-infected cells and to identify Hsp90 as an important host factor in the life cycle of this virus.

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) serves as virus-induced model of chronic progressive multiple sclerosis. Infection of susceptible SJL/J mice leads to life-long CNS virus persistence and a progressive autoimmune demyelinating disease mediated by myelin-specific T cells activated via epitope spreading. In contrast, virus is rapidly cleared by a robust CTL response in TMEV-IDD-resistant C57BL/6 mice. We investigated whether differential induction of regulatory T cells (Tregs) controls susceptibility to TMEV-IDD. Infection of disease-susceptible SJL/J, but not B6 mice, leads to rapid activation and expansion of Tregs resulting in an unfavorable CNS ratio of Treg:Teffector cells. In addition, anti-CD25-induced inactivation of Tregs in susceptible SJL/J, but not resistant B6, mice results in significantly decreased clinical disease concomitant with enhanced anti-viral CD4(+), CD8(+) and antibody responses resulting in decreased CNS viral titers. This is the first demonstration that virus-induced Treg activation regulates susceptibility to autoimmune disease differentially in susceptible and resistant strains of mice and provides a new mechanistic explanation for the etiology of infection-induced autoimmunity., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Cardioviruses disrupt nucleocytoplasmic transport through the activity of their leader (L) protein. We have shown that hyperphosphorylation of nuclear pore proteins (nucleoporins or Nups), including Nup62, Nup153, and Nup214, is central to this L protein function and requires one or more cytosolic kinases. In this study, potential cellular enzymes involved in encephalomyocarditis virus (EMCV) L-directed Nup phosphorylation were screened with a panel of specific, cell-permeating kinase inhibitors. Extracellular signal-regulated receptor kinase (ERK) and p38 mitogen-activated protein kinase inhibitors (U0126 and SB203580) were sufficient to block Nup hyperphosphorylation in EMCV-infected or L-expressing cells. Recombinant L alone, in the absence of infection, triggered activation of ERK and p38, independent of their upstream signaling cascades. Conserved residues within the L zinc finger (Cys(19)) and acidic domain (Asp(48),(51),(52),(55)) were essential for this activation and for the phosphorylation of Nups, suggesting that the phenomena are linked. Analysis of the hyperphosphorylated Nup species revealed only phosphoserine and phosphothreonine residues. The sizes of the tryptic phosphopeptides derived from Nup62 were compatible with sites in the Phe/Gly repeat domain which display common consensus sequences for ERK and p38 substrates. The results provide strong evidence that ERK and p38 are the probable effector kinases required for L-dependent inhibition of nuclear trafficking.

The promyelocytic leukemia (PML) protein is expressed in the diffuse nuclear fraction of the nucleoplasm and in matrix-associated structures, known as nuclear bodies (NBs). PML NB formation requires the covalent modification of PML to SUMO. The noncovalent interactions of SUMO with PML based on the identification of a SUMO-interacting motif within PML seem to be required for further recruitment within PML NBs of SUMOylated proteins. RNA viruses whose replication takes place in the cytoplasm and is inhibited by PML have developed various strategies to counteract the antiviral defense mediated by PML NBs. We show here that primary fibroblasts derived from PML knockout mice are more sensitive to infection with encephalomyocarditis virus (EMCV), suggesting that the absence of PML results in an increase in EMCV replication. Also, we found that EMCV induces a decrease in PML protein levels both in interferon-treated cells and in PMLIII-expressing cells. Reduction of PML was carried out by the EMCV 3C protease. Indeed, at early times postinfection, EMCV induced PML transfer from the nucleoplasm to the nuclear matrix and PML conjugation to SUMO-1, SUMO-2, and SUMO-3, leading to an increase in PML body size where the viral protease 3C and the proteasome component were found colocalizing with PML within the NBs. This process was followed by PML degradation occurring in a proteasome- and SUMO-dependent manner and did not involve the SUMO-interacting motif of PML. Together, these findings reveal a new mechanism evolved by EMCV to antagonize the PML pathway in the interferon-induced antiviral defense.

Substance P (SP) is widely expressed in the central nervous system and in peripheral tissues such as myocardial nerves. We examined SP in viral myocarditis in mice induced by encephalomyocarditis virus (EMCV). Localization of SP in the hearts was examined immunohistochemically, and concentrations of SP in hearts and sera were measured by enzyme immunoassay. Substance P levels and density of SP-containing cells in murine hearts on day 6 after EMCV inoculation were decreased compared with those in normal controls. There was a negative correlation between SP levels in the hearts and ratio of heart weight to body weight of the mice at 6 days. Circulating SP levels were decreased in mice on day 6 after EMCV inoculation, and further decreased on day 14. Substance P in hearts and sera is decreased in viral myocarditis in mice, suggesting that SP may play a role in the pathogenesis of viral myocarditis, and that interaction of the neuropeptide nervous system and mast-cell immune system is important in the pathogenesis of viral myocarditis.

Exposure to tobacco smoke is known to have deleterious cardiovascular effects. In this study, we tested whether exposure to tobacco smoke exacerbates the severity of viral myocarditis in mice. Viral myocarditis was generated in 4-week-old male BALB/c mice by injection of Encephalomyocarditis virus (EMCV). Four groups were studied: (1) control (C, no smoke and no virus); (2) smoke only (S, exposure to cigarette smoke for 90 min/day for 15 days); (3) virus only (V); and (4) exposure to smoke for 5 days before plus 10 days following virus injection (S+V). We found that viral inoculation preceded by smoke exposure increased mortality more than twofold compared with virus inoculation alone. In addition, the mRNA level of atrial natriuretic factor was significantly higher in S+V than among any of the other 3 groups. Virus injection significantly decreased cardiac function compared with controls, with further deterioration observed in the S+V group. We also observed a significantly increased rate of apoptosis, with an increased activation of apoptosis-inducing factor in hearts exposed to S+V compared with those exposed to V alone. Our results suggest that preexposure to smoke significantly exacerbates the severity of viral myocarditis, likely through increased viral load and increased cardiomyocyte cell death.

Ligation of CD27, a member of the tumor necrosis factor (TNF) receptor family, by its ligand CD70 is thought to be important in T cell activation, expansion and survival, B cell activation, and NK cell activation. We examined the role of CD70 in Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) mice. Blocking of CD70 in effector phase by anti-CD70 monoclonal antibody (mAb) suppressed the development of TMEV-IDD. The number of IFN-gamma- or TNF-alpha-producing cells in the spleen and mRNA levels of IFN-gamma and TNF-alpha in spinal cord were decreased in mice treated with anti-CD70 mAb at the effector phase. In contrast, treatment with anti-CD70 mAb in induction phase failed to reduce these responses, compared to nonspecific IgG-treated control mice. These data suggest that CD70 is critically involved in the pathogenesis of TMEV-IDD and that antibodies against CD70 could be a novel therapeutic approach in the clinical treatment of demyelinating diseases such as human multiple sclerosis., (2009 Elsevier B.V. All rights reserved.)