Mouse Hepatitis Virus Research Articles

Chemokine CXCL10 and Coronavirus-Induced Neurologic Disease.

Chemokines (chemotactic cytokines) are involved in a wide variety of biological processes. Following microbial infection, there is often robust chemokine signaling elicited from infected cells, which contributes to both innate and adaptive immune responses that control growth of the invading pathogen. Infection of the central nervous system (CNS) by the neuroadapted John Howard Mueller (JHM) strain of mouse hepatitis virus (JHMV) provides an excellent example of how chemokines aid in host defense as well as contribute to disease. Intracranial inoculation of the CNS of susceptible mice with JHMV results in an acute encephalomyelitis characterized by widespread dissemination of virus throughout the parenchyma. Virus-specific T cells are recruited to the CNS, and control viral replication through release of antiviral cytokines and cytolytic activity. Sterile immunity is not acquired, and virus will persist primarily in white matter tracts leading to chronic neuroinflammation and demyelination. Chemokines are expressed and contribute to defense as well as chronic disease by attracting targeted populations of leukocytes to the CNS. The T cell chemoattractant chemokine CXCL10 (interferon-inducible protein 10 kDa, IP-10) is prominently expressed in both stages of disease, and serves to attract activated T and B lymphocytes expressing CXC chemokine receptor 3 (CXCR3), the receptor for CXCL10. Functional studies that have blocked expression of either CXCL10 or CXCR3 illuminate the important role of this signaling pathway in host defense and neurodegeneration in a model of viral-induced neurologic disease.

γδ T Cells Contribute to the Outcome of Murine Fulminant Viral Hepatitis via Effector Cytokines TNF-α and IFN-γ.

The mechanisms involved in virus-induced severe hepatitis have not been fully elucidated. In this study, we investigated the role of gamma delta T cell receptors (γδ) T cells in the pathogenesis of fulminant viral hepatitis (FVH) induced by murine hepatitis virus strain 3 (MHV-3). The model of FVH was established by intraperitoneal injection of MHV-3 into Balb/cJ mice. The survival days of mice, and the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were examined. The proportions of γδ T cells in blood, spleen and liver, and cytokines secreted by hepatic γδ T cells were analyzed by flow cytometry. The function of hepatic γδ T cells was examined by cytotoxicity assay. Balb/cJ mice died in 3 to 6 days post MHV-3 infection, with severe hepatic necrosis and significant augmentation of serum ALT and AST levels. The proportions of γδ T cells in blood, spleen and liver were significantly increased post MHV-3 infection, while those of the early activating molecule CD69-expressing γδ T cells and productions of cytokines tumor necrosis factor-alpha (TNF-α) and interferon-γ (IFN-γ) increased remarkably in the liver. These highly activated liver γδ T cells were cytotoxic to MHV-3-infected hepatocytes in vitro and this effect of liver γδ T cells against hepatocytes might involve the TNF-α and IFN-γ pathway. These results demonstrated that γδ T cells might contribute to the pathogenesis of MHV-3-induced FVH through the effector cytokines TNF-α and IFN-γ.

Nasal priming by a murine coronavirus provides protective immunity against lethal heterologous virus pneumonia.

The nasal mucosa is an important component of mucosal immunity. Immunogenic particles in inspired air are known to activate the local nasal mucosal immune system and can lead to sinonasal inflammation; however, little is known about the effect of this activation on the lung immune environment. Here, we showed that nasal inoculation of murine coronavirus (CoV) in the absence of direct lung infection primes the lung immune environment by recruiting activated monocytes (Ly6C+ inflammatory monocytes) and NK cells into the lungs. Unlike infiltration of these cells into directly infected lungs, a process that requires type I IFN signaling, nasally induced infiltration of Ly6C+ inflammatory monocytes into the lungs is IFN-I independent. These activated macrophages ingested antigen and migrated to pulmonary lymph nodes, and enhanced both innate and adaptive immunity after heterologous virus infection. Clinically, such nasal-only inoculation of MHV-1 failed to cause pneumonia but significantly reduced mortality and morbidity of lethal pneumonia caused by severe acute respiratory syndrome CoV (SARS-CoV) or influenza A virus. Together, the data indicate that the nose and upper airway remotely prime the lung immunity to protect the lungs from direct viral infections.

Identification of H209 as Essential for pH 8-Triggered Receptor-Independent Syncytium Formation by S Protein of Mouse Hepatitis Virus A59.

The spike glycoprotein (S) of murine coronavirus mouse hepatitis virus (MHV) strain A59 uses murine carcinoembryonic antigen-related cell adhesion molecule 1a as its receptor for cell entry, but S protein can also be triggered in the absence of receptor by pH 8.0 alone at 37°C. The mechanism by which conformational changes of this S glycoprotein can be triggered by pH 8.0 has not yet been determined. Here, we show that MHV-A59 S protein is triggered by pH 8.0 at 37°C to induce receptor-independent syncytium (RIS) formation on 293T cells, and that the conformational changes in S proteins triggered by pH 8.0 are very similar to those triggered by receptor binding. We systemically mutated each of 15 histidine residues in S protein and found that H209 is essential for pH 8.0-triggered RIS formation, while H179, H441, H643, and H759 also play important roles in this process. Replacement of H209 with Ala had no effect on receptor binding, but in murine 17Cl.1 cells mutant H209A MHV-A59 showed delayed growth kinetics and was readily outcompeted by wild-type virus when mixed together, indicating that the H209A mutation caused a defect in virus fitness. Finally, the H209A mutation significantly increased the thermostability of S protein in its prefusion conformation, which may raise the energy barrier for conformational change of S protein required for membrane fusion and lead to a decrease in virus fitness in cell culture. Thus, MHV-A59 may have evolved to lower the stability of its S protein in order to increase virus fitness.IMPORTANCE Enveloped viruses enter cells through fusion of viral and cellular membranes, and the process is mediated by interactions between viral envelope proteins and their host receptors. In the prefusion conformation, viral envelope proteins are metastable, and activation to the fusion conformation is tightly regulated, since premature activation would lead to loss of viral infectivity. The stability of viral envelope proteins greatly influences their activation and virus fitness. Here, we report that, similar to the A82V mutation in Ebola glycoprotein, in the S glycoprotein of murine coronavirus MHV-A59, the histidine residue at position of 209 significantly affects the thermal stability of the S protein, determines whether S protein can be activated at 37°C by either pH 8.0 alone or by receptor binding, and affects viral fitness in cell culture. Thus, the spike glycoprotein of MHV-A59 has evolved to retain histidine at position 209 to optimize virus fitness.

Selective Packaging in Murine Coronavirus Promotes Virulence by Limiting Type I Interferon Responses.

ABSTRACTSelective packaging is a mechanism used by multiple virus families to specifically incorporate genomic RNA (gRNA) into virions and exclude other types of RNA. Lineage A betacoronaviruses incorporate a 95-bp stem-loop structure, the packaging signal (PS), into the nsp15 locus of ORF1b that is both necessary and sufficient for the packaging of RNAs. However, unlike other viral PSs, where mutations generally resulted in viral replication defects, mutation of the coronavirus (CoV) PS results in large increases in subgenomic RNA packaging with minimal effects on gRNA packaging in vitro and on viral titers. Here, we show that selective packaging is also required for viral evasion of the innate immune response and optimal pathogenicity. We engineered two distinct PS mutants in two different strains of murine hepatitis virus (MHV) that packaged increased levels of subgenomic RNAs, negative-sense genomic RNA, and even cellular RNAs. All PS mutant viruses replicated normally in vitro but caused dramatically reduced lethality and weight loss in vivo. PS mutant virus infection of bone marrow-derived macrophages resulted in increased interferon (IFN) production, indicating that the innate immune system limited the replication and/or pathogenesis of PS mutant viruses in vivo. PS mutant viruses remained attenuated in MAVS−/− and Toll-like receptor 7-knockout (TLR7−/−) mice, two well-known RNA sensors for CoVs, but virulence was restored in interferon alpha/beta receptor-knockout (IFNAR−/−) mice or in MAVS−/− mice treated with IFNAR-blocking antibodies. Together, these data indicate that coronaviruses promote virulence by utilizing selective packaging to avoid innate immune detection.

Alpha/Beta Interferon (IFN-α/β) Signaling in Astrocytes Mediates Protection against Viral Encephalomyelitis and Regulates IFN-γ-Dependent Responses.

ABSTRACTThe contribution of distinct central nervous system (CNS) resident cells to protective alpha/beta interferon (IFN-α/β) function following viral infections is poorly understood. Based on numerous immune regulatory functions of astrocytes, we evaluated the contribution of astrocyte IFN-α/β signaling toward protection against the nonlethal glia- and neuronotropic mouse hepatitis virus (MHV) strain A59. Analysis of gene expression associated with IFN-α/β function, e.g., pattern recognition receptors (PRRs) and interferon-stimulated genes (ISGs), revealed lower basal mRNA levels in brain-derived astrocytes than in microglia. Although astrocytes poorly induced Ifnβ mRNA following infection, they upregulated various mRNAs in the IFN-α/β pathway to a higher extent than microglia, supporting effective IFN-α/β responsiveness. Ablation of the IFN-α/β receptor (IFNAR) in astrocytes using mGFAPcre IFNARfl/fl mice resulted in severe encephalomyelitis and mortality, coincident with uncontrolled virus replication. Further, virus spread was not restricted to astrocytes but also affected microglia and neurons, despite increased and sustained Ifnα/β and ISG mRNA levels within the CNS. IFN-γ, a crucial mediator for MHV control, was not impaired in infected mGFAPcre IFNARfl/fl mice despite reduced T cell CNS infiltration. Unexpectedly however, poor induction of IFN-γ-dependent major histocompatibility complex (MHC) class II expression on microglia supported that defective IFN-γ signaling contributes to uncontrolled virus replication. A link between sustained elevated IFN-α/β and impaired responsiveness to IFN-γ supports the novel concept that temporally limited early IFN-α/β responses are critical for effective antiviral IFN-γ function. Overall, our results imply that IFN-α/β signaling in astrocytes is not only critical in limiting early CNS viral spread but also promotes protective antiviral IFN-γ function.IMPORTANCE An antiviral state established by IFN-α/β contains initial viral spread as adaptive immunity develops. While it is apparent that the CNS lacks professional IFN-α/β producers and that resident cells have distinct abilities to elicit innate IFN-α/β responses, protective interactions between inducer and responder cells require further investigation. Infection with a glia- and neuronotropic coronavirus demonstrates that astrocytes mount a delayed but more robust response to infection than microglia, despite their lower basal mRNA levels of IFN-α/β-inducing components. Lethal, uncontrolled viral dissemination following ablation of astrocyte IFN-α/β signaling revealed the importance of IFN-α/β responses in a single cell type for protection. Sustained global IFN-α/β expression associated with uncontrolled virus did not suffice to protect neurons and further impaired responsiveness to protective IFN-γ. The results support astrocytes as critical contributors to innate immunity and the concept that limited IFN-α/β responses are critical for effective subsequent antiviral IFN-γ function.

A novel mechanism of RNase L inhibition: Theiler's virus L* protein prevents 2-5A from binding to RNase L.

The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler’s murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L. Our current data show that L* binds the ankyrin repeats R1 and R2 of RNase L and inhibits 2’-5’ oligoadenylates (2-5A) binding to RNase L. Thereby, L* prevents dimerization and oligomerization of RNase L in response to 2-5A. Using chimeric mouse hepatitis virus (MHV) expressing TMEV L*, we showed that L* efficiently inhibits RNase L in vivo. Interestingly, those data show that L* can functionally substitute for the MHV-encoded phosphodiesterase ns2, which acts upstream of L* in the OAS/RNase L pathway, by degrading 2-5A.

SAT-406 - Depletion of RIPK1 in hepatocytes exacerbates PolyI:C and Murine Hepatitis Virus induced liver damage

SAT-406 - Depletion of RIPK1 in hepatocytes exacerbates PolyI:C and Murine Hepatitis Virus induced liver damage

Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease.

ABSTRACTEmerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs in vitro and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance associated with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concentration values (EC50) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as determined by EC50. The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same in vitro resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concentrations of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs.

Is regulation preventing the development of therapeutics that may prevent future coronavirus pandemics?

Is regulation preventing the development of therapeutics that may prevent future coronavirus pandemics?

Microglia are required for protection against lethal coronavirus encephalitis in mice.

Recent findings have highlighted the role of microglia in orchestrating normal development and refining neural network connectivity in the healthy CNS. Microglia are not only vital cells in maintaining CNS homeostasis, but also respond to injury, infection, and disease by undergoing proliferation and changes in transcription and morphology. A better understanding of the specific role of microglia in responding to viral infection is complicated by the presence of nonmicroglial myeloid cells with potentially overlapping function in the healthy brain and by the rapid infiltration of hematopoietic myeloid cells into the brain in diseased states. Here, we used an inhibitor of colony-stimulating factor 1 receptor (CSF1R) that depletes microglia to examine the specific roles of microglia in response to infection with the mouse hepatitis virus (MHV), a neurotropic coronavirus. Our results show that microglia were required during the early days after infection to limit MHV replication and subsequent morbidity and lethality. Additionally, microglia depletion resulted in ineffective T cell responses. These results reveal nonredundant, critical roles for microglia in the early innate and virus-specific T cell responses and for subsequent host protection from viral encephalitis.

Loss of Cx43-Mediated Functional Gap Junction Communication in Meningeal Fibroblasts Following Mouse Hepatitis Virus Infection.

Mouse hepatitis virus (MHV) infection causes meningoencephalitis by disrupting the neuro-glial and glial-pial homeostasis. Recent studies suggest that MHV infection alters gap junction protein connexin 43 (Cx43)-mediated intercellular communication in brain and primary cultured astrocytes. In addition to astrocytes, meningeal fibroblasts also express high levels of Cx43. Fibroblasts in the meninges together with the basal lamina and the astrocyte endfeet forms the glial limitans superficialis as part of the blood–brain barrier (BBB). Alteration of glial-pial gap junction intercellular communication (GJIC) in MHV infection has the potential to affect the integrity of BBB. Till date, it is not known if viral infection can modulate Cx43 expression and function in cells of the brain meninges and thus affect BBB permeability. In the present study, we have investigated the effect of MHV infection on Cx43 localization and function in mouse brain meningeal cells and primary meningeal fibroblasts. Our results show that MHV infection reduces total Cx43 levels and causes its intracellular retention in the perinuclear compartments reducing its surface expression. Reduced trafficking of Cx43 to the cell surface in MHV-infected cells is associated with loss functional GJIC. Together, these data suggest that MHV infection can directly affect expression and cellular distribution of Cx43 resulting in loss of Cx43-mediated GJIC in meningeal fibroblasts, which may be associated with altered BBB function observed in acute infection.

Leader sequences of coronavirus are altered during infection.

The life cycle of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) involves a unique process called discontinuous transcription by which a set of 3' coterminal subgenomic mRNAs (sgmRNA) with identical 5' leader sequences can be generated. The current study demonstrates that the replication intermediates of minus strand of subgenomic RNA (sgRNA) can be readily recovered from SARS-CoV infected cells. A novel sgmRNA (M-1) was identified as a short version of membrane (M) gene. Transcriptional regulatory sequences (TRS) of SARS-CoV and Mouse Hepatitis Virus (MHV) sgmRNAs contain a species specific core element (CE). The sizes of leader sequences in MHVs vary not only in different viral strains but also among different genes in the same strain. Leader alterations such as deletion and nucleotide substitution were observed in MHVs, while a dynamic one-orientation 'sequential deletion' was found among the leaders of 76 SARS-CoV isolates. These results imply that the leader sequence of coronavirus might be unstable and leader alterations during SARS-CoV transmission in humans might have negative impact on its viral infectivity.

Murine Hepatitis Virus nsp14 Exoribonuclease Activity Is Required for Resistance to Innate Immunity

Coronaviruses (CoVs) are positive-sense RNA viruses that infect numerous mammalian and avian species and are capable of causing severe and lethal disease in humans. CoVs encode several innate immune antagonists that counteract the host innate immune response to facilitate efficient viral replication. CoV nonstructural protein 14 (nsp14) encodes 3'-to-5' exoribonuclease activity (ExoN), which performs a proofreading function and is required for high-fidelity replication. Outside of the order Nidovirales, arenaviruses are the only RNA viruses that encode an ExoN, which functions to degrade double-stranded RNA (dsRNA) replication intermediates. In this study, we tested the hypothesis that CoV ExoN also functions to antagonize the innate immune response. We demonstrate that viruses lacking ExoN activity [ExoN(-)] are sensitive to cellular pretreatment with interferon beta (IFN-β) in a dose-dependent manner. In addition, ExoN(-) virus replication was attenuated in wild-type bone marrow-derived macrophages (BMMs) and partially restored in interferon alpha/beta receptor-deficient (IFNAR-/-) BMMs. ExoN(-) virus replication did not result in IFN-β gene expression, and in the presence of an IFN-β-mediated antiviral state, ExoN(-) viral RNA levels were not substantially reduced relative to those of untreated samples. However, ExoN(-) virus generated from IFN-β-pretreated cells had reduced specific infectivity and decreased relative fitness, suggesting that ExoN(-) virus generated during an antiviral state is less viable to establish a subsequent infection. Overall, our data suggest murine hepatitis virus (MHV) ExoN activity is required for resistance to the innate immune response, and antiviral mechanisms affecting the viral RNA sequence and/or an RNA modification act on viruses lacking ExoN activity.IMPORTANCE CoVs encode multiple antagonists that prevent or disrupt an efficient innate immune response. Additionally, no specific antiviral therapies or vaccines currently exist for human CoV infections. Therefore, the study of CoV innate immune antagonists is essential for understanding how CoVs overcome host defenses and to maximize potential therapeutic interventions. Here, we sought to determine the contributions of nsp14 ExoN activity in the induction of and resistance to the innate immune response. We show that viruses lacking nsp14 ExoN activity are more sensitive than wild-type MHV to restriction by exogenous IFN-β and that viruses produced in the presence of an antiviral state are less capable of establishing a subsequent viral infection. Our results support the hypothesis that murine hepatitis virus ExoN activity is required for resistance to the innate immune response.

The coronavirus nucleocapsid protein is ADP-ribosylated

ADP-ribosylation is a common post-translational modification, although how it modulates RNA virus infection is not well understood. While screening for ADP-ribosylated proteins during coronavirus (CoV) infection, we detected a ~55kDa ADP-ribosylated protein in mouse hepatitis virus (MHV)-infected cells and in virions, which we identified as the viral nucleocapsid (N) protein. The N proteins of porcine epidemic diarrhea virus (PEDV), severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV were also ADP-ribosylated. ADP-ribosylation of N protein was also observed in cells exogenously expressing N protein by transduction using Venezuelan equine encephalitis virus replicon particles (VRPs). However, plasmid-derived N protein was not ADP-ribosylated following transient transfection but was ADP-ribosylated after MHV infection, indicating that this modification requires virus infection. In conclusion, we have identified a novel post-translation modification of the CoV N protein that may play a regulatory role for this important structural protein.

Macrophage scavenger receptor 1 contributes to pathogenesis of fulminant hepatitis via neutrophil-mediated complement activation

The macrophage scavenger receptor 1 (Msr1, also called SRA) is a pattern recognition receptor primarily expressed on myeloid cells, which plays an important role in the maintenance of immune homeostasis. Since MSR1 expression was upregulated in the livers of patients with fulminant hepatitis (FH), we investigated the functional mechanism of Msr1 in FH pathogenesis. Msr1-deficient (Msr1-/-) mice and their wild-type (WT) littermates were infected with mouse hepatitis virus strain-A59 (MHV-A59) to induce FH, and the levels of tissue damage, serum alanine aminotransferase, inflammatory cytokines and complement component 5a (C5a) were measured and compared. Liver injury was studied after MHV infection with or without neutrophil depletion. Our results showed that Msr1-/- mice were resistant to MHV-induced hepatitis. Treatment with the C5a receptor antagonist (C5aRa) diminished the differences in inflammatory responses and liver injury between MHV-infected wild-type and Msr1-/- mice, suggesting that C5a-induced pro-inflammatory response plays a critical role in the Msr1-mediated regulation of FH pathogenesis. We demonstrated that Msr1 efficiently enhanced transforming growth factor-activated kinase-1 phosphorylation in neutrophils upon MHV-A59 stimulation, thereby promoting the activation of the extracellular signal-regulated kinase pathway and subsequent NETosis formation. Moreover, we provided evidence that blockage of Msr1 attenuated the liver damage caused by MHV-A59 infection. Msr1 promotes the pathogenesis of virus-induced FH by enhancing induction of neutrophil NETosis and subsequent complement activation. Targeting Msr1 may be employed as a new immunotherapeutic strategy for FH. Virus-induced fulminant hepatitis (FH) is a disease with a high mortality worldwide. Enhanced levels of macrophage scavenger receptor 1 (Msr1) in the liver of patients with FH and of murine experimental FH indicated Msr1 plays a role in the pathogenesis of FH. Herein, we demonstrate that mice deficient in Msr1 are resistant to FH induced by MHV-A59, and the Msr1 inhibitor fucoidan suppresses the progression of FH in mice. Our study suggests that use of drugs inhibiting MSR1 function could be beneficial to patients with FH.

Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations.

ABSTRACTThe coronavirus (CoV) RNA genome is the largest among the single-stranded positive-sense RNA viruses. CoVs encode a proofreading 3′-to-5′ exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is responsible for CoV high-fidelity replication. Alanine substitution of ExoN catalytic residues [ExoN(-)] in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and murine hepatitis virus (MHV) disrupts ExoN activity, yielding viable mutant viruses with defective replication, up to 20-fold-decreased fidelity, and increased susceptibility to nucleoside analogues. To test the stability of the ExoN(-) genotype and phenotype, we passaged MHV-ExoN(-) 250 times in cultured cells (P250), in parallel with wild-type MHV (WT-MHV). Compared to MHV-ExoN(-) P3, MHV-ExoN(-) P250 demonstrated enhanced replication and increased competitive fitness without reversion at the ExoN(-) active site. Furthermore, MHV-ExoN(-) P250 was less susceptible than MHV-ExoN(-) P3 to multiple nucleoside analogues, suggesting that MHV-ExoN(-) was under selection for increased replication fidelity. We subsequently identified novel amino acid changes within the RNA-dependent RNA polymerase and nsp14 of MHV-ExoN(-) P250 that partially accounted for the reduced susceptibility to nucleoside analogues. Our results suggest that increased replication fidelity is selected in ExoN(-) CoVs and that there may be a significant barrier to ExoN(-) reversion. These results also support the hypothesis that high-fidelity replication is linked to CoV fitness and indicate that multiple replicase proteins could compensate for ExoN functions during replication.

VSIG4 inhibits proinflammatory macrophage activation by reprogramming mitochondrial pyruvate metabolism

Exacerbation of macrophage-mediated inflammation contributes to pathogenesis of various inflammatory diseases, but the immunometabolic programs underlying regulation of macrophage activation are unclear. Here we show that V-set immunoglobulin-domain-containing 4 (VSIG4), a B7 family-related protein that is expressed by resting macrophages, inhibits macrophage activation in response to lipopolysaccharide. Vsig4−/− mice are susceptible to high-fat diet-caused obesity and murine hepatitis virus strain-3 (MHV-3)-induced fulminant hepatitis due to excessive macrophage-dependent inflammation. VSIG4 activates the PI3K/Akt–STAT3 pathway, leading to pyruvate dehydrogenase kinase-2 (PDK2) upregulation and subsequent phosphorylation of pyruvate dehydrogenase, which results in reduction in pyruvate/acetyl-CoA conversion, mitochondrial reactive oxygen species secretion, and macrophage inhibition. Conversely, interruption of Vsig4 or Pdk2 promotes inflammation. Forced expression of Vsig4 in mice ameliorates MHV-3-induced viral fulminant hepatitis. These data show that VSIG4 negatively regulates macrophage activation by reprogramming mitochondrial pyruvate metabolism.

Murine Olfactory Bulb Interneurons Survive Infection with a Neurotropic Coronavirus.

Viral infection of the central nervous system (CNS) is complicated by the mostly irreplaceable nature of neurons, as the loss of neurons has the potential to result in permanent damage to brain function. However, whether neurons or other cells in the CNS sometimes survive infection and the effects of infection on neuronal function is largely unknown. To address this question, we used the rJHM strain (rJ) of mouse hepatitis virus (MHV), a neurotropic coronavirus that causes acute encephalitis in susceptible strains of mice. To determine whether neurons or other CNS cells survive acute infection with this virulent virus, we developed a recombinant JHMV that expresses Cre recombinase (rJ-Cre) and infected mice that universally expressed a silent (floxed) version of tdTomato. Infection of these mice with rJ-Cre resulted in expression of tdTomato in host cells. The results showed that some cells were able to survive the infection, as demonstrated by continued tdTomato expression after virus antigen could no longer be detected. Most notably, interneurons in the olfactory bulb, which are known to be inhibitory, represented a large fraction of the surviving cells. In conclusion, our results indicated that some neurons are resistant to virus-mediated cell death and provide a framework for studying the effects of prior coronavirus infection on neuron function.IMPORTANCE We developed a novel recombinant virus that allows the study of cells that survive an infection by a central nervous system-specific strain of murine coronavirus. Using this virus, we identified neurons and, to a lesser extent, nonneuronal cells in the brain that were infected during the acute phase of the infection and survived for approximately 2 weeks until the mice succumbed to the infection. We focused on neurons and glial cells within the olfactory bulb because the virus enters the brain at this site. Our results show that interneurons of the olfactory bulb were the primary cell type able to survive infection. Further, these results indicate that this system will be useful for functional and gene expression studies of cells in the brain that survive acute infection.

Role of the inflammasome-related cytokines Il-1 and Il-18 during infection with murine coronavirus.

The inflammasome, a cytosolic protein complex that mediates the processing and secretion of pro-inflammatory cytokines, is one of the first responders during viral infection. The cytokines secreted following inflammasome activation, which include IL-1 and IL-18, regulate cells of both the innate and adaptive immune system, guiding the subsequent immune responses. In this study, we used murine coronavirus, mouse hepatitis virus (MHV), infection of the central nervous system and liver to assess of the role of the inflammasome and its related cytokines on pathogenesis and host defense during viral infection. Mice lacking all inflammasome signaling due to the absence of caspase-1 and -11 were more vulnerable to infection, with poor survival and elevated viral replication compared to wild-type mice. Mice lacking IL-1 signaling experienced elevated viral replication but similar survival compared to wild-type controls. In the absence of IL-18, mice had elevated viral replication and poor survival, and this protective effect of IL-18 was found to be due to promotion of interferon gamma production in αβ T cells. These data suggest that inflammasome signaling is largely protective during murine coronavirus infection, in large part due to the pro-inflammatory effects of IL-18.