[show abstract][hide abstract] ABSTRACT: Infection of mouse oligodendrocytes with a recombinant mouse hepatitis virus (MHV) expressing a green fluorescence protein facilitated specific selection of virus-infected cells and subsequent establishment of persistence. Interestingly, while viral genomic RNAs persisted in infected cells over 14 subsequent passages with concomitant synthesis of viral subgenomic mRNAs and structural proteins, no infectious virus was isolated beyond passage 2. Further biochemical and electron microscopic analyses revealed that virions, while assembled, contained little spike in the envelope, indicating that lack of infectivity during persistence was likely due to deficiency in spike incorporation. This type of non-lytic, non-productive persistence in oligodendrocytes is unique among animal viruses and resembles MHV persistence previously observed in the mouse central nervous system. Thus, establishment of such a culture system that can recapitulate the in vivo phenomenon will provide a powerful approach for elucidating the mechanisms of coronavirus persistence in glial cells at the cellular and molecular levels.
[show abstract][hide abstract] ABSTRACT: The murine coronavirus mouse hepatitis virus (MHV) induced the expression of type I interferon (alpha/beta interferon [IFN-alpha/beta]) in mouse oligodendrocytic N20.1 cells. This induction is completely dependent on virus replication, since infection with UV light-inactivated virus could no longer induce IFN-alpha/beta. We show that MHV infection activated both transcription factors, the IFN regulatory factor 3 (IRF-3) and nuclear factor kappaB (NF-kappaB), as evidenced by phosphorylation and nuclear translocation of IRF-3 and an increased promoter binding activity for IRF-3 and NF-kappaB. Furthermore, the cytoplasmic pattern recognition receptor retinoic acid-inducible gene I (RIG-I) was induced by MHV infection. Knockdown of RIG-I by small interfering RNAs blocked the activation of IRF-3 and subsequent IFN-alpha/beta production induced by MHV infection. Knockdown of another cytoplasmic receptor, the melanoma-differentiation-associated gene 5 (MDA5), by small interfering RNAs also blocked IFN-beta induction. These results demonstrate that MHV is recognized by both RIG-I and MDA5 and induces IFN-alpha/beta through the activation of the IRF-3 signaling pathway. However, knockdown of RIG-I only partially blocked NF-kappaB activity induced by MHV infection and inhibition of NF-kappaB activity by a decoy peptide inhibitor had little effect on IFN-alpha/beta production. These data suggest that activation of the NF-kappaB pathway might not play a critical role in IFN-alpha/beta induction by MHV infection in oligodendrocytes.
Journal of Virology 07/2010; 84(13):6472-82. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Previous studies have shown that proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6), are differentially induced in primary mouse astrocytes by mouse hepatitis virus strain A59 (MHV-A59) and MHV-2. However, the signaling events that trigger TNF-alpha and IL-6 induction in these cells upon MHV infection remain unknown. In this study, we explored the potential signaling events. We found that induction of TNF-alpha and IL-6 occurred as early as 2 h postinfection and was completely dependent on viral replication. Using inhibitors specific for three mitogen-activated protein kinases, we showed that induction of TNF-alpha and IL-6 by MHV-A59 infection was mediated through activation of the Janus N-terminal kinase signaling pathway, but not through the extracellular signal-regulated kinase or p38 signaling pathway. This finding was further confirmed with knockdown experiments using small interfering RNAs specific for Janus N-terminal kinase. Interestingly, while nuclear factor kappaB (NF-kappaB), a key transcription factor required for the expression of proinflammatory cytokines in most cell types, was activated in astrocytes during MHV-A59 infection, disruption of the NF-kappaB pathway by peptide inhibitors did not significantly inhibit TNF-alpha and IL-6 expression. Furthermore, experiments using chimeric viruses demonstrated that the viral spike and nucleocapsid proteins, which play important roles in MHV pathogenicity in mice, are not responsible for the differential induction of the cytokines. These results illustrate the complexity of the regulatory mechanism by which MHV induces proinflammatory cytokines in primary astrocytes.
Journal of Virology 09/2009; 83(23):12204-14. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: We previously showed that infection of rat oligodendrocytes by ultraviolet light-inactivated mouse hepatitis virus (MHV) resulted in apoptosis, suggesting that the apoptosis is triggered during cell entry. To further characterize the earliest apoptotic signaling events, here we treated cells with an antibody specific to the MHV receptor prior to and during virus infection or with an antibody specific to MHV spike protein following virus binding. Both treatments blocked virus infection and apoptosis, indicating that virus-receptor binding is necessary but not sufficient for the apoptosis induction. Furthermore, virus infection significantly increased the formation of the "death-receptor complexes" consisting of Fas, Fas-associated death domain and procaspase-8, but did not induce the complexes involving the tumor necrosis factor receptor and its associated death domain, demonstrating the specific activation of the Fas signaling pathway. Moreover, virus infection did not alter the abundance of the individual proteins of the complexes, suggesting that the activation of the Fas signaling pathway was at the post-translational level. Treatment with a Fas/Fc chimera, which blocks Fas-Fas ligand-mediated apoptosis, inhibited the formation of the complexes and blocked the activation of caspase-8 and apoptosis in MHV-infected cells. It also inhibited the release of cytochrome c from mitochondria and the activation of caspase-9. These results demonstrate that oligodendrocyte apoptosis is triggered by MHV infection during cell entry through the activation of the Fas signaling pathway.
[show abstract][hide abstract] ABSTRACT: We previously demonstrated that infection of cultured cells with murine coronavirus mouse hepatitis virus (MHV) resulted in activation of the mitogen-activated protein kinase (Raf/MEK/ERK) signal transduction pathway (Y. Cai et al., Virology 355:152-163, 2006). Here we show that inhibition of the Raf/MEK/ERK signaling pathway by the MEK inhibitor UO126 significantly impaired MHV progeny production (a reduction of 95 to 99% in virus titer), which correlated with the phosphorylation status of ERK1/2. Moreover, knockdown of MEK1/2 and ERK1/2 by small interfering RNAs suppressed MHV replication. The inhibitory effect of UO126 on MHV production appeared to be a general phenomenon since the effect was consistently observed in all six different MHV strains and in three different cell types tested; it was likely exerted at the postentry steps of the virus life cycle because the virus titers were similarly inhibited from infected cells treated at 1 h prior to, during, or after infection. Furthermore, the treatment did not affect the virus entry, as revealed by the virus internalization assay. Metabolic labeling and reporter gene assays demonstrated that translation of cellular and viral mRNAs appeared unaffected by UO126 treatment. However, synthesis of viral genomic and subgenomic RNAs was severely suppressed by UO126 treatment, as demonstrated by a reduced incorporation of [3H]uridine and a decrease in chloramphenicol acetyltransferase (CAT) activity in a defective-interfering RNA-CAT reporter assay. These findings indicate that the Raf/MEK/ERK signaling pathway is involved in MHV RNA synthesis.
Journal of Virology 02/2007; 81(2):446-56. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mouse hepatitis virus (MHV) causes encephalitis and demyelination in the central nervous system (CNS) of susceptible rodents. Astrocytes are one of the major targets for MHV infection in the CNS, and respond to MHV infection by expressing diverse molecules that may contribute to CNS pathogenesis. Here we characterized the activation of an immediate-early transcription factor Egr-1 by MHV infection in an astrocytoma cell line. We found that the expression of Egr-1 was dramatically increased following virus infection. Using various inhibitors of mitogen-activated protein kinases, we identified that the extracellular signal-regulated kinases 1/2 were involved in the activation of Egr-1 transcription by MHV infection. Experiments with ultraviolet light-inactivated virus revealed that the induction of Egr-1 did not require virus replication and was likely mediated during cell entry. We further found that over-expression of Egr-1 suppressed the expression of BNip3, a pro-apoptotic member of the Bcl-2 family. This finding may provide an explanation for our previously observed down-regulation of BNip3 by MHV infection in astrocytoma cells (Cai, Liu, Yu, and Zhang, Virology 316:104-115, 2003). Furthermore, knockdown of Egr-1 by an siRNA inhibited MHV propagation, suggesting the biological relevance of Egr-1 induction to virus replication. In addition, the persistence/demylinating-positive strains (JHM and A59) induced Egr-1 expression, whereas the persistence/demylinating-negative strain (MHV-2) did not. These results indicate a correlation between the ability of MHVs to induce Egr-1 expression and their ability to cause demyelination in the CNS, which may suggest a potential role for the induction of Egr-1 in viral pathogenesis.
[show abstract][hide abstract] ABSTRACT: 3However, infectious virus can no longer be isolated from the CNS at this time, although viral RNAs continue to persist in the CNS for more than one year, during which time period demyelination is concomitantly detectable. 4,5 The correlation between viral RNA persistence and demyelination in the CNS suggests that viral persistence may be a prerequisite for the development of CNS demyelination. However, virtually nothing is known as to how viral persistence contributes to demyelination. Previous studies attempted to establish an in vitro system of glial or fibroblast cell culture for viral persistence. 6,7 Unfortunately, the persistent infection established in these cells is productive, i.e., generation of infectious viruses with significant virus titers. This type of persistence does not reflect on the infection of animal CNS. Recently we established a persistent MHV infection in a progenitor rat oligodendrocyte. We showed that MHV RNAs were continuously detected in infected cells of more than 20 passages. However, no infectious virus could be isolated from these cells. This phenomenon resembles the persistent, nonproductive infection in animal CNS. To understand the molecular basis of viral persistence in host cells, we analyzed the gene expression profiles of the persistently infected cells by using DNA microarray technology and RTPCR. We found that the expression of a substantial number of cellular genes was altered by viral persistence. Interestingly, although persistently infected progenitor cells could be induced to differentiate into mature oligodendrocytes, the number of dendrites and level of myelin basic protein were markedly reduced in persistent cells. This finding indicates
Advances in experimental medicine and biology 02/2006; 581:379-84. · 1.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: A previous study demonstrated that infection of rat oligodendrocytes by mouse hepatitis virus (MHV) resulted in apoptosis, which is caspase dependent (Y. Liu, Y. Cai, and X. Zhang, J. Virol. 77:11952-11963, 2003). Here we determined the involvement of the mitochondrial pathway in MHV-induced oligodendrocyte apoptosis. We found that caspase-9 activity was 12-fold higher in virus-infected cells than in mock-infected cells at 24 h postinfection (p.i.). Pretreatment of cells with a caspase-9 inhibitor completely blocked caspase-9 activation and partially inhibited the apoptosis mediated by MHV infection. Analyses of cytochrome c release further revealed an activation of the mitochondrial apoptotic pathway. Stable overexpression of the two antiapoptotic proteins Bcl-2 and Bcl-xL significantly, though only partially, blocked apoptosis, suggesting that activation of the mitochondrial pathway is partially responsible for the apoptosis. To identify upstream signals, we determined caspase-8 activity, cleavage of Bid, and expression of Bax and Bad by Western blotting. We found a drastic increase in caspase-8 activity and cleavage of Bid at 24 h p.i. in virus-infected cells, suggesting that Bid may serve as a messenger to relay the signals from caspase-8 to mitochondria. However, treatment with a caspase-8 inhibitor only slightly blocked cytochrome c release from the mitochondria. Furthermore, we found that Bax but not Bad was significantly increased at 12 h p.i. in cells infected with both live and UV-inactivated viruses and that Bax activation was partially blocked by treatment with the caspase-8 inhibitor. These results thus establish the involvement of the mitochondrial pathway in MHV-induced oligodendrocyte apoptosis.
Journal of Virology 02/2006; 80(1):395-403. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Murine coronavirus mouse hepatitis virus (MHV) causes persistent infection of the central nervous system (CNS) in rodents, which has been associated with demyelination. However, the precise mechanism of MHV persistence in the CNS remains elusive. Here we show that the progenitor oligodendrocytes (central glial 4 [CG-4] cells) derived from newborn rat brain were permissive to MHV infection, which resulted in cell death, although viral replication was restricted. Interestingly, treatment with fetal bovine serum or exogenous expression of cellular oncogene Bcl-xL prevented CG-4 cells from MHV-induced cell death. Significantly, overexpression of Bcl-xL alone was sufficient to convert acute to persistent, nonproductive infection in CG-4 cells. This finding indicates that intracellular factors rather than viral components play a critical role in establishing viral persistence in CNS cells. Although viral genomic RNAs continuously persisted in Bcl-xL-expressing CG-4 cells over 10 passages, infectious virus could no longer be isolated beyond 2 passages of the cell. Such a phenomenon resembles the persistent MHV infection in animal CNS. Thus, the establishment of a persistent, nonproductive infection in CG-4 cells may provide a useful in vitro model for studying viral persistence in animal CNS. The data also suggest that direct virus-host cell interaction is one of the underlying mechanisms that regulate viral persistence in CNS cells.
Journal of Virology 02/2005; 79(1):47-56. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Murine coronavirus mouse hepatitis virus (MHV) causes demyelination of the central nervous system (CNS) in rats and mice. Apoptotic oligodendrocytes have been detected in the vicinity of the CNS demyelinating lesions in these animals. However, whether MHV can directly induce oligodendrocyte apoptosis has not been documented. Here, we established a rat oligodendrocyte culture that is morphologically and phenotypically indistinguishable from the primary rat oligodendrocytes. Using this culture, we showed that mature rat oligodendrocytes were permissive to MHV infection but did not support productive virus replication. Significantly, oligodendrocytes infected with both live and ultraviolet light-inactivated viruses underwent apoptosis to a similar extent, which was readily detectable at 24 h postinfection as revealed by apoptotic bodies and DNA fragmentation, indicating that MHV-induced apoptosis is mediated during the early stages of the virus life cycle and does not require virus replication. Prior treatment of cells with the lysosomotropic agents NH(4)Cl and chloroquine as well as the vacuolar proton pump-ATPase inhibitor bafilomycin A1, all of which block the acidification of the endosome, prevented oligodendrocytes from succumbing to apoptosis induced by MHV mutant OBLV60, which enters cells via endocytosis, indicating that fusion between the viral envelope and cell membranes triggers the apoptotic cascade. Treatment with the pan-caspase inhibitor Z-VAD-fmk blocked MHV-induced apoptosis, suggesting an involvement of the caspase-dependent pathway. Our results, thus, for the first time provide unequivocal evidence that infection of oligodendrocytes with MHV directly results in apoptosis. This finding provides an explanation for the destruction of oligodendrocytes and the damage of myelin sheath in MHV-infected CNS and suggests that oligodendrocyte apoptosis may be one of the underlying mechanisms for the pathogenesis of MHV-induced demyelinating diseases in animals.
Journal of Virology 12/2003; 77(22):11952-63. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Murine coronavirus mouse hepatitis virus (MHV) causes encephalitis and demyelination in the central nervous system of susceptible rodents. Astrocytes are the major target for MHV persistence. However, the mechanisms by which astrocytes survive MHV infection and permit viral persistence are not known. Here we performed DNA microarray analysis on differential gene expression in astrocyte DBT cells by MHV infection and found that the mRNA of the proapoptotic gene BNip3 was significantly decreased following MHV infection. This finding was further confirmed by quantitative reverse transcription-polymerase chain reaction, Western blot analysis, and BNip3-promoter-luciferase reporter system. Interestingly, infection with live and ultraviolet light-inactivated viruses equally repressed BNip3 expression, indicating that the down-regulation of BNip3 expression does not require virus replication and is mediated during cell entry. Furthermore, treatment of cells with chloroquine, which blocks the acidification of endosomes, significantly inhibited the repression of the BNip3 promoter activity induced by the acidic pH-dependent MHV mutant OBLV60, which enters cells via endocytosis, indicating that the down-regulation of BNip3 expression is mediated by fusion between viral envelope and cell membranes during entry. Deletion analysis showed that the sequence between nucleotides 262 and 550 of the 588-base-pair BNip3 promoter is necessary and sufficient for driving the BNip3 expression and that it contains signals that are responsible for MHV-induced down-regulation of BNip3 expression in DBT cells. These results may provide insights into the mechanisms by which MHV evades host antiviral defense and promotes cell survival, thereby allowing its persistence in the host astrocytes.