Most strains of murine coronavirus mouse hepatitis virus (MHV) express a cleavable spike glycoprotein that mediates viral entry and pH-independent cell-cell fusion. The MHV type 2 (MHV-2) strain of murine coronavirus differs from other strains in that it expresses an uncleaved spike and cannot induce cell-cell fusion at neutral pH values. We show here that while infection of the prototype MHV-A59 strain is not sensitive to pretreatment with lysosomotropic agents, MHV-2 replication is significantly inhibited by these agents. By use of an A59/MHV-2 chimeric virus, the susceptibility to lysosomotropic agents is mapped to the MHV-2 spike, suggesting a requirement of acidification of endosomes for MHV-2 spike-mediated entry. However, acidification is likely not a direct trigger for MHV-2 spike-mediated membrane fusion, as low-pH treatment is unable to overcome ammonium chloride inhibition, and it also cannot induce cell-cell fusion between MHV-2-infected cells. In contrast, trypsin treatment can both overcome ammonium chloride inhibition and promote cell-cell fusion. Inhibitors of the endosomal cysteine proteases cathepsin B and cathepsin L greatly reduce MHV-2 spike-mediated entry, while they have little effect on A59 entry, suggesting that there is a proteolytic step in MHV-2 entry. Finally, a recombinant virus expressing a cleaved MHV-2 spike has the ability to induce cell-cell fusion at neutral pH values and does not require low pH and endosomal cathepsins during infection. These studies demonstrate that endosomal proteolysis by cathepsins is necessary for MHV-2 spike-mediated entry; this is similar to the entry pathway recently described for severe acute respiratory syndrome coronavirus and indicates that coronaviruses may use multiple pathways for entry.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS.Recent studies have demonstrated that significant axonal injury also occurs in MS patients and correlates with neurological dysfunction, but it is not known whether this neuronal damage is a primary disease process, or occurs only secondary to demyelination. In the current studies, neurotropic strains of mouse hepatitis virus (MHV) that induce meningitis, encephalitis, and demyelination in the CNS, an animal model of MS, were used to evaluate mechanisms of axonal injury. The pathogenic properties of genetically engineered isogenic spike protein recombinant demyelinating and nondemyelinating strains of MHV were compared. Studies demonstrate that a demyelinating strain of MHV causes concomitant axonal loss and macrophage-mediated demyelination. The mechanism of axonal loss and demyelination in MHV infection is dependent on successful transport of virus from gray matter to white matter using the MHV host attachment spike glycoprotein. Our data show that axonal loss and demyelination can be independent direct viral cytopathic events, and suggest that similar direct axonal damage may occur in MS. These results have important implications for the design of neuroprotective strategies for CNS demyelinating disease, and our model identifies the spike protein as a therapeutic target to prevent axonal transport of neurotropic viruses.

Infection of primary mouse glial cell cultures with mouse hepatitis virus strain A59 results in a productive, persistent infection, but without any obvious cytopathic effect. Mutant viruses isolated from infected glial cultures 16 to 18 weeks postinfection replicate with kinetics similar to those of wild-type virus but produce small plaques on fibroblasts and cause only minimal levels of cell-to-cell fusion under conditions in which wild type causes nearly complete cell fusion. However, since extensive fusion is present in mutant-infected cells at late times postinfection, the defect is actually a delay in kinetics rather than an absolute block in activity. Addition of trypsin to mutant-infected fibroblast cultures enhanced cell fusion a small (two- to fivefold) but significant degree, indicating that the defect could be due to a lack of cleavage of the viral spike (fusion) protein. Sequencing of portions of the spike genes of six fusion-defective mutants revealed that all contained the same single nucleotide mutation resulting in a substitution of aspartic acid for histidine in the spike cleavage signal. Mutant virions contained only the 180-kDa form of spike protein, suggesting that this mutation prevented the normal proteolytic cleavage of the 180-kDa protein into the 90-kDa subunits. Examination of revertants of the mutants supports this hypothesis. Acquisition of fusion competence correlates with the replacement of the negatively charged aspartic acid with either the wild-type histidine or a nonpolar amino acid and the restoration of spike protein cleavage. These data confirm and extend previous reports concluding cleavage of S is required for efficient cell-cell fusion by mouse hepatitis virus but not for virus-cell fusion (infectivity).

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Optic neuritis (ON), an inflammatory demyelinating optic nerve disease, occurs in multiple sclerosis (MS).Pathological mechanisms and potential treatments for ON have been studied via experimental autoimmune MS models. However, evidence suggests that virus-induced inflammation is a likely etiology triggering MS and ON; experimental virus-induced ON models are therefore required. We demonstrate that MHV-A59, a mouse hepatitis virus (MHV) strain that causes brain and spinal cord inflammation and demyelination, induces ON by promoting mixed inflammatory cell infiltration. In contrast, MHV-2, a nondemyelinating MHV strain, does not induce ON. Results reveal a reproducible virus-induced ON model important for the evaluation of novel therapies.Significant neuronal damage, with loss of retinal ganglion cell (RGC) axons that comprise the optic nerve, occurs following inflammatory optic neuritis (ON) and correlates with permanent vision loss (2,6,28). Experimental ON is a useful model to examine mechanisms of neuronal damage in multiple sclerosis (MS) because RGCs can readily be labeled and quantified (23). Rodents immunized with myelin proteins develop experimental autoimmune encephalomyelitis (EAE), a model of MS with inflammation in the brain, spinal cord, and optic nerves (12). Mechanisms of neuronal damage during ON in EAE have been examined but vary between chronic (16, 19) and relapsing (23) EAE models, suggesting that different causes of ON may mediate vision-threatening neuronal damage by distinct mechanisms.MS may be caused by a viral infection triggering an immune response against myelin (1, 26). Studies of virus-mediated models of MS are therefore important for our understanding of disease mechanisms and the development of novel therapies. While virus-induced models of MS exist (15, 26), the incidence of ON has not been characterized.Mouse hepatitis virus (MHV) infection in mice has been used as a model for virus-induced demyelination that mimics many pathological features of MS (9,13,15,27,29). Some neurotropic strains of MHV induce a biphasic neurological disease with acute meningoencephalitis, followed by chronic demyelination (15). Similarly to results for autoimmune mod- els of MS, Dandekar et al. recently demonstrated that axonal damage occurs in mice with MHV-induced demyelination (3).It is not known whether the inflammatory demyelination and axonal loss observed to occur in the MHV-infected mouse brain and spinal cord also affect the optic nerve.In the current study, we inoculated mice with plaque-purified demyelinating strain MHV-A59 (14, 15) and nondemyelinating strain MHV-2 (10). MHV-A59 infects a variety of cell types, including neurons, astrocytes, oligodendrocytes, microglia, and ependymal cells (11,13,15,30), in the central nervous system (CNS) and causes acute encephalitis, meningitis, hepatitis, and chronic demyelination. In contrast, MHV-2, a strain closely related to MHV-A59, has a limited ability to invade the brain and spinal cord, causing meningitis without encephalitis or demyelination ...

The coronavirus mouse hepatitis virus (MHV) contains a large open reading frame embedded entirely within the 5 half of its nucleocapsid (N) gene. This internal gene (designated I) is in the ؉1 reading frame with respect to the N gene, and it encodes a mostly hydrophobic 23-kDa polypeptide. We have found that this protein is expressed in MHV-infected cells and that it is a previously unrecognized structural protein of the virion. To analyze the potential biological importance of the I gene, we disrupted its expression by site-directed mutagenesis using targeted RNA recombination. The start codon for I was replaced by a threonine codon, and a stop codon was introduced at a short interval downstream. Both alterations created silent changes in the N reading frame. In vitro translation studies showed that these mutations completely abolished synthesis of I protein, and immunological analysis of infected cell lysates confirmed this conclusion. The MHV I mutant was viable and grew to high titer. However, the I mutant had a reduced plaque size in comparison with its isogenic wild-type counterpart, suggesting that expression of I confers some minor growth advantage to the virus. The engineered mutations were stable during the course of experimental infection in mice, and the I mutant showed no significant differences from wild type in its ability to replicate in the brains or livers of infected animals. These results demonstrate that I protein is not essential for the replication of MHV either in tissue culture or in its natural host.

Recombinant mouse hepatitis viruses (MHV) differing only in the spike gene, containing A59, MHV-4, and MHV-2 spike genes in the background of the A59 genome, were compared for their ability to replicate in the liver and induce hepatitis in weanling C57BL/6 mice infected with 500 PFU of each virus by intrahepatic injection. Penn98-1, expressing the MHV-2 spike gene, replicated to high titer in the liver, similar to MHV-2, and induced severe hepatitis with extensive hepatocellular necrosis. S A59 R13, expressing the A59 spike gene, replicated to a somewhat lower titer and induced moderate to severe hepatitis with zonal necrosis, similar to MHV-A59. S 4 R21, expressing the MHV-4 spike gene, replicated to a minimal extent and induced few if any pathological changes, similar to MHV-4. Thus, the extent of replication and the degree of hepatitis in the liver induced by these recombinant viruses were determined largely by the spike protein.Various strains of mouse hepatitis virus (MHV) induce different patterns of pathogenesis, including enteritis, hepatitis, encephalitis, and demyelination in the mouse (20,21). We are considering three strains here, MHV-A59, MHV-2, and MHV-4 (an isolate of MHV-JHM). The MHV-A59 strain is dualtropic, producing moderate to severe hepatitis as well as mild to moderate acute meningoencephalitis and chronic demyelination in C57BL/6 weanling mice (29, 30). The MHV-4 strain causes severe acute encephalitis, chronic demyelination, and only minimal levels of hepatitis (6, 23). The MHV-2 strain causes severe hepatitis and meningitis but is unable to cause encephalitis (7,20,42). There are previous studies demonstrating a relationship between attenuation of neurovirulence (6, 10, 13, 42) or hepatitis (14, 28) and the presence of mutations and variations in the spike (S) gene. The S protein, found on the virion envelope and on the plasma membrane of infected cells, is responsible for attachment to viral receptor and viruscell fusion during viral entry and for cell-to-cell fusion later during infection. S is a 180-kDa glycoprotein, which (in the case of most MHV spike proteins) is cleaved into two noncovalently associated 90-kDa subunits, the amino-terminal S1 and carboxy-terminal S2 subunits (14,33). It is speculated that the S1 subunit forms the globular head of the spike and the S2 subunit forms the membrane-bound stalk (8). Recently, a receptor-binding activity has been demonstrated using a recombinant protein containing the amino-terminal 330 residues of the S1 subunit of MHV-JHM (25, 41). S2 is believed to contain the domain that mediates fusion of viral and cell membranes (5, 8). The MHV-2 spike, while highly homologous in sequence to the spike proteins of other MHV strains, remains uncleaved and does not mediate fusion (44,45).Using targeted recombination technology (11,12,35), we have directly demonstrated that the spike protein is a major determinant of the neuropathogenic properties of MHV (39). When the S gene of MHV-4 was introduced into the background of MHV-A59, the resulting recombi...

C12, an attenuated, fusion delayed, very weakly hepatotropic mutant of mouse hepatitis virus strain A59 (MHV-A59( has been further characterized. We have previously shown that C12 has two amino acid substitutions relative to wild type virus in the spike protein, Q159L (within a region of S1 shown to bind to viral receptor in an in vitro assay) and H716D (in the proteolytic cleavage recognition site). We have sequenced the rest of the 31-kb genome of C-12 and compared it to wild type virus. Only three additional amino acids substitutions were found, all encoded within the replicase gene. Analysis of C12 in vivo in C57Bl/6 mice has shown that despite the fact that this virus replicates in the brain to titers at least as high as wild type and causes acute encephalitis similar to wild-type, this virus causes a minimal level of demyelination and only at very high levels of virus inoculation. Thus acute encephalitis is not sufficient for the induction of demyelination by MHV-A59. Analysis of mutants isolated at earlier times from the same persistently infected glial cell culture as C12, as well as mutants isolated from a second independent culture of persistently infected glial cells, suggests that both the weakly demyelinating and the weakly hepatotropic phenotypes of C12 are associated with the Q159L amino acid substitution.