Sequence analysis of the S gene of recombinant MHV-2/A59 coronaviruses reveals three candidate mutations associated with demyelination and hepatitis.

Coronaviruses, mouse hepatitis virus (MHV) strains, exhibit various degrees of neurotropism and hepatotropism following intracerebral (IC) infection of 4-week-old C57Bl/6 mice. Whereas MHV-A59 produces acute meningitis, encephalitis, hepatitis, and chronic demyelination, a closely related strain, MHV-2, produces only acute meningitis and hepatitis. We previously reported that the spike glycoprotein gene of MHV contains determinants of demyelination and hepatitis. To further investigate the site of demyelination and hepatitis determinants within the S gene, we sequenced the S gene of several nondemyelinating recombinant viruses. We found that three encephalitis-positive, demyelination-negative, hepatitis-negative recombinant viruses have an MHV-A59-derived S gene, which contains three identical point mutations (I375M, L652I, and T1087N). One or more of the sites of these mutations in the MHV-A59 genome are likely to contribute to demyelination and hepatitis.

Mouse hepatitis virus type-2 infection in mice: an experimental model system of acute meningitis and hepatitis.

Infection with mouse hepatitis virus (MHV) strain A59 produces acute hepatitis, encephalitis, and chronic demyelination in mice. However, little is known about a closely related strain, MHV-2, which is only weakly neurotropic. To better understand the molecular basis of neurotropism of MHVs, we compared the pathogenesis and genomic sequence of MHV-2 with that of MHV-A59. Intracerebral injection of MHV-2 into 4-week-old C57B1/6 mice produces acute meningitis and hepatitis without encephalitis or chronic inflammatory demyelination. Sequence comparison between MHV-2 and MHV-A59 reveals 94-98% sequence identity of the replicase gene, 83-95% sequence identity of genes 2a, 3, 5b, 6, and 7, and marked difference in the sequence of genes, 2b, 4, and 5a. This information provides the basis for further studies exploring the mechanism of viral neurotropism and virus-induced demyelination.

Murine coronavirus spike protein determines the ability of the virus to replicate in the liver and cause hepatitis.

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. S4R21, 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.

Targeted recombination within the spike gene of murine coronavirus mouse hepatitis virus-A59: Q159 is a determinant of hepatotropism.

Previous studies of a group of mutants of the murine coronavirus mouse hepatitis virus (MHV)-A59, isolated from persistently infected glial cells, have shown a strong correlation between a Q159L amino acid substitution in the S1 subunit of the spike gene and a loss in the ability to induce hepatitis and demyelination. To determine if Q159L alone is sufficient to cause these altered pathogenic properties, targeted RNA recombination was used to introduce a Q159L amino acid substitution into the spike gene of MHV-A59. Recombination was carried out between the genome of a temperature-sensitive mutant of MHV-A59 (Alb4) and RNA transcribed from a plasmid (pFV1) containing the spike gene as well as downstream regions, through the 3' end, of the MHV-A59 genome. We have selected and characterized two recombinant viruses containing Q159L. These recombinant viruses (159R36 and 159R40) replicate in the brains of C57BL/6 mice and induce encephalitis to a similar extent as wild-type MHV-A59. However, they exhibit a markedly reduced ability to replicate in the liver or produce hepatitis compared to wild-type MHV-A59. These viruses also exhibit reduced virulence and reduced demyelination. A recombinant virus containing the wild-type MHV-A59 spike gene, wtR10, behaved essentially like wild-type MHV-A59. This is the first report of the isolation of recombinant viruses containing a site-directed mutation, encoding an amino acid substitution, within the spike gene of any coronavirus. This technology will allow us to begin to map the molecular determinants of pathogenesis within the spike glycoprotein.

The mouse hepatitis virus A59 spike protein is not cleaved in primary hepatocyte and glial cell cultures.

Mouse hepatitis virus strain A59 (MHV-A59) produces mild meningoencephalitis and severe hepatitis during acute infection. To determine whether an in vitro system could be established which would mimic in vivo replication of the virus, we examined the ability of MHV-A59 to replicate in primary cultures of hepatocytes derived from C57BL/6 mice. Infection of hepatocytes with MHV-A59 resulted in low levels of replication, with virus remaining cell associated. Maximum viral yield was observed at 24 hours postinfection, while occasional syncytia were observed at 48 hours postinfection. Primary glial cell culture represents a potential in vitro system representing the second main target of MHV-A59, namely the brain. It is known that MHV-A59 produces a productive, but nonlytic infection in these cultures. Since cell-to-cell fusion is associated with the cleavage of S, the observation of little or no syncytia following MHV-A59 infection of both hepatocytes and glial cells prompted us to examine the cleavage of the spike protein (S) by Western blot analysis. The cleavage of S is inefficient in MHV-A59 infected hepatocytes and in glial cells. Furthermore, no cleavage of this protein was detected in liver homogenates from C57BL/6 mice infected with MHV-A59. These data suggest that cleavage of the MHV-A59 S protein, and by inference cell-to-cell fusion, does not seem to be essential for entry and spread of the virus in vivo and in vitro.

The C12 mutant of MHV-A59 is very weakly demyelinating and has five amino acid substitutions restricted to the spike and replicase genes.

C12, an attenuated, fusion defective, very weakly hepatotropic mutant of MHV-A59 has been further characterized. 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 minimal demyelination. Thus acute encephalitis is not sufficient for induction of demyelination by wild type MHV-A59. We have previously shown that C12 has two amino acid substitutions relative to wild type virus in the spike gene, Q159L (in the receptor binding domain of S1) and H716D (in the signal sequence for cleavage of S). We have now sequenced the rest of the 31 kb genome of C12 and compared it to wild type virus. Only three additional amino acids substitutions were found, all within the replicase gene, one in the predicted papain like proteinase (PLP)-2 domain and one in the predicted helicase domain. Thus, determinants of virulence, hepatotropism, and demyelination may map to the replicase gene as well as to the spike gene.

Protein phosphorylation associated with epipodophyllotoxin-induced apoptosis of lymphoid cells: role of a serine/threonine protein kinase.

We have previously shown that apoptosis induced in thymocytes by dexamethasone or teniposide (VM-26) could be inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and sangivamycin, both relatively specific inhibitors for protein kinase C, but not by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a more specific inhibitor for cAMP-dependent protein kinases. Apoptosis in this model system was not blocked by EGTA and no increase in cytosolic Ca2+ was observed during apoptosis induced by either dexamethasone or VM-26, suggesting that this kinase was Ca2+-independent. In the present study, we demonstrate that addition of 10 microM sangivamycin to thymocyte cultures up to 2 h after addition of either inducer resulted in virtually complete inhibition of apoptosis. Addition of 10 microM sangivamycin at 3 or 4 h after addition of inducer resulted in partial inhibition of apoptosis. Computerized image analysis of two-dimensional PAGE analyses of whole-cell lysates demonstrated that treatment of mouse thymocytes with VM-26 resulted in a limited number of de novo phosphorylation events within 1 h of treatment. The most prominent phosphorylation events associated with VM-26-induced apoptosis were that two intracellular protein species (Protein 1: m.w. = 22.9 kDa, pI, 5.11; and Protein 2: m.w. = 22.9 kDa, pI, 4.98). Similar phosphorylation events were seen in cells treated with dexamethasone. Finally, Western blot analysis suggests that de novo protein phosphorylation induced by VM-26 is on serine/threonine residues. These results provide further evidence that the mechanism of VM-26-induced apoptosis of murine thymocytes involves the action of one or more serine/threonine kinases.

The spike protein of murine coronavirus mouse hepatitis virus strain A59 is not cleaved in primary glial cells and primary hepatocytes.

Mouse hepatitis virus strain A59 (MHV-A59) produces meningoencephalitis and severe hepatitis during acute infection. Infection of primary cells derived from the central nervous system (CNS) and liver was examined to analyze the interaction of virus with individual cell types derived from the two principal sites of viral replication in vivo. In glial cell cultures derived from C57BL/6 mice, MHV-A59 produces a productive but nonlytic infection, with no evidence of cell-to-cell fusion. In contrast, in continuously cultured cells, this virus produces a lytic infection with extensive formation of syncytia. The observation of few and delayed syncytia following MHV-A59 infection of hepatocytes more closely resembles infection of glial cells than that of continuously cultured cell lines. For MHV-A59, lack of syncytium formation correlates with lack of cleavage of the fusion glycoprotein, or spike (S) protein. The absence of cell-to-cell fusion following infection of both primary cell types prompted us to examine the cleavage of the spike protein. Cleavage of S protein was below the level of detection by Western blot analysis in MHV-A59-infected hepatocytes and glial cells. Furthermore, no cleavage of this protein was detected in liver homogenates from C57BL/6 mice infected with MHV-A59. Thus, cleavage of the spike protein does not seem to be essential for entry and spread of the virus in vivo, as well as for replication in vitro.

The internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication.

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.

Altered pathogenesis of a mutant of the murine coronavirus MHV-A59 is associated with a Q159L amino acid substitution in the spike protein.

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.

Genomic regions associated with neurotropism identified in MHV by RNA-RNA recombination.

Infection of mice with a neurotropic strain of MHV (MHV-A59), a non-neurotropic strain of MHV (MHV-2), and a set of recombinant viruses (kindly provided by Dr. Michael Lai) were used to map genetic determinants of viral neurotropism and demyelination. Following intracerebral (IC) inoculation of 4-week old C57B1/6 mice, 1LD50 of MHV-A59 produced acute meningoencephalitis and hepatitis, and subsequently chronic CNS demyelinating disease. IC inoculation of 1LD50 of MHV-2 produced acute hepatitis without CNS disease. Recombinants ML-3, ML-11, ML-7, ML-8, ML-9 and ML-10 produced acute encephalitis similar to MHV-A59. According to previous oligonucleotide fingerprinting analysis the only common denominator of the neurotropic recombinant viruses was an M gene derived from MHV-A59. Sequencing of PCR-amplified viral S and M genes confirmed that the M genes of neurotropic viruses are derived from A59 while the S genes of neurotropic viruses are either derived from MHV-2 or from A59. In tissue culture, ML-11, ML-3 and MHV-2 are fusion negative, while A59, ML-7, ML-8 and ML-10 are fusion positive. Thus, neurotropism in MHVs is not linked to fusion or the S gene. Moreover, the M gene may be a significant determinant of neurotropism and acute encephalitis.

Hepatitis mutants of mouse hepatitis virus strain A59.

MHV-A59 causes acute meningoencephalitis and hepatitis in susceptible mice, and a persistent productive, but nonlytic, infection of cultured glial cells. We have shown previously that viruses isolated from persistently infected glial cell cultures have a fusion-defective phenotype and were impaired in their abilities to cause hepatitis compared to wild-type MHV-A59. Two mutants chosen for detailed study, B11 and C12, display two distinct hepatitis phenotypes. The ability of B11 to replicate in the liver was dependent on infectious dose and route of inoculation, while C12 consistently displayed decreased liver titers regardless of dose and route of inoculation. Sequence analysis of wild-type, mutant and revertant S proteins indicates that 1) a mutation in the N terminal subunit of S, resulting in a glutamine to leucine amino acid substitution (Q159L), may affect ability to cause hepatitis and 2) a cleavage site mutation (H716D) which determines fusogenicity is not responsible for the altered hepatitis phenotype. Sequence analysis indicated that hepatitis-producing revertants did not revert at mutation Q159L, although it is possible that a mutation in the heptad repeat domain of S2 may compensate for the mutation in S1. Since B11, C12 and a nonattenuated fusion mutant (B12) have identical S protein sequences, there must be additional mutations outside of S which influence both virulence and ability to replicate in the liver.

MHV-A59 fusion mutants are attenuated and display altered hepatotropism.

Mouse hepatitis virus strain A59 causes a persistent productive, but nonlytic, infection of cultured glial cells. We have mutants isolated from persistently infected glial cell cultures which have been shown to be fusion-defective due to a histidine to aspartic acid mutation (H716D) near the cleavage site of the peplomer protein, S. Here, we examine the pathogenicity of these mutants and show differences in hepatotropism and virulence compared to wild-type virus (WT). Two mutants chosen for detailed study, B11 and C12, were impaired in their abilities to cause hepatitis and/or replicate in the liver of susceptible mice. Furthermore, B11 and C12 display two separate hepatotropic phenotypes. The ability of B11 to replicate in the liver was dependent on infectious dose and route of inoculation, while C12 consistently displayed decreased hepatotropism regardless of dose and route of inoculation. However, B11 and C12 were shown to replicate in the CNS of infected animals similarly to WT. Like WT, the mutants produced meningoencephalitis during acute infection, with viral antigen exhibiting a similar distribution in the brain, and demyelination during chronic infection. Sequence analysis of wild-type, mutant, and revertant S proteins indicates that (1) a mutation in the N terminal subunit of S (S1), resulting in a glutamine to leucine amino acid substitution (Q159L), may affect hepatotropism and (2) a cleavage site mutation which determines fusogenicity is not responsible for altered hepatotropism. Furthermore, since B11, C12, and a nonattenuated fusion mutant (B12) have identical S protein sequences, there must be additional mutations outside of S which influence both virulence and hepatotropism.

Fusion-defective mutants of mouse hepatitis virus A59 contain a mutation in the spike protein cleavage signal.

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).

Identification of peplomer cleavage site mutations arising during persistence of MHV-A59.

Primary mouse glial cell cultures were infected with mouse hepatitis virus strain A59 (MHV-A59) and maintained over an 18 week period. Viruses isolated from these cultures 16-18 weeks postinfection produce small plaques on fibroblasts and cause only minimal levels of cell-to-cell fusion at times when wild type causes nearly complete cell fusion. However, when mutant-infected cultures were examined 24-36 hours postinfection approximately 90% of the cells were in syncytia showing that the fusion defect is not absolute but rather delayed. Addition of trypsin to mutant-infected cultures enhanced cell fusion a small (2- to 5-fold) but significant degree. 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. Replacement of the negatively-charged aspartic acid with either the wild type histidine or a non-polar amino acid was associated with the restoration of spike protein cleavage and cell fusion.

Disruption of respiratory cilia by proteases including those of Pseudomonas aeruginosa.

Pseudomonad proteases disrupted the function and structure of demembranated cilia (axonemes) extracted from porcine tracheae. Proteolytic degradation by the two pseudomonad proteases elastase and alkaline protease and by trypsin and subtilisin impaired motility of ATP-activated axonemes. In addition, electron microscopic observation of negatively stained axonemes indicated that exposure to proteases caused dissociation into individual doublet or singlet microtubules. Inhibition of motility and axonemal fraying occurred when axonemes were treated with less than 5 U of proteolytic activity of any of the four proteases tested. When the effects of 2 U of each protease were compared, trypsin and subtilisin were able to produce immotility in less time than pseudomonad elastase and alkaline protease, while alkaline protease and subtilisin caused the most axonemal fraying in 10 min. Proteolytic digestion of axonemal proteins was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All four proteases cleaved dynein proteins (proteins necessary for motility), though treatment with trypsin resulted in the most extensive solubilization of axonemal proteins. Trypsin and subtilisin both produced more changes in the protein profiles of treated axonemes, using fewer units of proteolytic activity, than the pseudomonad proteases. However, the limited alteration of only a few axonemal proteins by pseudomonad proteases indicates that cleavage need not be extensive to produce dysfunction. Thus, ciliary axonemes are susceptible to proteolytic attack. Degradation of axonemal proteins by pseudomonad proteases, which are released during active infection, may contribute to the impaired ciliary function associated with pseudomonad colonization of the respiratory tract.

Effect of ciliostatic factors from Pseudomonas aeruginosa on rabbit respiratory cilia.

Heat-stable factors released by Pseudomonas aeruginosa in culture supernatants inhibit functional cilia of rabbit tracheal epithelium. Chloroform extraction removed heat-stable factors from stationary-phase culture supernatants. The extracts contained at least seven components separable by thin-layer chromatography (TLC). Cilioinhibitory components were identified as a phenazine derivative, pyo compounds (2-alkyl-4-hydroxyquinolines), and a rhamnolipid, also known as a hemolysin. Fluorescence and absorption spectra, relative migration on TLC, staining characteristics, and gas chromatography were the basis for identification. Inhibitory concentrations of each active component were established by quantitative measures of percent motility and beat frequency. Corresponding damage to ciliary ultrastructure was examined by electron microscopy. The pyo compounds produced ciliostasis at concentrations of 50 micrograms/ml, but without obvious ultrastructural lesions. The phenazine derivative also inhibited ciliary motility and caused some membrane disruption, although at substantially greater concentrations of 400 micrograms/ml. Limited exposure of tracheal explants to the rhamnolipid resulted in ciliostasis which was associated with altered ciliary membranes. More extensive exposure to rhamnolipid was associated with removal of dynein arms from axonemes. Pyocyanin at a concentration of 0.5 mg/ml did not inhibit ciliary beating under our conditions. The data suggest that the pyo compounds are the most effective per weight ciliostatic factors released by P. aeruginosa and rhamnolipid is the most destructive of cilia ultrastructure. By interfering with normal ciliary function, these ciliostatic factors may enable P. aeruginosa to more easily colonize the respiratory tract.

Isolation of cilia from porcine tracheal epithelium and extraction of dynein arms.

Milligram amounts of mammalian ciliary axonemes were isolated from porcine tracheas. These were reactivated upon addition of ATP, indicating intact functional capability with a mean beat frequency at 37 degrees C of 8.2 Hz. Electron microscopy showed typical ultrastructure of the isolated demembranated axonemes. Electrophoresis into polyacrylamide gradient gels containing sodium dodecyl sulfate revealed reproducible protein profiles from ten different tracheal preparations. Four major protein bands were observed in the 300-330 K molecular weight region, as well as tubulin at 51-54K. Extraction of the isolated tracheal axonemes with 0.6M KCl removed the outer dynein arms seen in electron microscopic cross-section of axonemes, preferentially solubilized two of the high molecular weight proteins at 320 and 330 K, and resulted in a three- to four-fold increase in ATPase specific activity. Sedimentation of the dialyzed salt extract on a 5-30% sucrose density gradient and subsequent fractionation yielded two peaks of ATPase activity. The faster migrating, 19S major ATPase peak correlated with the 320 and 330 K proteins, and two other proteins at 81 and 67 K. The slower sedimenting, 12S minor ATPase peak corresponded to a 308 K protein and two smaller proteins at 33 and 48 K. Thus, the outer dynein arm of tracheal cilia appeared to be associated with at least two high molecular weight proteins. These results demonstrate that adequate quantities of functionally intact axonemes can be reproducibly isolated from porcine tracheas, allowing further fractionation and analysis of mammalian cilia.