Substitutions of conserved amino acids in the receptor-binding domain of the spike glycoprotein affect utilization of murine CEACAM1a by the murine coronavirus MHV-A59

The host range of the murine coronavirus (MHV) is limited to susceptible mice and murine cell lines by interactions of the spike glycoprotein (S) with its receptor, mCEACAM1a. We identified five residues in S (S33, L79, T82, Y162 and K183) that are conserved in the receptor-binding domain of MHV strains, but not in related coronaviruses. We used targeted RNA recombination to generate isogenic viruses that differ from MHV-A59 by amino acid substitutions in S. Viruses with S33R and K183R substitutions had wild type growth, while L79A/T82A viruses formed small plaques. Viruses with S33G, L79M/T82M or K183G substitutions could only be recovered from cells that over-expressed a mutant mCEACAM1a. Viruses with Y162H or Y162Q substitutions were never recovered, while Y162A viruses formed minute plaques. However, viruses with Y162F substitutions had wild type growth, suggesting that Y162 may comprise part of a hydrophobic domain that contacts the MHV-binding site of mCEACAM1a.     

Primary Structures of Hemagglutinin-esterase and Spike Glycoproteins of Murine Coronavirus DVIM

Diarrhea virus of infant mice (DVIM) is a member of murine hepatitis viruses (MHVs). The nucleotide sequences of the genes encoding the hemagglutinin-esterase (HE) and the spike (S) glycoproteins from DVIM were determined and compared with those of other MHVs. The deduced amino acid sequence of the HE protein was most similar to that of MHV-S strain (94% identity), and the S protein sequence was most similar to that of MHV-Y strain (90% identity). The DVIM HE protein has a unique N-linked glycosylation site in addition to other glycosylation sites common to many MHV strains. Unlike in some typical MHV strain, such as MHV-A59 and MHV-JHM, the vast majority of the S glycoprotein molecules in DVIM exist an uncleaved form probably due to several amino acid substitutions around the cleavage site. This revised version was published online in July 2006 with corrections to the Cover Date.     

Coronaviruses SD and SK share extensive nucleotide homology with murine coronavirus MHV-A59, more than that shared between human and murine coronaviruses

A cDNA probe representing the genome of mouse hepatitis virus (MHV) strain A59 (MHV-A59) was used to measure nucleotide sequence homologies among murine and human coronaviruses and the SD and SK coronaviruses isolated by Burks et al. Since SD and SK were isolated by inoculation of multiple sclerosis (MS) central nervous system (CNS) tissue into mice or cultured mouse cells, it is important to determine their relationships to other murine and human coronavirus isolates. Our results indicate that SD and SK share almost complete nucleotide homology (approximately 90%) with MHV-A59 and generate subgenomic RNAs of the same sizes as MHV-A59. The human coronavirus (HCV) strains tested show less homology with MHV-A59. The immunologically unrelated HCV-229E shows no nucleotide homology with MHV-A59. The immunologically cross-reactive HCV-OC43 shows nucleotide homology with MHV-A59 by blot hybridization but not when hybridized in solution and assayed by S1 nuclease digestion.     

Difference in Bgp-independent fusion activity among mouse hepatitis viruses

Summary.  Mouse hepatitis virus (MHV) utilizes a mouse biliary glycoprotein (Bgp) as a receptor. Co-cultivation of MHV-nonpermissive hamster BHK cells devoid of mouse Bgp with mouse DBT cells infected with MHV-A59 or JHMV induces syncytia formation on BHK cells (Bgp-independent fusion). This study shows the difference in Bgp-independent fusion activity among various MHV strains. Under a phase contrast microscopy, JHMV (cl-2, sp-4) induced the Bgp-independent syncytia on BHK cells similar to those observed on DBT cells, while such syncytia were not seen with the infection of other MHV strains (MHV-1, MHV-3, MHV-A59, MHV-S, srr7, srr11 and srr18). Tiny syncytia detectable only by immunofluorescence were produced with the latter MHV strains except for srr7 which failed to produce syncytia. MHVs except for srr7 grew in BHK cells after Bgp-independent infection. The Bgp-independent fusion by JHMV was inhibited either by anti-S1 or anti-S2 antibodies. These results showed that the JHMV spike protein had a remarkably high Bgp-independent fusion activity. Accepted May 18, 1999 Received February 15, 1999     

Protein synthesis in cells infected by murine hepatitis viruses JHM and A59: Tryptic peptide analysis

Summary The structural and intracellular proteins of the murine hepatitis viruses MHV-JHM and MHV-A59 were studied by tryptic peptide mapping. The results demonstrated that the virions contained three distinct proteins: the two related chains of the E2 complex, the nucleocapsid protein and the heterogeneous E1 complex. Five distinct virus-specific proteins were synthesized by infected cells. Three of the five intracellular proteins contained tryptic-peptides with properties similar to the three structural proteins. Models describing the evolution of the proteins are proposed. Although the pathogenic properties of MHV-JHM and MHV-A59 differ greatly, the tryptic peptide maps of the corresponding proteins of these MHV strains were remarkably similar.With 8 Figures     

Differential regulation of innate and adaptive immune responses in viral encephalitis

Viral encephalitis is a global health concern. The ability of a virus to modulate the immune response can have a pivotal effect on the course of disease and the fate of the infected host. In this study, we sought to understand the immunological basis for the fatal encephalitis following infection with the murine coronavirus, mouse hepatitis virus (MHV)-JHM, in contrast with the more attenuated MHV-A59. Distinct glial cell cytokine and chemokine response patterns were observed within 3 days after infection, became progressively more polarized during the course of infection and with the infiltration of leukocytes. In the brain, MHV-JHM infection induced strong accumulation of IFNβ mRNA relative to IFNγ mRNA. This trend was reversed in MHV-A59 infection and was accompanied by increased CD8 T cell infiltration into brain compared to MHV-JHM infection. Increased apoptosis appeared to contribute to the diminished presence of CD8 T cells in MHV-JHM-infected brain with the consequence of a lower potential for IFNγ production and antiviral activity. MHV-JHM infection also induced sustained mRNA accumulation of the innate immune response products interleukin (IL)-6 and IL-1. Furthermore, high levels of macrophage-inflammatory protein (MIP)-1α, MIP-1β, and MIP-2 mRNA were observed at the onset of MHV-JHM infection and correlated with a marked elevation in the number of macrophages in the brain on day 7 compared to MHV-A59 infection. These observations indicate that differences in the severity of viral encephalitis may reflect the differential ability of viruses to stimulate innate immune responses within the CNS and subsequently the character of infiltrating leukocyte populations.     

Genetic heterogeneity of murine coronaviruses

Several mouse hepatitis viruses (MHV) with different pathogenicity were studied by oligonucleotide fingerprinting. Two strains, MHV-K and MHV-D, which were isolated in Japan and, which cause anaplasia and necrosis of bone marrow and diarrhea, respectively, were found to be closely related to MHV-A59, the prototype MHV. Two other MHV strains, isolated from nude mice, were found to have diverged extensively from the known MHV strains. The MHVs isolated from separate cloned neuroblastoma cell lines persistently infected with JHM strain were also found to have diverged more markedly than the corresponding virus maintained under the conditions of lytic infection. Genetic divergence during persistent infection may be one of the mechanisms by which the MHV diverges.     

MHV S peplomer protein expressed by a recombinant vaccinia virus vector exhibits IgG Fc-receptor activity.

We have previously shown that cells infected with mouse hepatitis virus (MHV) bind rabbit, mouse, and rat IgG by the Fc portion of the IgG molecule. This Fc-binding activity appeared to be mediated by the MHV S protein. S protein could also be precipitated from MHV-infected cells by a monoclonal antibody directed against the murine Fc gamma receptor (Fc gamma R). To prove definitively that the S protein mediates Fc-binding activity, we have expressed the MHV S protein utilizing recombinant vaccinia viruses. The anti-Fc gamma R monoclonal antibody, 2.4G2, precipitated recombinant S protein in cells of murine, human, and rabbit origin. Since the anti-Fc receptor monoclonal antibody does not react with human and rabbit Fc receptors these results demonstrate that the epitope recognized by this antibody is carried on the MHV S protein and is not murine in origin. Examination of various MHV isolates and escape mutants failed to identify the precise sequences in S responsible for the molecular mimicry of the murine Fc gamma R. These data are consistent with the hypothesis that a previously identified region of similarity between the S protein and the Fc gamma R mediates this activity. The Fc binding activity of S was expressed on the cell surface, since MHV-JHM-infected cells, but not uninfected cells, formed rosettes with anti-sheep red blood cell (SRBC) antibody-coated SRBC. The anti-Fc gamma R monoclonal antibody neutralized MHV-JHM and inhibited syncytium formation induced by the MHV S protein.     

Sequence analysis of the nucleoprotein genes of three enterotropic strains of murine coronavirus

Summary The nucleotide sequences of the nucleoprotein genes of three enterotropic strains of the murine coronavirus mouse hepatitis virus (MHV-Y, MHV-RI and DVIM) were determined and compared with previously reported sequences of three polytropic (respiratory) strains (MHV-A59, MHV-JHM and MHV-S). Greater than 92% homology was found among the six strains by pair-wise comparison at the nucleotide level. The genes encoded proteins of 451 to 455 residues and the deduced amino acid sequences were more than 91% homologous. A unique deletion of twelve nucleotides was found at the carboxy terminus of MHV-Y and a three nucleotide deletion was found in MHV-RI, which corresponded to the one previously reported in MHV-A59 and MHV-S. Two internal open reading frames were found within the coding region of the nucleoprotein, the smaller one was specific for the enterotropic strains. It could potentially encode a truncated version of the hypothetical protein described for MHV-A59 and MHV-S. Sequence relationship of the N gene showed no correlation with tissue tropism and no sequence or even single amino acid change unique to either tropism group was found. This indicates that the nucleoprotein of MHV probably has no part in the determination of the primary tissue tropism of an MHV strain. The role of the potential internal protein warrants further investigation.     

Communication between S1N330 and a region in S2 of murine coronavirus spike protein is important for virus entry into cells expressing CEACAM1b receptor

The soluble receptor-resistant (srr) mutants, srr7 and srr11, isolated from a murine coronavirus, mouse hepatitis virus (MHV) JHMV, have an amino acid mutation at positions 1114 (Leu to Phe) and 65 (Leu to His), respectively, in the spike (S) protein. These mutants failed to efficiently infect BHK cells expressing CEACAM1b (BHK-R2), due to their low entry into this cell line, although they infected cells expressing CEACAM1a (BHK-R1) in a manner similar to that of wild-type (wt) JHMV cl-2 (Matsuyama and Taguchi, Virology 273, 80-89, 2000). Following the repeated passage of these mutants through BHK-R2 cells, viruses were no longer isolated from srr11-infected cells, while two distinct mutants, srr7A and srr7B, were obtained from srr7-infected cells. Srr7A and srr7B grew 2 log10 higher than srr7 and induced fusion in BHK-R2 cells, being similar to wt virus. In addition to the amino acid change at position 1114 that stemmed from parental srr7, srr7A and srr7B had mutations around position 280, corresponding to the third region of the S1N330 receptor-binding site (S1N330-III) common to all MHV strains examined thus far. Srr7A and srr7B S proteins showed high fusogenicity in both BHK-R1 and BHK-R2 cells, like the wt virus, while srr7Aa and srr7Ba S proteins, which had mutations in S1N330-III but not at amino acid 1114, exhibited profoundly reduced fusion activity in these cell lines. These findings suggest that communication between S1N330-III and the amino acid at position 1114 is important for efficient fusion activity in BHK-R2 cells. S1N330-III is a possible region in the S1 involved in viral entry into cells.     

Mouse hepatitis virus A59 increases steady-state levels of MHC mRNAs in primary glial cell cultures and in the murine central nervous system

Infection of mixed glial cell cultures with mouse hepatitis virus (MHV)-A59 results in an approximately six-fold increase in the level of major histocompatibility complex (MHC) class I mRNA. In situ hybridization of glial cell cultures infected with MHV-A59 again showed enhanced MHC mRNA expression, both in infected and uninfected cells. These results extend our earlier finding that MHC surface antigens are enhanced on astrocytes and oligodendrocytes after MHV-A59 infection and suggest that this enhancement is a result of an increase in the steady-state level of MHC mRNA. We further demonstrate that increases in MHC mRNA occur in the murine central nervous system (CNS) following infection in vivo. Northern blot analysis of RNA from the brains of infected animals showed transient expression of both MHC class I and class II mRNA over the first 14 days of infection. Expression coincided with viral replication and clearance. In situ hybridization of brain sections from infected animals showed that class I and class II expression was widespread throughout all portions of the brain and in uninfected as well as infected cells. Viral RNA, in contrast, was observed in small foci of cells and mostly within the limbic system. Thus enhancement of MHC mRNA was not restricted either to areas of infection or inflammation. The spatial relationship between viral and MHC expression supports our hypothesis that a soluble mediator is involved in the mechanism of the increase in MHC levels. The fact that MHC induction occurs in vivo as well as in vitro suggests MHC may be important in the mechanism of MHV-induced disease.     

Inactivation of expression of gene 4 of mouse hepatitis virus strain JHM does not affect virulence in the murine CNS.

The protein encoded by ORF 4 of mouse hepatitis virus (MHV) is not required for growth of some strains in tissue culture cells, but its role in pathogenesis in the murine host has not been defined previously in a controlled manner. MHV strain JHM causes acute and chronic neurological diseases in susceptible strains of rodents. To genetically manipulate the structural proteins of this and other strains of MHV, we have generalized an interspecies-targeted RNA recombination selection that was originally developed for the A59 strain of MHV. Using this approach, a recombinant MHV-JHM was constructed in which gene 4 was genetically inactivated. Virus lacking gene 4 expression replicated in tissue culture cells with similar kinetics to recombinant virus in which gene 4 expression was not disrupted. Both types of viruses exhibited similar virulence when analyzed in a murine model of encephalitis. These results establish a targeted recombination system for inserting mutations into MHV-JHM. Furthermore, the protein encoded by ORF 4 is not essential for growth in tissue culture cells or in the CNS of the infected host.     

RNA-binding proteins of coronavirus MHV: Detection of monomeric and multimeric N protein with an RNA overlay-protein blot assay

RNA-binding proteins of coronavirus MHV-A59 were identified using an RNA overlay-protein blot assay (ROPBA). The major viral RNA-binding protein in virions and infected cells was the phosphorylated nucleocapsid protein N (50K). A new 140K virus structural protein was identified as a minor RNA-binding protein both in virions and in infected cells. The 140K protein was antigenically related to N, and upon reduction, yielded only 50K N. Thus, the 140K protein is probably a trimer of N subunits linked by intermolecular disulfide bonds. Several cellular RNA-binding proteins were also detected. RNA-binding of N was not nucleotide sequence specific. Single-stranded RNA of MHV, VSV, or cellular origin, a DNA probe of the MHV leader sequence, and double-stranded bovine rotavirus RNA could all bind to N. Binding of MHV RNA was optimal between pH 7 and 8, and the RNA could be eluted in 0.1 M NaCl. The ROPBA is a useful method for the initial identification of RNA-binding proteins, such as N and the 140K protein of murine coronavirus.     

Mouse hepatitis virus strain JHM infects a human hepatocellular carcinoma cell line

Mouse hepatitis virus (MHV) strain JHM is a coronavirus that causes encephalitis and demyelination in susceptible rodents. The known receptors for MHV are all members of the carcinoembryonic antigen family. Although human forms of the MHV receptor can function as MHV receptors in some assays, no human cell line has been identified that can support wild-type MHV infection. Here we describe the infection of a human hepatocellular carcinoma cell line, HuH-7, with MHV. HuH-7 cells were susceptible to strains JHM-DL and JHM-DS, yielding virus titers nearly identical to those seen in mouse DBT cells. In contrast, HuH-7 cells were only marginally susceptible or completely resistant to infection by other MHV strains, including A59. JHM produced a strong cytopathic effect in HuH-7 cells with the formation of round plaques. Studies of various recombinant viruses between JHM and A59 strains suggested that the ability of JHM to infect HuH-7 cells was determined by multiple viral genetic elements. Blocking the viral spike (S) protein with a neutralizing antibody or a soluble form of the MHV receptor inhibited infection of HuH-7 cells, suggesting that infection is mediated through the S protein. Transfection with the prototype mouse receptor, biliary glycoprotein, rendered HuH-7 cells susceptible to infection by other MHV strains as well, suggesting that JHM uses a receptor distinct from the classical MHV receptor to infect HuH-7 cells. Possible implications for human disease are discussed.     

The biological relationship of mouse hepatitis virus (MHV) strains and interferon:In vitro induction and sensitivities

Summary Five prototype strains of mouse hepatitis virus (MHV) -1,-3, -S,- A59 and -JHM were analyzed for their ability to induce interferon (IFN) in seven cell lines of rodent origin. Induction of IFN by all of the prototype MHV strains was infrequent and unpredictable, while IFN was produced consistently by five cell lines treated with known inducers. Priming and/or aging of cells did not enhance IFN induction by the MHV strains except in the case of MHV-A59 which consistently induced moderate levels of IFN on L-cells which were both primed and aged. Kinetic studies of MHV-A59-induced IFN on primed and aged L-cells demonstrated that detectable levels of IFN were not produced until 24 hours post-inoculation (p.i.). Peak levels were attained at 30 hours p.i. with no additional IFN produced through 48 hours p.i. MHV-induced IFN was similar in composition and properties to Newcastle disease virus-induced IFN.The sensitivities of the five MHV strains to eight concentrations of preformed L-cell IFN were also assessed. All strains except MHV-S fit a linear model with MHV-3, MHV-A59 and MHV-JHM having similar slopes. At most concentrations MHV-3 was less sensitive than MHV-1, -A59 or -JHM to IFN. The response curve for MHV-S was non-linear. This strain was more sensitive to the antiviral effects of the pre-formed IFN except at the highest concentrations of IFN used.With 1 Figure     

New strain of mouse hepatitis virus as the cause of lethal enteritis in infant mice.

A new strain of mouse hepatitis virus (MHV) was isolated from pooled gut suspensions from an epizootic of lethal enteritis in newborn mice. Negative-contrast electron microscopy showed an abundance of coronavirus particles in the intestinal contents and intestinal epithelium of moribund mice. We found no other virus in the epizootic. Dams seroconverted to MHV polyvalent antigen and to the agent isolated, but did not develop antibodies to other known mouse pathogens. Virus propagated in NCTC-1469 tissue culture produced enteric disease in suckling mice but not fatal diarrhea; the dams of these mice also developed antibodies to MHV and to the isolates. By complement fixation, single radial hemolysis, and quantal neutralization tests, we found the isolates antigenically most closely related to MHV-S, unilaterally related to MHV-JHM, and more distantly related to MHV-1, MHV-3, MHV-A59, and human coronavirus OC-43. We also studied cross-reactions among the murine and human coronaviruses in detail. Tissues of infected newborn mice were examined by light microscopy, thin-section electron microscopy, and frozen-section indirect immunofluorescence, revealing that viral antigen, virus particles, and pathological changes were limited to the intestinal tract. We have designated our isolates as MHV-S/CDC.     

The ns 4 gene of mouse hepatitis virus (MHV), strain A 59 contains two ORFs and thus differs from ns 4 of the JHM and S strains

Summary The sequence of the MHV-A 59 non-structural gene 4 (ns 4) reveals two open reading frames. The upstream ORF potentially encodes a 19 amino acid (2.2 kDa) polypeptide and the downstream ORF potentially encodes a 106 amino acid (11.7 kDa) polypeptide. This is in contrast to MHV-JHM gene 4 which expresses a 15 kDa protein. Cell free translation of a synthetic mRNA containing both ORFs of MHV-A 59 ns 4 results in the synthesis of a 2.2 kDa poylpeptide; the predicted 11.7 kDa product of the MHV-A 59 downstream ORF is not detected during cell free translation nor in infected cells. These results add to the recent data suggesting that expression of some of the ns proteins of MHV is not necessary for efficient growth in tissue culture.     

Murine coronavirus spike glycoprotein mediates degree of viral spread,inflammation, and virus-induced immunopathology in the central nervous system

The mouse hepatitis virus (MHV) spike glycoprotein is a major determinant of neurovirulence. We investigated how alterations in spike affect neurovirulence using two isogenic recombinant viruses differing exclusively in spike. S(4)R, containing the MHV-4 spike gene, is dramatically more neurovirulent than S(A59)R, containing the MHV-A59 spike gene (J. J. Phillips, M. M. Chua, E. Lavi, and S. R. Weiss, 1999, J. Virol. 73, 7752-7760). We examined the contribution of differences in cellular tropism, viral spread, and the immune response to infection to the differential neurovirulence of S(4)R and S(A59)R. MHV-4 spike-mediated neurovirulence was associated with extensive viral spread in the brain in both neurons and astrocytes. Infection of primary hippocampal neuron cultures demonstrated that S(4)R spread more rapidly than S(A59)R and suggested that spread may occur between cells in close physical contact. In addition, S(4)R infection induced a massive influx of lymphocytes into the brain, a higher percentage of CD8(+) T cells, and a higher frequency of MHV-specific CD8(+) T cells relative S(A59)R infection. Despite this robust and viral-specific immune response to S(4)R infection, infection of RAG1-/- mice suggested that immune-mediated pathology also contributes to the high neurovirulence of S(4)R.