Unique N-linked glycosylation of murine coronavirus MHV-2 membrane protein at the conserved O-linked glycosylation site

The membrane (M) proteins of murine coronavirus (MHV) strains have been reported to contain only O-linked oligosaccharides. The predicted O-glycosylation site consisting of four amino acid residues of Ser-Ser-Thr-Thr is located immediately adjacent to the initiator Met and is well conserved among MHV strains investigated so far. We analyzed the nucleotide sequence of a highly virulent strain MHV-2 M-coding region and demonstrated that MHV-2 had a unique amino acid, Asn, at position 2 at the conserved O-glycosylation site. We also demonstrated that this substitution added N-linked glycans to MHV-2 M protein resulting in increment of molecular mass of MHV-2 M protein compared with JHM strain having only O-linked glycans.     

Common RNA replication signals exist among group 2 coronaviruses: evidence for in vivo recombination between animal and human coronavirus molecules

5' and 3' UTR sequences on the coronavirus genome are known to carry cis-acting elements for DI RNA replication and presumably also virus genome replication. 5' UTR-adjacent coding sequences are also thought to harbor cis-acting elements. Here we have determined the 5' UTR and adjacent 289-nt sequences, and 3' UTR sequences, for six group 2 coronaviruses and have compared them to each other and to three previously reported group 2 members. Extensive regions of highly similar UTR sequences were found but small regions of divergence were also found indicating group 2 coronaviruses could be subdivided into those that are bovine coronavirus (BCoV)-like (BCoV, human respiratory coronavirus-OC43, human enteric coronavirus, porcine hemagglutinating encephalomyelitis virus, and equine coronavirus) and those that are murine hepatitis virus (MHV)-like (A59, 2, and JHM strains of MHV, puffinosis virus, and rat sialodacryoadenitis virus). The 3' UTRs of BCoV and MHV have been previously shown to be interchangeable. Here, a reporter-containing BCoV DI RNA was shown to be replicated by all five BCoV-like helper viruses and by MHV-H2 (a human cell-adapted MHV strain), a representative of the MHV-like subgroup, demonstrating group 2 common 5' and 3' replication signaling elements. BCoV DI RNA, furthermore, acquired the leader of HCoV-OC43 by leader switching, demonstrating for the first time in vivo recombination between animal and human coronavirus molecules. These results indicate that common replication signaling elements exist among group 2 coronaviruses despite a two-cluster pattern within the group and imply there could exist a high potential for recombination among group members.     

Primary structure and translation of a defective interfering RNA of murine coronavirus

An intracellular defective-interfering (DI) RNA, DIssE, of mouse hepatitis virus (MHV) obtained after serial high multiplicity passage of the virus was cloned and sequenced. DIssE RNA is composed of three noncontiguous genomic regions, representing the first 864 nucleotides of the 5' end, an internal 748 nucleotides of the polymerase gene, and 601 nucleotides from the 3' end of the parental MHV genome. The DIssE sequence contains one large continuous open reading frame. Two protein products from this open reading frame were identified both by in vitro translation and in DI-infected cells. Sequence comparison of DIssE and the corresponding parts of the parental virus genome revealed that DIssE had three base substitutions within the leader sequence and also a deletion of nine nucleotides located at the junction of the leader and the remaining genomic sequence. The 5' end of DIssE RNA was heterogeneous with respect to the number of UCUAA repeats within the leader sequence. The parental MHV genomic RNA appears to have extensive and stable secondary structures at the regions where DI RNA rearrangements occurred. These data suggest that MHV DI RNA may have been generated as a result of the discontinuous and nonprocessive manner of MHV RNA synthesis.     

Multiple recombination sites at the 5'-end of murine coronavirus RNA

Mouse hepatitis virus (MHV), a murine coronavirus, contains a nonsegmented RNA genome. We have previously shown that MHV could undergo RNA-RNA recombination in crosses between temperature-sensitive mutants and wild-type viruses at a very high frequency (S. Makino, J.G. Keck, S.A. Stohlman, and M.M.C. Lai (1986) J. Virol. 57, 729-737). To better define the mechanism of RNA recombination, we have performed additional crosses involving different sets of MHV strains. Three or possibly four classes of recombinants were isolated. Recombinants in the first class, which are similar to the ones previously reported, contain a single crossover in either gene A or B, which are the 5'-most genes. The second class of recombinants contain double crossovers in gene A. The third class of recombinants have crossovers within the leader sequence located at the 5'-end of the genome. The crossover sites of the third class have been located between 35 and 60 nucleotides from the 5'-end of the leader RNA. One of these recombinants has double crossovers within the short region comprising the leader sequences. Finally, we describe one recombinant which may contain a triple crossover. The presence of so many recombination sites within the 5'-end of the genome of murine coronaviruses confirms that RNA recombination is a frequent event during MHV replication and is consistent with our proposed model of "copy-choice" recombination in which RNA replication occurs in a discontinuous and nonprocessive manner.     

Sequence analysis reveals extensive polymorphism and evidence of deletions within the E2 glycoprotein gene of several strains of murine hepatitis virus

Direct RNA sequence analysis of the E2 gene of wild-type MHV-4 and of neutralization resistant, neuroattenuated variants has identified a polymorphic region with respect to deletions. These variants had large deletions of 142 to 159 amino acids mapping to a localized region in the amino-terminal domain of the peplomer glycoprotein. The nucleotide sequence of the E2 gene for wild-type strain MHV-4 was found to be very similar to that of MHV-JHM but had an insertion of 423 nucleotides resulting in the addition of a stretch of 141 unique amino acids in the amino-terminal domain of E2. We propose that deletions reflect a major source of heterogeneity in the E2 protein of MHV.     

Correlation between growth potential of mouse hepatitis viruses in macrophages and their virulence for mice.

Correlation between the virulence of mouse hepatitis virus (MHV) for mice and the growth potential of the virus in peritoneal adherent cells was observed for highly virulent MHV-2 and avirulent MHV-1, JHM, and MHV-S strains. However, this phenomenon was not observed in strain MHV-3, which multiplied to almost the same degree in peritoneal adherent cells from susceptible and resistant mouse strains.     

RNA and polypeptide homology among murine coronaviruses

Using a ³²P complementary DNA (cDNA) prepared against the A59 nucleocapsid protein messenger RNA, we have investigated the extent of homology between A59 and four other strains of murine hepatitis virus (MHV). Analysis by hybridization kinetics of the annealing between A59 [³²P]cDNA and infected cell RNA from the other four MHV strains demonstrated 70–80% homology. By gel transfer analysis, the A59 [³²P]cDNA was able to detect subgenomic-size virus-specific RNAs in cells infected with all of the five MHV strains. A similar pattern of seven viral RNAs was detected in cells infected with A59, MHV-1, MHV-3, and JHM. In contrast, cells infected with MHV-S contained seven virus-specific RNAs, of which only the two smallest species comigrated with RNAs from the other four strains. The results suggest that as previously shown with A59 (S. Cheley, R. Anderson, M. J. Cupples, E. C. M. Lee Chan, and V. L. Morris (1981)Virology, 112, 596–604), all MHV strains tested encode a nested set of subgenomic RNAs. Analysis of [³⁵S]methionine-labeled viral proteins by SDS-polyacrylamide gel electrophoresis revealed that each strain of MHV specified four major viral polypeptides with apparent molecular weights very similar to those previously reported for the E2, N, El, and PEI polypeptides of A59. The strong degree of interstrain homology among the five MHV strains investigated was confirmed by comparative chymotryptic peptide mapping of the viral N proteins. A majority of the chymotryptic peptides from each of the [³⁵Sknethionine-labeled N proteins was found to coelute by high-performance liquid chromotography. Moreover, this technique of peptide mapping indicated a particularly strong relatedness between MHV-1 and MHV-S and among MHV-3, JHM, and A59.     

Sequence analysis of the leader RNA of two porcine coronaviruses: Transmissible gastroenteritis virus and porcine respiratory coronavirus

The leader RNA sequence was determined for two pig coronaviruses, tranmissible gastroenteritis virus (TGEV), and porcine respiratory coronavirus (PRCV). Primer extension, of a synthetic oligonucleotide complementary to the 5 end of the nucleoprotein gene of TGEV was used to produce a single-stranded DNA copy of the leader RNA from the nucleoprotein mRNA species from TGEV and PRCV, the sequences of which were determined by Maxam and Gilbert cleavage. Northern blot analysis, using a synthetic oligonucleotide complementary to the leader RNA, showed that the leader RNA sequence was present on all of the subgenomic mRNA species. The porcine coronavirus leader RNA sequences were compared to each other and to published coronavirus leader RNA sequences. Sequence homologies and secondary structure similarities were identified that may play a role in the biological function of these RNA sequences.The nucleotide sequence data reported in this paper have been submitted to the EMBL/Genbank/DDBJ nucleotide sequence databases and have been assigned the accession numbers X52157, X52668.     

Mutagenesis of the murine hepatitis virus nsp1-coding region identifies residues important for protein processing, viral RNA synthesis, and viral replication

Despite ongoing research investigating mechanisms of coronavirus replication, functions of many viral nonstructural proteins (nsps) remain unknown. In the current study, a reverse genetic approach was used to define the role of the 28-kDa amino-terminal product (nsp1) of the gene 1 polyprotein during replication of the coronavirus murine hepatitis virus (MHV) in cell culture. To determine whether nsp1 is required for MHV replication and to identify residues critical for protein function, mutant viruses that contained deletions or point mutations within the nsp1-coding region were generated and assayed for defects in viral replication, viral protein expression, protein localization, and RNA synthesis. The results demonstrated that the carboxy-terminal half of nsp1 (residues K(124) through L(241)) was dispensable for virus replication in culture but was required for efficient proteolytic cleavage of nsp1 from the gene 1 polyprotein and for optimal viral replication. Furthermore, whereas deletion of nsp1 residues amino-terminal to K(124) failed to produce infectious virus, point mutagenesis of the nsp1 amino-terminus allowed recovery of several mutants with altered replication and RNA synthesis. This study identifies nsp1 residues important for protein processing, viral RNA synthesis, and viral replication.     

Coronavirus defective-interfering RNA as an expression vector: the generation of a pseudorecombinant mouse hepatitis virus expressing hemagglutinin-esterase

We have developed an expression vector system using a defective-interfering (DI) RNA of mouse hepatitis virus (MHV), a prototype coronavirus, to deliver and express a foreign gene in MHV-infected cells. This vector contains an MHV intergenic sequence to promote the expression of foreign genes. In this study, we used this vector to introduce a hemagglutinin-esterase (HE) protein, an optional MHV structural protein, into the MHV-infected cells. The engineered HE protein could be efficiently incorporated into the virion which did not synthesize its own HE protein, thus generating a pseudorecombinant virus that expresses an exogenous HE protein. The engineered HE protein could be made distinguishable from the native protein by attaching an 8-amino-acid peptide tag at the carboxyl-terminus. Both the engineered and native HE proteins from the HE-producing virus train could be incorporated into the virion, thus generating phenotypically mixed virus particles. We also showed that the HE-expressing DI RNA could be incorporated into viruses, and the engineered HE protein expressed in the infected cells for at least three serial virus passages. Furthermore, we have made two mutants, in which parts of the external domain of the HE protein have been deleted, to study the sequence requirements for the stable expression of HE and its incorporation into MHV virions. Although both of the mutant HE proteins could be expressed in the MHV-infected cells, they failed to be incorporated into virions, suggesting the importance of the extracellular domain of HE protein for its incorporation into virus particles. This vector system enabled the first successful incorporation of a selected coronaviral protein into virions and demonstrates its utility as an expression vector for studying the molecular biology of coronaviruses.     

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.     

Sequence comparison of the N genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein

To obtain information about the structure and evolution of the nucleocapsid (N) protein of the coronavirus mouse hepatitis virus (MHV), we determined the entire nucleotide sequences of the N genes of MHV-A59, MHV-3, MHV-S, and MHV-1 from cDNA clones. At the nucleotide level, the N gene sequences of these viral strains, and that of MHV-JHM, were more than 92% conserved overall. Even higher nucleotide sequence identity was found in the 3' untranslated regions (3' UTRs) of the five strains, which may reflect the role of the 3' UTR in negative-strand RNA synthesis. All five N genes were found to encode markedly basic proteins of 454 or 455 residues having at least 94% sequence identity in pairwise comparisons. However, amino acid sequence divergences were found to be clustered in two short segments of N, putative spacer regions that, together, constituted only 11% of the molecule. Thus, the data suggest that the MHV N protein is composed of three highly conserved structural domains connected to each other by regions that have much less constraint on their amino acid sequences. The first two conserved domains contain most of the excess of basic amino acid residues; by contrast, the carboxy-terminal domain is acidic. Finally, we noted that four of the five N genes contain an internal open reading frame that potentially encodes a protein of 207 amino acids having a large proportion of basic and hydrophobic residues.     

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.