The glycosylation status of the murine hepatitis coronavirus M protein affects the interferogenic capacity of the virus in vitro and its ability to replicate in the liver but not the brain

Abstract

The coronavirus M protein, the most abundant coronaviral envelope component, is invariably glycosylated, which provides the virion with a diffuse, hydrophilic cover on its outer surface. Remarkably, while the group 1 and group 3 coronaviruses all have M proteins with N-linked sugars, the M proteins of the group 2 coronaviruses [e.g., mouse hepatitis virus (MHV)] are O-glycosylated. The conservation of the N- and O-glycosylation motifs suggests that each of these types of carbohydrate modifications is beneficial to their respective virus. Since glycosylation of the M protein is not required for virus assembly, the oligosaccharides are likely to be involved in the virus-host interaction. In order to investigate the role of the M protein glycosylation in the host, two genetically modified MHVs were generated by using targeted RNA recombination. The recombinant viruses carried M proteins that were either N-glycosylated or not glycosylated at all, and these were compared with the parental, O-glycosylated, virus. The M protein glycosylation state did not influence the tissue culture growth characteristics of the recombinant viruses. However, it affected their interferogenic capacity as measured using fixed, virus-infected cells. Viruses containing M proteins with N-linked sugars induced type I interferons to higher levels than viruses carrying M proteins with O-linked sugars. MHV with unglycosylated M proteins appeared to be a poor interferon inducer. In mice, the recombinant viruses differed in their ability to replicate in the liver, but not in the brain, whereas their in vivo interferogenic capacity did not appear to be affected by their glycosylation status. Strikingly, their abilities to replicate in the liver correlated with their in vitro interferogenic capacity. This apparent correlation might be explained by the functioning of lectins, such as the mannose receptor, which are abundantly expressed in the liver but also play a role in the induction of interferon-alpha by dendritic cells.

Fig. 1

Construction of MHV M protein glycosylation mutants. (A) Schematic of transcription vector pFV1 (Fischer et al., 1997), which contains a cDNA copy of the 5′ 467 nt of the MHV genome fused to the 3′ 7406 nt of the MHV genome. The T7 RNA polymerase promoter is denoted by an arrow. The EcoRV and BssHII sites, used for mutant plasmid construction, and the HindIII site, used for transcription vector truncation, are indicated. The expanded segments of sequence show the wild-type region encompassing the start of the M gene in pFV1, and, below this, the corresponding sequences of mutant vectors pCM1 to pCM4. Nucleotide point mutations or insertions are marked by stars, and the resulting changed amino acids are circled. The transcriptional regulatory sequence preceding the M gene is underlined. (B) Strategy for targeted recombination between recipient virus Alb4 and synthetic donor RNA transcribed from pFV1 or a mutant derivative of pFV1. The star indicates mutations transduced from the donor RNA into the recombinant genome. (C) Portions of sequence of purified genomic RNA isolated from virions of recombinants Alb244 and Alb246, which were derived from pCM1 and pCM2, respectively, and Alb248 and Alb250, which were derived from pCM3 and pCM4, respectively. Sequence was obtained using a primer complementary to nt 113 to 131 of the M protein coding region, and lanes labeled U, C, G, and A were from reactions terminated with ddATP, ddGTP, ddCTP, and ddTTP, respectively.

Fig. 2

Analysis of the synthesis of recombinant MHV proteins. LR7 cells were infected with recombinant viruses as indicated on the top of the gel. Cells were labeled for 3 h with ³⁵S-labeled amino acids starting 5 h postinfection. When indicated, tunicamycin (5 μg/ml) was added to the culture media from 2 h postinfection. At the end of the labeling period, culture media were collected and prepared for immunoprecipitation with the anti-MHV serum k134 The immunoprecipitates were analyzed by SDS–15%PAGE. The positions of the different proteins are indicated on the left.

Fig. 3

Induction of type I interferon by formaldehyde-fixed, coronavirus-infected cells. LMR cells were infected in duplicate with each recombinant virus or TGEV at a multiplicity of 0.1 or 10 PFU per cell. At 9.5 h postinfection cells were fixed with 3% formaldehyde. PBM cells were induced to produce IFNα by overnight incubation at 37°C on the TGEV- or MHV-infected, fixed cells. Supernatants were collected and serial twofold dilutions were assayed for IFN in an infection inhibition assay on LMR cells using VSV as a challenge virus. For each type of recombinant the mean value is shown, and the standard deviation is indicated. The IFN produced by the porcine PBM cells has been characterized as type I IFN, as it was neutralized by more than 90% by an antitype I IFN-antiserum Charley and Laude 1988, La Bonnardiere et al 1986.

Fig. 4

Replication of MHV recombinants in the brain. C57BI/6 mice were inoculated with the indicated recombinant intranasally at a dose of 1.4 × 10⁵ PFU. Mice were sacrificed on days 1, 3, 5, and 7 postinfection and virus titers in the brains were determined by plaque-assay on L2 cell monolayers.

Fig. 5

Replication of MHV recombinants in the liver. C57BI/6 mice were inoculated with the indicated recombinant viruses by injection into the intraperitoneal cavity. Mice were sacrificed and the livers were removed at day 4 postinfection. (A) Virus titers were determined by plaque-assay on LR7 cell monolayers following homogenization of the organs. P values determined by Student’s t test are indicated. (B) For immunohistochemical analysis, the liver samples were fixed in phosphate-buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin. Viral antigen was detected with the anti-MHV serum k134, using peroxidase conjugated swine immunoglobulins to rabbit immunoglobulins as secondary antibodies. Peroxidase was visualized using diaminobenzidine. Magnification: ×25 (left side) and ×50 (right side).

Fig. 6

Induction of IFNα in liver. C57BI/6 mice were inoculated with the indicated recombinant viruses by injection into the intraperitoneal cavity. Mice were sacrificed and the livers were removed at day 1 and 3 postinfection. IFNα levels were determined using a mouse IFNα ELISA kit and standard deviations are indicated.