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. 2005 Mar 30;334(1):98-110.
doi: 10.1016/j.virol.2005.01.016.

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

Larissa B Thackray  1 , Brian C Turner, Kathryn V Holmes
Affiliations

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

Larissa B Thackray et al. Virology. .

Abstract

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.

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Figures

Fig. 1
Fig. 1
(A) Residues conserved in spike glycoproteins of murine coronaviruses. Four residues (black diamonds) are conserved in the N-terminal 330 aa of S proteins of MHV strains (MHV-A59, MHV-JHM, MHV-1, MHV-2, MHV-3, MHV-S and MHV-U) and an extended host range variant of MHV-A59 (MHV/BHK), but not in the N-termini of S proteins of related rat (RCoV), bovine (BCoV) and human (HCoV-OC43) coronaviruses in group II. Two residues (grey diamonds) conserved in S proteins of murine, rat, bovine and human coronaviruses in group II are also indicated. (B) Composition of S construct used to introduce mutations into MHV-A59 using targeted RNA recombination. Mutations were engineered into the S/pBC SK+ plasmid and subcloned into the pMH54 plasmid. The minimal receptor binding domain (RBD) for murine CEACAM1a is shaded in gray.
Fig. 2
Fig. 2
Growth of EGFP viruses in murine 17 Cl 1 or hamster BHK + mCEACAM1a(I41R) cells. EGFP was visualized in cell monolayers overlaid with fMHV-inoculated Fcwf cells transfected with wild type (SA59) or mutant pMH54-EGFP RNAs. None of the 17 Cl 1 or BHK + mCEACAM1a(I41R) cells overlaid with mock-inoculated Fcwf cells transfected with wild type or mutant pMH54-EGFP RNAs exhibited EGFP expression. Magnification, 200×.
Fig. 3
Fig. 3
Plaque morphologies of EGFP viruses on murine 17 Cl 1 cell monolayers. Plaques of the SA59, Y162F and Y162A-EGFP viruses were visualized at 72 h post-inoculation (p.i.) by neutral red staining (neutral red) or immunoperoxidase labeling of viral nucleocapsid protein (N antigen) in cell monolayers. Plaques formed by the Y162A-EGFP virus are indicated by black arrows.
Fig. 4
Fig. 4
Plaque morphologies of recombinant viruses on murine 17 Cl 1 cell monolayers. Plaques of MHV-A59 or the plaque-purified recombinant viruses were visualized at 72 h p.i. by neutral red staining (neutral red) or immunoperoxidase labeling of viral nucleocapsid protein (N antigen) in cell monolayers.
Fig. 5
Fig. 5
Neutralization of recombinant viruses with soluble, murine CEACAM1a. MHV-A59 or the recombinant viruses (5000 PFU) were pre-incubated with 70 nM of soluble mCEACAM1a[1,4]. Virus survival was determined and percent neutralization calculated as described in Materials and methods. Averages of percent neutralization ± SEM of 2 independent experiments are shown.
Fig. 6
Fig. 6
Growth of recombinant viruses during anti-receptor antibody blockade of murine 17 Cl 1 cells. Yields of MHV-A59 or the recombinant viruses at 24 h p.i. from cells treated with 0.05 mg/ml of anti-CEACAM1a MAb-CC1 or isotype matched MAb-Ctrl before, during and after inoculation. The yield of the L79A/T82A B virus was assayed at 72 h p.i. due the low titer of this virus. Virus yields shown are representative of two independent experiments.
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