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. 2004 Sep;78(17):9073-83.
doi: 10.1128/JVI.78.17.9073-9083.2004.

The N-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells

Jeanne H Schickli  1 , Larissa B Thackray, Stanley G Sawicki, Kathryn V Holmes
Affiliations

The N-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells

Jeanne H Schickli et al. J Virol. 2004 Sep.

Abstract

Although murine coronaviruses naturally infect only mice, several virus variants derived from persistently infected murine cell cultures have an extended host range. The mouse hepatitis virus (MHV) variant MHV/BHK can infect hamster, rat, cat, dog, monkey, and human cell lines but not the swine testis (ST) porcine cell line (J. H. Schickli, B. D. Zelus, D. E. Wentworth, S. G. Sawicki, and K. V. Holmes, J. Virol. 71:9499-9507, 1997). The spike (S) gene of MHV/BHK had 63 point mutations and a 21-bp insert that encoded 56 amino acid substitutions and a 7-amino-acid insert compared to the parental MHV strain A59. Recombinant viruses between MHV-A59 and MHV/BHK were selected in hamster cells. All of the recombinants retained 21 amino acid substitutions and a 7-amino-acid insert found in the N-terminal region of S of MHV/BHK, suggesting that these residues were responsible for the extended host range of MHV/BHK. Flow cytometry showed that MHV-A59 bound only to cells that expressed the murine glycoprotein receptor CEACAM1a. In contrast, MHV/BHK and a recombinant virus, k6c, with the 21 amino acid substitutions and 7-amino-acid insert in S bound to hamster (BHK) and ST cells as well as murine cells. Thus, 21 amino acid substitutions and a 7-amino-acid insert in the N-terminal region of the S glycoprotein of MHV/BHK confer the ability to bind and in some cases infect cells of nonmurine species.

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Figures

FIG. 1.
FIG. 1.
MHV/pi23 inefficiently infects hamster BHK cells. 17 Cl 1 cells and BHK cells were inoculated with MHV/pi23 at a multiplicity of infection of 3 to 5 (as determined by plaque assay in 17 Cl 1 cells) and fixed at 24 h postinoculation Viral nucleocapsid protein was detected by immunolabeling with anti-N MAb followed by fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin antibody. Magnification, 300×.
FIG. 2.
FIG. 2.
Comparison of the 12-bp insert of MHV/pi23 with the 21-bp insert of MHV/BHK. Compared to MHV-A59, MHV/pi23 has a 12-bp insert (bold). Eight of the nine nucleotides immediately upstream of the 12-bp inset of MHV/pi23 (italicized) match the 5′-most nine nucleotides (italicized) of the 21-bp insert (bold) of MHV/BHK.
FIG. 3.
FIG. 3.
Plaque morphology differs between recombinant viruses. Twice-plaque-purified recombinant viruses were plaqued in 17 Cl 1 cells at 37°C and BHK cells at 37 or 39.5°C. Plaques were visualized by neutral red staining and photographed 4 days postinoculation.
FIG. 4.
FIG. 4.
Expression of viral nucleocapsid protein differs between recombinant viruses. Following plaque purification, recombinant viruses were inoculated onto 17 Cl 1 or BHK cells at a multiplicity of infection of 3 to 5 PFU/cell, and the cells were fixed 24 h postinoculation. Viral nucleocapsid protein was visualized by labeling with anti-N MAb followed by fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin antibody. Magnification, 300×.
FIG. 5.
FIG. 5.
Restriction enzyme digestion analysis of recombinant viruses with HaeIII, AvrII, and NcoI. RT-PCR of the S genes of recombinant viruses was performed with primers 8 and 19 (Table 1). The RT-PCR products were digested with either HaeIII (A) or AvrII (B). In addition, RT-PCR of the S genes was performed with primers 9 and 16 (Table 1), and the RT-PCR products were digested by NcoI (C). MHV-A59 has one HaeIII site that yielded two fragments of 256 and 190 bp. MHV/BHK has one AvrII site that yielded two fragments of 349 and 97 bp and one NcoI site that yielded two fragments of 391 and 161 bp.
FIG. 6.
FIG. 6.
Recombinant viruses that infect BHK cells have the 5′ region of the S gene from MHV/BHK. Recombinant viruses had a single crossover event in one of three regions of the S gene, as determined by restriction enzyme digestion analysis and sequencing of RT-PCR products. Each of the representative recombinants shown retained the 21-bp insert (triangles) and 24 point mutations upstream of the insert found in the S gene of MHV/BHK.
FIG. 7.
FIG. 7.
Binding of viruses to cells of different species. Expression of mCEACAM1a on cells treated with the blocking anti-mCEACAM1a MAb CC1 or an isotype control MAb followed by phycoerythrin-conjugated goat anti-mouse immunoglobulin antibody is illustrated in panels A, B, C, D, and E. The percentage of cells positive for mCEACAM1a expression compared to the isotype control is also indicated. Binding of medium alone (no virus), MHV-A59, MHV/BHK, or the recombinant virus k6c to cells was detected with anti-S2 MAb 5B19 or an isotype control MAb followed by phycoerythrin-conjugated goat anti-mouse immunoglobulin antibody (F through Y). The percentage of cells positive for bound virus compared to the isotype control is also indicated. Flow cytometry data shown are representative of two or more independent experiments.
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References

    1. Baric, R. S., E. Sullivan, L. Hensley, B. Yount, and W. Chen. 1999. Persistent infection promotes cross-species transmissibility of mouse hepatitis virus. J. Virol. 73:638-649. - PMC - PubMed
    1. Baric, R. S., B. Yount, L. Hensley, S. A. Peel, and W. Chen. 1997. Episodic evolution mediates interspecies transfer of a murine coronavirus. J. Virol. 71:1946-1955. - PMC - PubMed
    1. Beauchemin, N., T. Chen, P. Draber, G. Dveksler, P. Gold, S. Gray-Owen, F. Grunert, S. Hammarstrom, K. V. Holmes, A. Karlson, M. Kuroki, S. H. Lin, L. Lucka, S. M. Najjar, M. Neumaier, B. Obrink, J. E. Shively, K. M. Skubitz, C. P. Stanners, P. Thomas, J. A. Thompson, M. Virji, S. von Kleist, C. Wagener, S. Watt, and W. Zimmermann. 1999. Redefined nomenclature for members of the carcinoembryonic antigen family. Exp. Cell Res. 252:243-249. - PubMed
    1. Bos, E. C., L. Heijnen, W. Luytjes, and W. J. Spaan. 1995. Mutational analysis of the murine coronavirus spike protein: effect on cell-to-cell fusion. Virology 214:453-463. - PMC - PubMed
    1. Bosch, B. J., R. van der Zee, C. A. de Haan, and P. J. Rottier. 2003. The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex. J. Virol. 77:8801-8811. - PMC - PubMed

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