Expression, Glycosylation, and Modification of the Spike (S) Glycoprotein of SARS CoV

  • Shuo Shen
  • Timothy H. P. Tan
  • Yee-Joo Tan
Part of the Methods in Molecular Biology book series (MIMB, volume 379)


The spike (S) glycoprotein of coronaviruses is known to be essential in the binding of the virus to the host cell at the advent of the infection process. To study the maturation pathway of the S glycoprotein of the severe acute respiratory syndrome (SARS)-coronavirus (CoV) within the host cell, a T7/vaccinia virus-based expression system coupled to immunoprecipitation with anti-S antibodies was used to test and analyze different forms of the S glycoprotein. The state of maturity of the S glycoprotein can be deduced from its sensitivity to hydrolysis by endoglycosidase H (EndoH) or N-glycosidase F (N-Gly F). A fully matured S glycoprotein will be modified with complex oligosaccharides which makes it resistant to cleavage by EndoH but not by N-Gly F. By exploiting this characteristic, it is then possible to determine which forms of the immunoprecipitated S protein are properly processed by the host cell. With this system, many different constructs of the S glycoprotein can be analyzed in parallel thus providing another method by which to study the functional domains of S involved in membrane fusion event that occurs during viral infection.

Key Words

Severe acute respiratory syndrome (SARS) coronavirus spike glycoprotein maturation membrane fusion endoglycosidase H 


  1. 1.
    Matsuyama, S., Ujike, M., Morikawa, S., Tashiro, M., and Taguchi, F. (2005) Protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection. Proc. Natl. Acad. Sci. USA 102, 12,543–12,547.CrossRefPubMedGoogle Scholar
  2. 2.
    Lip, K. M., Shen, S., Yang, X., et al. (2006) Monoclonal antibodies targeting the HR2 domain and the region immediately upstream of the HR2 of the S protein neutralize in vitro infection of severe acute respiratory syndrome coronavirus. J. Virol. 80, 941–950.CrossRefPubMedGoogle Scholar
  3. 3.
    Hebert, D. N., Zhang, J. X., Chen, W., Foellmer, B., and Helenius, A. (1997) The number and location of glycans on influenza hemagglutinin determine folding and association with calnexin and calreticulin. J. Cell Biol. 139, 613–623.CrossRefPubMedGoogle Scholar
  4. 4.
    Shen, S. Y. C., Law, Y. C., and Liu, D. X. (2004) Single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature. Virology 326, 288–298.CrossRefPubMedGoogle Scholar
  5. 5.
    Ruan, Y. J., Wei, C. L., Ee, A. L., et al. (2003) Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection. Lancet 316, 1779–1785.CrossRefGoogle Scholar
  6. 6.
    Liu, D. X., Brierley, I., Tibbies, K. W., and Brown, T. D. (1994) A 100-kilodalton polypeptide encoded by open reading frame (ORF) 1b of the coronavirus infectious bronchitis virus is processed by ORF 1a products. J. Virol. 68, 5772–5780.PubMedGoogle Scholar
  7. 7.
    Keng, C. T., Zhang, A., Shen, S., et al. (2005) Amino acids 1055 to 1192 in the S2region of severe acute respiratory syndrome coronavirus S protein induce neutral-izing antibodies: implications for the development of vaccines and antiviral agents. J. Virol. 79, 3289–3296.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Shuo Shen
    • 1
  • Timothy H. P. Tan
    • 1
  • Yee-Joo Tan
    • 1
  1. 1.Collaborative Antiviral Research Group, Institute of Molecular and Cell BiologyProteosSingapore

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