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Journal of Virology, February 2003, p. 2530-2538, Vol. 77, No. 4
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.4.2530-2538.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Identification of a Receptor-Binding Domain of the Spike Glycoprotein of Human Coronavirus HCoV-229E

Aurelio Bonavia,1,{dagger} Bruce D. Zelus,1 David E. Wentworth,1 Pierre J. Talbot,2 and Kathryn V. Holmes1,3*

Department of Microbiology,1 Molecular Biology Program, University of Colorado Health Sciences Center, Denver, Colorado 80262,3 INRS-Institut Armand-Frappier, Laval, Québec, Canada H7V 1B72

Received 2 April 2002/ Accepted 19 November 2002

Human coronavirus HCoV-229E uses human aminopeptidase N (hAPN) as its receptor (C. L. Yeager et al., Nature 357:420-422, 1992). To identify the receptor-binding domain of the viral spike glycoprotein (S), we expressed soluble truncated histidine-tagged S glycoproteins by using baculovirus expression vectors. Truncated S proteins purified by nickel affinity chromatography were shown to be glycosylated and to react with polyclonal anti-HCoV-229E antibodies and monoclonal antibodies to the viral S protein. A truncated protein (S547) that contains the N-terminal 547 amino acids bound to 3T3 mouse cells that express hAPN but not to mouse 3T3 cells transfected with empty vector. Binding of S547 to hAPN was blocked by an anti-hAPN monoclonal antibody that inhibits binding of virus to hAPN and blocks virus infection of human cells and was also blocked by polyclonal anti-HCoV-229E antibody. S proteins that contain the N-terminal 268 or 417 amino acids did not bind to hAPN-3T3 cells. Antibody to the region from amino acid 417 to the C terminus of S blocked binding of S547 to hAPN-3T3 cells. Thus, the data suggest that the domain of the spike protein between amino acids 417 and 547 is required for the binding of HCoV-229E to its hAPN receptor.

* Corresponding author. Mailing address: Department of Microbiology, Campus Box B-175, University of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80220. Phone: (303) 315-7903. Fax: (303) 315-6785. E-mail: kathryn.holmes{at}uchsc.edu.

{dagger} Present address: Department of Comparative Medicine, Johns Hopkins University School of Medicine, Retrovirus Laboratory, Baltimore, MD 21287.

Journal of Virology, February 2003, p. 2530-2538, Vol. 77, No. 4
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.4.2530-2538.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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