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Journal of Virology, November 2006, p. 10315-10324, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.01138-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identifying Epitopes Responsible for Neutralizing Antibody and DC-SIGN Binding on the Spike Glycoprotein of the Severe Acute Respiratory Syndrome Coronavirus

Yi-Ping Shih,1,2 Chia-Yen Chen,1,2 Shih-Jen Liu,3 Kuan-Hsuan Chen,1 Yuan-Ming Lee,2,4 Yu-Chan Chao,5 and Yi-Ming Arthur Chen1,2*

AIDS Prevention and Research Center, National Yang-Ming University,1 Division of Preventive Medicine, Institute of Public Health, School of Medicine National Yang-Ming University,2 Vaccine Research and Development Center, National Health Research Institutes,3 Department of Pathology and Laboratory Diagnosis, Taipei Veterans General Hospital,4 Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China5

Received 2 June 2006/ Accepted 5 August 2006

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) uses dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) to facilitate cell entry via cellular receptor-angiotensin-converting enzyme 2. For this project, we used recombinant baculoviruses expressing different lengths of SARS-CoV spike (S) protein in a capture assay to deduce the minimal DC-SIGN binding region. Our results identified the region location between amino acid (aa) residues 324 to 386 of the S protein. We then generated nine monoclonal antibodies (MAbs) against the S protein to map the DC-SIGN-binding domain using capture assays with pseudotyped viruses and observed that MAb SIa5 significantly blocked S protein-DC-SIGN interaction. An enhancement assay using the HKU39849 SARS-CoV strain and human immature dendritic cells confirmed our observation. Data from a pepscan analysis and M13 phage peptide display library system mapped the reactive MAb SIa5 epitope to aa residues 363 to 368 of the S protein. Results from a capture assay testing three pseudotyped viruses with mutated N-linked glycosylation sites of the S protein indicate that only two pseudotyped viruses (N330Q and N357Q, both of which lost glycosylation sites near the SIa5 epitope) had diminished DC-SIGN-binding capacity. We also noted that MAb SIb4 exerted a neutralizing effect against HKU39849; its reactive epitope was mapped to aa residues 435 to 439 of the S protein. We offer the data to facilitate the development of therapeutic agents and preventive vaccines against SARS-CoV infection.

* Corresponding author. Mailing address: AIDS Prevention and Research Center, National Yang-Ming University, Taipei 111, Taiwan, Republic of China. Phone: 886 2 28267304. Fax: 886 2 28270576. E-mail: arthur{at}ym.edu.tw.

Journal of Virology, November 2006, p. 10315-10324, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.01138-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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  • Han, D. P., Lohani, M., Cho, M. W. (2007). Specific Asparagine-Linked Glycosylation Sites Are Critical for DC-SIGN- and L-SIGN-Mediated Severe Acute Respiratory Syndrome Coronavirus Entry. J. Virol. 81: 12029-12039 [Abstract] [Full Text]  


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