A novel cell-based binding assay system reconstituting interaction between SARS-CoV S protein and its cellular receptor
Date
2005Author
Chou, Chih-Fong
Shen, Shuo
Tan, Yee-Joo
Fielding, Burtram C.
Tan, Timothy H.P.
Fu, Jianlin
Xu, Qiurong
Lim, Seng Gee
Hong, Wanjin
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Show full item recordAbstract
Severe acute respiratory syndrome (SARS), a life-threatening disease, is caused by the newly identified virus SARS coronavirus (SARSCoV).
In order to study the spike (S) protein of this highly contagious virus, we established a clonal cell-line, CHO-SG, from the Chinese
hamster ovary cells that stably expresses C-terminally EGFP-tagged SARS-CoV S protein (S-EGFP). The ectodomain of the S glycoprotein
is localized on the surface of CHO-SG cells with N-acetyl-glucosamine-terminated carbohydrate structure. CHO-SG cells associated tightly
with Vero E6 cells, a SARS-CoV receptor (ACE2) expressing cell-line, and the interaction remained stable under highly stringent condition
(1MNaCl). This interaction could be blocked by either the serum from a SARS convalescent patient or a goat anti-ACE2 antibody, indicating
that the interaction is specific. A binding epitope with lesser degree of glycosylation and native conformation was localized by using rabbit
anti-sera raised against five denatured recombinant S protein fragments expressed in Escherichia coli. One of the sera obtained from the
fragment encompassing amino acids 48-358 significantly blocked the interaction between CHO-SG and Vero E6 cells. The region is useful for
studying neutralizing antibodies in future vaccine development. This paper describes an easy and safe cell-based assay suitable for studying
the binding between SARS-CoV S protein and its receptor.