A novel cell-based binding assay system reconstituting interaction between SARS-CoV S protein and its cellular receptor.
Severe acute respiratory syndrome (SARS), a life-threatening disease, is caused by the newly identified virus SARS coronavirus (SARS-CoV). 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 (1M NaCl). 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.
Chou
Chih-Fong
CF
Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore. mcbccf@imcb.a-star.edu.sg
Shen
Shuo
S
Tan
Yee-Joo
YJ
Fielding
Burtram C
BC
Tan
Timothy H P
TH
Fu
Jianlin
J
Xu
Qiurong
Q
Lim
Seng Gee
SG
Hong
Wanjin
W
D000818:Animals; D016466:CHO Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D011991:Receptors, Virus / Q000378:metabolism*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000378:metabolism*; D064370:Spike Glycoprotein, Coronavirus; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
D008562:Membrane Glycoproteins; D011991:Receptors, Virus; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D049452:Green Fluorescent Proteins