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. 2005 Jan;123(1):41-8.
doi: 10.1016/j.jviromet.2004.09.008.

A novel cell-based binding assay system reconstituting interaction between SARS-CoV S protein and its cellular receptor

Chih-Fong Chou  1 , Shuo Shen, Yee-Joo Tan, Burtram C Fielding, Timothy H P Tan, Jianlin Fu, Qiurong Xu, Seng Gee Lim, Wanjin Hong
Affiliations

A novel cell-based binding assay system reconstituting interaction between SARS-CoV S protein and its cellular receptor

Chih-Fong Chou et al. J Virol Methods. 2005 Jan.

Abstract

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.

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Figures

Fig. 1
Fig. 1
The construction and expression of S-EGFP. (A) The construct of C-terminal EGFP tagged SARS-CoV S fusion gene (S-EGFP) in the multiple cloning site (MCS) of pMMTC (Tan and Hong, 1999). The expressions were driven by the promoter (MTI) of mouse metallothionein 1 and an enhancer (MSV) from moloney murine sarcoma virus, and the Neo gene was responsible for the selection of G418 resistant cells. (B) The regular culture of CHO-SG, EGFP fluorescence of S-EGFP is localized along the plasma membrane shown as fluorescenced curves.
Fig. 2
Fig. 2
Western blot analysis to detect S-EGFP from CHO-SG cell lysate and samples prepared by using WGA affinity chromatography. Protein samples were resolved by 8% SDS PAGE and then transferred to a nitrocellulose membrane. Lanes A–C are the identification of S-EGFP in CHO-SG cell lysate. The same membrane was probed with three antibodies separately. Lane A, mouse monoclonal anti-GFP antibody; lane B, rabbit anti-S serum; and lane C, horse anti-S serum. S-EGFP is indicated by the arrowhead. Lanes D–F are the results of WGA chromatography. Lane D, CHO-SG cell lysate; lane E, the flow-through from the WGA column; and lane F, sample eluted from the WGA column. Rabbit anti-S antibody was used for lanes D–F.
Fig. 3
Fig. 3
CHO-SG expresses S-EGFP at cell surface with expected orientation. Non-permeable IFA using the serum (P6 in Tan et al., 2004a) from a recovered SARS patient showed cell surface staining of S-EGFP expressing CHO-SG cells. The control is normal human serum. The green fluorescence seen in the upper panels was from the EGFP tag and the red fluorescence observed in the lower panels was from rhodamine labeled secondary antibody.
Fig. 4
Fig. 4
CHO-SG and Vero E6 binding assay. In all panels, Vero E6 cells were first cultured to a confluent layer. (A) EDTA dislodged CHO-G (upper panels) and CHO-SG (lower panels) were incubated with the Vero E6 cell layers in the cold room for 2 h. Cells were washed once with PBS and followed by three washes with PBS containing different amounts of NaCl as indicated at the bottom of the figure. (B) The photos shown in the upper panels were taken under tungsten lamp and these in the lower panels were taken under green fluorescence. CHO-SG cells were pre-treated with either human normal serum (a) or SARS recovered patient's serum (P8 in Tan et al., 2004a) (b) before being added to the Vero E6 cell layer. The cells were washed with 500 mM NaCl after the incubation with Vero E6 cells. Vero E6 cultures were pre-treated with goat anti-ACE2 antibody (d) or the PBS control (c) and then incubated with the CHO-SG cells followed by 500 mM NaCl washes.
Fig. 5
Fig. 5
Identification of the receptor-binding region of SARS-CoV S gene. (A) Five fragments of SARS-CoV S protein were expressed in E. coli. Rabbits were immunized with these five recombinant S protein fragments. The rabbit anti-sera were tested for blocking CHO-SG and Vero E6 cell interaction. (B) The CHO-SG cells were first treated with pre-immune serum or one of five rabbit anti-sera described in (A), and then added to the Vero E6 cell layer. After incubation, the cells were washed with 500 mM NaCl. The rabbit serum from SΔ1 had the most significant blocking effect. (C) Western blot analysis using rabbit anti-S antibody to show the amount of S-EGFP in (B) samples.
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