Preparation of cells coexpressing the A antigen and SARS-CoV S protein for the study of the ACE2/S protein interaction in a cell adhesion assay
The interaction between the SARS-CoV spike protein and its cellular receptor ACE2 can be studied using a cell-based assay, as described previously (Chou et al. 2005). In this assay, the viral S protein expressed by transfection into Chinese hamster ovary (CHO) cells mediates adhesion to Vero E6 cells that possess ACE2. CHO cells do not express ABH antigens because of the lack of an α1,2-fucosyltransferase activity and of either the A or B histo-blood group enzymes. In order to obtain cells able to express the A antigen, parental CHO cells were stably transfected successively with the rat Fut2 cDNA and a rat A enzyme cDNA. Unlike mock-transfected cells, these double transfectants strongly express cell surface A antigen as detected by flow cytometry. Transfection of the S protein–EGFP fusion construction (S–EGFP) into these cells allowed the expression of the S protein together with the histo-blood group A antigen (Figure 1A). Observation of the triple transfectants by confocal microscopy revealed that, as expected, the A antigen and the S–EGFP fusion protein partially colocalized at the cell surface (Figure 1B). In addition, western blot analysis revealed that among various A antigen positive glycoproteins, a band at the expected size of the S–EGFP fusion protein, between 210—and 230 kDa (Chou et al. 2005), was present in the extract from the triple transfectant Fut2/A/SP but absent from the double Fut2/A transfectant cell extract. It indicated that the S-protein expressed by A-positive CHO cells carried A histo-blood group epitopes. Specificity of the anti-A labeling was ensured since no band was detected in extracts from CHO Fut2 only transfectants (Figure 1C). A stable A antigen and S-EGFP expressing clone showed significantly higher adhesion to Vero cells than either mock transfectants or the A expressing clone devoid of the S protein (Figure 2A and B). Similar results were obtained after transient transfection of the S-EGFP construct (not shown). The presence of the A and/or H antigens on the S protein expressing cells did not affect adhesion since CHO cells only transfected with the S-EGFP construct, as well as CHO cells transfected with both the S-EGFP and Fut2 cDNAs, adhered to Vero cells at a similar level as the A antigen S protein triple transfectants (Figure 2A). In order to control that the cell adhesion was dependent upon the ACE2/S protein interaction, blocking experiments with either an anti-ACE2 or an anti-S protein were performed. Both antibodies significantly inhibited adhesion, although the anti-ACE2 mAb proved more efficient (Figure 2C).
Fig. 1  CHO cells coexpressing the A histo-blood group antigen and the SARS-CoV S protein. (A) Flow cytometry analysis of the expression of A antigen and the S protein–EFGP construct on CHO mock-transfected cells (mock), double transfectants either with the Fut2 and S protein constructs (Fut2/SP) or the Fut2 and A enzyme cDNAs (Fut2/A), triple transfectants with the Fut2, A enzyme and S protein constructs (Fut2/A/SP). Fluorescence of the S–EFGP molecule was directly recorded on the FL1 channel. Detection of the A antigen was performed using an anti-A mAb followed by Cy5-labeled anti-mouse IgG and recorded on the FL4 channel. (B) Confocal microscopy analysis of the A antigen (A TRITC) and the S protein–EFGP construct coexpression on CHO cells triple transfectants. Detection of the A antigen was performed using an anti-A mAb followed by TRITC-labeled anti-mouse IgG. (C) Western blot analysis of transfected CHO cells glycoproteins. Total protein extracts from CHO Fut2 simple transfectants, CHO Fut2/A double transfectants, and CHO Fut2/A/SP triple transfectants were submitted to SDS–PAGE and Western blotting. Glycoproteins carrying A histo-blood group epitopes were detected with an anti-A mAb. The arrow shows the expected molecular size of the EGFP–SP fusion protein.
Fig. 2  S Protein/ACE2-dependent adhesion of CHO cells to Vero cells. The binding assay between CHO cells and Vero E6 cells was performed as described in Material and methods. Adherent cells were counted under a fluorescence microscope. Cells from a total of 36 fields from 6 wells were counted. (A) The results shown correspond to the mean ± SD of one representative experiment out of four obtained with CHO cells mock transfectants (mock), double transfectants with the Fut2 and A histo-blood group glycosyltransferases (Fut2/A), simple transfectants with the SARS-CoV S protein construct (SP), double transfectants with the Fut2 enzyme and the SARS-CoV S protein construct (Fut2/SP), and triple transfectants with the Fut2, A glycosyltransferases and the SARS-CoV S protein cDNAs (Fut2/A/SP). Adhesion of SP, Fut2/SP, and Fut2/A/SP cells is significantly higher than that of either mock or Fut2/A cells (P < 0.001, Student's t-test). (B) Representative fields illustrating the adhesion of either mock-transfected CHO cells or triple transfectants. (C) Inhibition of the adhesion of triple CHO transfectants to Vero cells by anti-ACE2 or anti-S protein mAbs. The mAbs were added to the CHO cells suspension at 20 and 25 μg/mL, respectively prior to incubation on the Vero cell layer. Adhesion in the presence of the anti-ACE2 and anti-SP are significantly lower than that of control cells (P < 0.001 and P < 0.01, respectively). (D) Inhibition of the adhesion to Vero cells of S protein-transfected CHO cells coexpressing either the H (Fut2/SP) or the A antigen (Fut2/A/SP) by an anti-A mAb or a control isotype matched antibody used at 4 μg/mL. Only the adhesion of the triple transfectants in the presence of the anti-A differs significantly from other conditions (P < 0.01).