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).