Cell culture and transfections
Cell lines CHO-K1 and Vero E6 were purchased from American Type Cell Collection (Manassas, VA). Vero-E6 cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% decomplemented fetal-calf serum, 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, Paisley, UK).
CHO cells are devoid of α1,2-fucosyltransferase activity and therefore of ABH antigens. To obtain the expression of the A antigen, they were transfected first with the rat Fut2 cDNA, and then with an A enzyme cDNA (Bureau et al. 2001; Cailleau-Thomas et al. 2002). The rat Fut2 cDNA was inserted into the pDR2 eukaryotic expression vector (Clontech, St Germain en Laye, France) deleted of the sequences lying between the EcoRV and ClaI sites. This vector possesses a hygromycin selection marker. The rat A enzyme cDNA was inserted into the pcDNA3.1 vector (Invitrogen, Paisley, UK) with a zeocin selection marker. Cells were transfected with the rat Fut2 cDNA using Lipofectamin 2000TM according to the manufacturer's instructions (Invitrogen). After selection in hygromycin containing medium (0.4 mg/mL), cells were cloned by limiting dilution, and a clone strongly expressing cell surface H antigen, as detected using the FITC-labeled UEA-I lectin, was selected. This clone was further transfected with the rat A enzyme cDNA. Transfectants were then selected in a zeocin-containing medium (0.6 mg/mL). After cloning by limiting dilutions, a clone strongly expressing the A histo-blood group antigen was selected. Control transfected cells were prepared by transfection with the empty vectors. These stable transfectants were cultured in RPMI 1640 supplemented with 10% decomplemented fetal-calf serum, 2 mM l-glutamine, 10 μg/mL free nucleotides, 100 U/mL penicillin, and 100 μg/mL streptomycin, 0.4 mg/mL hygromycin, and 0.6 mg/mL zeocin. To obtain the expression of the SARS-CoV S protein, control CHO cells, transfected CHO cells expressing the H antigen, and double transfected CHO cells expressing the A antigen were transfected with the previously described pEGFP-N1 vector containing an S protein–EGFP construction (CMV-SG) (Chou et al. 2005). Stable transfectants were obtained after selection with 1 mg/mL G418 (Gibco). Since the expression of the S protein was progressively lost, even when cells were continuously cultured in the presence of G418, part of the experiments were performed 48 h after transient transfection with the CMV-SG vector.
Cells were passaged at confluence after dispersal with 0.025% trypsin in 0.02% EDTA and routinely checked for mycoplasma contamination by Hoechst 33258 (Sigma, St Louis, MO) labeling.