Fig. 2.

Serine mutation at R667, but not K672, reduces the ability of the S glycoprotein to mediate cell–cell fusion. (A) Wild-type SARS-CoV S glycoprotein and the R667S and K672S mutants were metabolically labeled in COS-7 cells and isolated using the C-terminal S-peptide (Spep) affinity tag (Kim and Raines, 1993). The mock lane represents cells transfected without vTF7-3 infection. Proteins were resolved by SDS polyacrylamide gel electrophoresis in NuPAGE 3–8% Tris–acetate gels (Invitrogen) either as the isolated glycoproteins (above) or following deglycosylation using PNGase F (below). The Endo-H-resistant (S_r) and Endo-H-sensitive (S_s) forms of the S glycoprotein are indicated, as is the fully deglycosylated S polypeptide (S). [¹⁴C]-methylated Rainbow molecular weight markers are indicated. The images are printed dark to highlight the absence of proteolytic cleavage. (B) The ability of the S glycoproteins to promote ACE2-dependent cell–cell fusion was detected using the recombinant vaccinia virus-based β-galactosidase reporter assay (Nussbaum et al., 1994, York et al., 2004). COS-7 cells expressing the wild-type and mutant glycoproteins were co-cultured with COS-7 cells transiently expressing the SARS-CoV cellular receptor ACE2 and infected with the fusion reporter recombinant vaccinia virus vCB21R-lacZ (Nussbaum et al., 1994, York et al., 2004). X-gal was used to detect β-galactosidase activity arising from cell–cell fusion. The number of blue syncytia is shown from two experiments, and error bars represent one standard deviation. Transfection efficiencies were comparable in all cases.