FIG. 2.
Expression and maturation of S and S chimeras. (a) CMV-driven S expression plasmids were transfected into VeroE6R cells. After a 24-h incubation, cells were pulsed for 2 h with [35S]Met-Cys and chased for the indicated periods (in hours). After the chase, cells were lysed and the S proteins were immunoprecipitated with anti-SARS serum. Samples were analyzed on an 8% PAA gel. Endo H-resistant S (SRes) and EndoH-sensitive S (SSens) are indicated. Lanes: 1, Swt; 2, Svsv-cyt; 3, Svsv-tmdcyt; 4, Smhv-tmdcyt; 5, VSV-G. (b) Cell surface expression of S was demonstrated by biotinylation of S proteins. Transfected 293T cells were incubated with sulfo-NHS-LC-biotin 24 h posttransfection. Subsequently, S-specific immunoprecipitation was carried out, followed by Western blot analysis using HRP-conjugated streptavidin. Bands were visualized by chemiluminescence. S-specific bands are indicated. Lanes: 1, Swt; 2, Svsv-cyt; 3, Svsv-tmdcyt; 4, Smhv-tmdcyt; 5, mock.