Fig. 1.

Role of the S1–S2 boundary in trypsin-mediated activation of SARS-CoV S membrane fusion. (A) BHK cells transiently expressing SARS-CoV S wild type (SARS wt) or the SARS-R667N mutant were treated with 1 μg/mL trypsin for 30 min or 1 h. Samples were analyzed by Western blot analysis, with the C-terminal part of the spike protein detected by using an anti-C9 antibody. (B) VeroE6 cells expressing SARS-CoV S wild type (SARS wt) or the SARS-R667N mutant were treated with 2 μg/mL trypsin for 20 min, then incubated for 40 min in absence of trypsin and analyzed by immunofluorescence microscopy using an anti-S monoclonal antibody (green), with nuclei counterstained with Hoechst 33548 (blue). (Magnification: 200×.) (C) BHK cells cotransfected with plasmids encoding the SARS-CoV S wild type (SARS wt) or SARS-R667N, as well as a plasmid encoding luciferase under the control of the T7 polymerase were overlaid with VeroE6 cells expressing the T7 polymerase. Cell–cell fusion was induced by treating the cells with increasing amount of trypsin for 20 min. At 5 h after fusion induction, the cells were lysed to monitor luciferase activity. Results are presented as relative light units (RLU). Error bars represent the standard error of the mean for 3 independent experiments.