FIG 2.

SARS2-S-mediated cell-cell fusion depends on ACE2 receptor expression, whereas SARS1-S-mediated fusion depends on TMPRSS2 activity in 293T cells. (A) Cell-cell fusion assay. Effector cells (293T cells transfected with either an empty vector or expression plasmids for the indicated spike variants and Vp16-Gal4 transactivator) were cultured together with target cells (293T cells transfected with ACE2 or TMPRSS2 expression plasmids at the indicated ratios and the Gal4-TurboGFP-Luc reporter plasmid). After 24 h, luciferase activity was measured. The data show averaged relative luminescence units, and error bars represent the standard deviations from one representative experiment performed in triplicate. Comparisons were made against the condition with maximum activation using two-way ANOVA, and P values were corrected for multiple comparisons by Sidak’s method (P > 0.05, ns; P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0.001, ***; P ≤ 0.0001, ****). (B) The expression of proteins in target cells and effector cells after cocultivation was analyzed by Western blotting from lysates harvested for determination of the luciferase activity in panel A. The unprocessed spike (S0) and the S1/S2-site processed spike (S2) are indicated by arrows. An additional cleavage product marked with an asterisk was observed. The predominant, processed, low-molecular-weight TMPRSS2 fragment is shown. The expression of GAPDH served as a loading control. One representative Western blot is shown.