FIG 4.

Arginine and lysine residues with ACE2 amino acids 697 to 716 are essential for TMPRSS2- and HAT-dependent augmentation of entry mediated by the SARS-CoV spike protein. (A) Plasmids encoding the ACE2 wt or the indicated ACE2 mutants were transiently transfected into 293T cells, and ACE2 surface expression was detected by FACS. Results of a single experiment are shown and were confirmed in two separate experiments. (B) Plasmids encoding the ACE2 wt or the indicated ACE2 mutants were transiently transfected into 293T cells and the cells transduced with a lentiviral vector pseudotyped with SARS-S. Cells transfected with empty plasmid (pcDNA) served as a negative control, while a vector pseudotyped with VSV-G served as a control for susceptibility to ACE2-independent transduction. Luciferase activities in cell lysates were determined at 72 h postransduction. The results of a representative experiment performed with triplicate samples are shown; error bars indicate standard deviations (SD). Similar results were obtained in two additional experiments. (C) The experiment was carried out as described for panel B, but transduction of target cells expressing the ACE2 wt or the indicated ACE2 mutants jointly with the indicated proteases was assessed. The results are shown as fold enhancement of transduction upon expression of the proteases TMPRSS2 and HAT. The results represent the averages from two to six independent experiments, and error bars indicate SEM.