Fig. 1.

Effect of trypsin on SARS-CoV infection. (A) Trypsin treatment bypasses ammonium chloride inhibition. HIV-luc(SARS S) or HIV-luc(VSV-G) were bound to mock (black and gray bars) or ammonium chloride-treated (third set of bars and white bars) 293T/ACE2 cells. The cells were incubated with either PBS (black bars and third set of bars) or TPCK-trypsin (gray and white bars). The results are presented as a percentage of no-ammonium-chloride (NH₄Cl), no-trypsin (Tryp.) controls (≈4,000 and 10,000 RLU for SARS S and VSV-G, respectively) and represent the means of samples run in triplicate (±SD). Similar results were seen in two subsequent assays. (B) Trypsin pretreatment of S protein inactivates infectivity. HIV-luc(SARS S) infection of 293T/ACE2 cells was assessed as luciferase activity, presented as a percentage of no-trypsin control (≈40,000 RLU). The results represent the means of samples run in triplicate (±SD). (C) Trypsin treatment bypasses ammonium chloride inhibition of SARS-CoV. Mock- (Center) or 25 mM ammonium chloride-pretreated (Right) Vero E6 cells were spin-infected with replication-competent SARS-CoV at a multiplicity of infection of 0.5 and incubated with either DMEM (Upper) or DMEM containing TPCK-trypsin (Lower). After 48 h, the cells were immunostained for S protein.