Fig. 3.

Time-of-drug-addition experiment of viral protease inhibitors. Time-of-drug-addition experiment of lopinavir and ritonavir (A) or rupintrivir and AG7404 (B). For “full-time” treatment, Vero E6 cells were pre-treated with indicated compounds for 1 h, and infected with SARS-CoV-2. Two hours later, the virus–drug mixture was removed, and the cells were cultured with compound-containing medium until the end of the experiment. For “Entry” treatment, Vero E6 cells were pre-treated with indicated compounds for 1 h, and infected with SARS-CoV-2. Two hours later, the virus–drug mixture was removed, and the cells were cultured with fresh culture medium until the end of the experiment. For “Post-entry” experiment, Vero E6 cells were infected with SARS-CoV-2. Two hours later, the virus-containing medium was removed, and the cells were cultured with compound-containing medium until the end of the experiment. For all treatments, an MOI of 0.2 was used for lopinavir and ritonavir groups, and an MOI of 0.05 was used for rupintrivir and AG7404 groups. At 14 h p.i., virus yield in the infected cell supernatants was quantified by qRT-PCR (left) and NP expression in infected cells was analyzed by Western blot (right).