Fig. 5

The polymerase activity of SARS-CoV-2 nsp12 is not required for suppression of type I IFN activation. a Effects of SARS-CoV-2 nsp12 and its mutants on SeV-induced IFN-β promoter activation. HEK293T cells were transfected with an IFN-β reporter plasmid along with a control plasmid or plasmids expressing wild-type SARS-CoV-2 nsp12 or the indicated SARS-CoV-2 nsp12 variants. The cells were infected with SeV for 12 h and assayed for luciferase activity. The expression levels of the indicated proteins were analyzed by western blotting. b Effect of SARS-CoV-2 nsp12 and its mutants on SeV-induced nuclear translocation of IRF3. HEK293T cells were transfected with a control plasmid or plasmids expressing wild-type SARS-CoV-2 nsp12 or the indicated SARS-CoV-2 nsp12 variants. At 24 h post transfection, the cells were infected with SeV for 4 h and then immunostained with the indicated antibodies. Merge 1 and Merge 2 indicate the merged red and green channels and the merged red, green, and blue channels, respectively. Scale bar, 20 μM. The enlarged image is a magnified view of the corresponding box. c Quantitation of the nuclear translocation of IRF3. d Vero cells were infected with SARS-CoV-2 at an MOI of 0.1 with or without 10 μM remdesivir for 24 h. The viral copies in the supernatants were measured using real-time RT-PCR (left panel). HEK293T cells were transfected with an IFN-β reporter plasmid along with a control plasmid or nsp12 expression plasmid and a plasmid expressing IRF3–5D. Remdesivir (10 μM) was added to the culture medium at 5 h post transfection. At 24 h post transfection, luciferase activity was measured (right panel). All experiments were performed at least twice, and one representative is shown. The error bars indicate the SDs of technical triplicates. The two-tailed unpaired t-test was used for two-group comparisons compared to the vector group, **P < 0.01, ***P < 0.001