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. 2020 Sep 19;33(1):108234. doi: 10.1016/j.celrep.2020.108234

Figure 4.

Figure 4

Comparison of IFN-I Inhibition by Different Coronaviruses

(A) IFN-β promoter luciferase assay. Viral proteins from SARS-CoV-2 (2019-nCoV/USA_WA1/2020, GenBank: MN985325), SARS-CoV (SARS MA15 strain, GenBank: DQ497008), and MERS-CoV (MERS EMC/2012 strain, GenBank: JX869059) were compared for their inhibition of IFN-I production. RIG-I-induced IFN-β promoter luciferase assay was performed by co-transfection of HEK293T cells as described in Figure 1.

(B) ISRE promoter luciferase assay. HEK293T cells were co-transfected with pISRE-luc luciferase reporter plasmid, phRluc-TK control plasmid, and viral protein expressing plasmid. At 24 hpt, the cells were treated with 1,000 U/mL IFN-α and assayed for luciferase activities after 8 h. Error bars represent mean ± SD from three independent experiments. Statistical significance was determined by comparing with SARS-CoV-2 and two-way ANOVA with Dunnett’s correction, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

(C) Western blot of phosphorylated STAT1 and STAT2. HEK293T cells were transfected with viral protein expressing plasmid and then treated with 1,000 U/mL IFN-α at 24 hpt for 30 min. Western blot was performed to analyze the cell lysates using anti-phosphorylated STAT1 (Y701) and STAT2 (Y690), and anti-total STAT1 and STAT2 antibodies. Protein band intensities were quantitated by Image Lab software.