Figure 3.

SARS-CoV-2 Proteins Block IFN-I Signaling

(A) ISRE promoter luciferase assay. HEK293T cells were co-transfected with an ISRE promoter-driven Firefly luciferase reporter plasmid pISRE-luc, Renilla luciferase control plasmid phRluc-TK, and viral protein expressing plasmid. At 24 hpt, cells were treated 1,000 U/mL IFN-α for 8 h, followed by dual-luciferase reporter assays. Data processing was the same as described in Figure 1. Error bars indicate SDs from three independent experiments. Statistical values were determined by comparing with EGFP control and one-way ANOVA with Dunnett’s correction, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.

(B) Inhibition of STAT1 and STAT2 phosphorylation. HEK293T cells were transfected with viral protein expressing plasmids. At 24 hpt, cells were treated with 1,000 U/mL IFN-α for 30 min and analyzed by western blot using anti-phosphorylated STAT1 at Y701, anti-total STAT1, anti-phosphorylated STAT2 at Y690, and anti-total STAT2 antibodies. Protein band intensity was quantitated using Image Lab software.

(C) Nuclear translocation of STAT1. Vero cells were transfected with ORF6 expressing plasmids for 24 h, treated with 1,000 U/mL IFN-α for 30 min, fixed and permeabilized, and probed with anti-STAT1 and anti-FLAG as primary antibodies and anti-Alexa Fluor 488 and anti-Alexa Fluor 568 as secondary antibodies. Images were obtained through fluorescence microscope and analyzed using ImageJ. Scale bar, 10 μm.

(D) Summary of antagonism of IFN-I signaling. The inhibitory steps are indicated for individual viral proteins.