Figure 2.
SARS-CoV-2 Proteins Inhibit TBK1 and IRF3 Activation
(A) Analysis of IRF3 phosphorylation. HEK293T cells were transfected with viral protein-encoding plasmid (1 μg); treated with poly(I:C) (10 μg); and analyzed for phosphorylated IRF3 (anti-pIRF3 at S396), total IRF3 (anti-IRF3), viral protein (anti-FLAG), and GAPDH (anti-GAPDH) by western blot.
(B) Analysis of TBK1 phosphorylation. HEK293T cells were co-transfected with TBK1-expressing plasmid and varying amounts of nsp6- or nsp13-encoding plasmids. At 24 hpt, western blot was used to analyze the cell lysates for phosphorylated TBK1 (anti-pTBK1 at S172), total TBK1 (anti-TBK1), phosphorylated IRF3 (S396; anti-pIRF3), total IRF3 (anti-IRF3), viral nsp 6 or nsp13 (anti-FLAG), and GAPDH (anti-GAPDH). Protein band intensity was quantitated using Image Lab software.
(C) Co-immunoprecipitation (coIP) of TBK1 and nsp6 or nsp13. HEK293T cells were co-transfected with plasmids expressing TBK1 and hemagglutinin (HA)-tagged nsp6 or nsp13. At 24 hpt, whole-cell lysate (WCL) was incubated with anti-HA beads for immunoprecipitation and TBK1 was detected by western blot.
(D) Nuclear translocation of IRF3. A549 cells were transfected with ORF6-expressing plasmid. At 24 hpt, cells were treated with poly(I:C) and fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with PBS containing 2% fetal bovine serum (FBS) and 0.1% Tween 20, the cells were probed with primary antibodies (anti-FLAG and anti-IRF3) and secondary antibodies (anti-Alexa Fluor 488 and anti-Alexa Fluor 568). Images were obtained using a fluorescence microscope and analyzed by ImageJ. Scale bar, 10 μm.
(E) coIP of ORF6 and KPNA1–6. HEK293T cells were co-transfected with FLAG-tagged ORF6-expressing plasmid and HA-tagged KPNA1–6 plasmid or empty plasmid. At 24 hpt, coIP was performed by incubating anti-HA antibody overnight, followed by addition of magnetic beads. After extensively washing, the eluate was analyzed by western blot with indicated antibodies.
(F) Summary of antagonism of IFN-I production. The inhibitory steps are indicated for individual viral proteins.