Fig. 5.

Identification of NF-κB antagonists of PEDV. (A), Suppression of TNFα-stimulated NF-κB activity by PEDV. MARC-145 cells were co-transfected with pNF-κB-Luc along with pRL-TK in 12-well plates for 6 h followed by PEDV infection at an MOI of 1 for 12 h. Cells were then stimulated with TNFα at the concentration of 15 ng/ml for 9 h, and cell lysates were prepared for dual-luciferase reporter assays. Results from three independent experiments were presented as the mean relative luciferase values with standard deviation. Asterisks indicate statistical significance calculated by the Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. (B) and (C), Regulation of TNFα-induced NF-κB activity by individual PEDV proteins. HeLa cells were grown in 48-well plates and co-transfected with pNF-κB-Luc along with individual PEDV genes and pRL-TK at a ratio of 10:10:1. For nsp3, nsp5, and nsp16, transfection was conducted as described previously (Zhang et al., 2016). PRRSV nsp1α (P-nsp1α) is a known NF-κB promoter suppressor, and its mutant P-nsp1α(m) reverts its suppressive function. Both constructs were included as controls. Cells were stimulated with poly(I:C) (0.5 μg/ml) at 12 h post-transfection for 12 h and dual luciferase activities were determined. Results from three independent experiments were presented as the mean relative luciferase values with standard deviation. Asterisks indicate statistical significance calculated by the Student's t-test using GST as a control. *, P<0.05; **, P<0.01; ***, P<0.001.