Fig. 4.

PDCoV N protein only suppressed poIRF7 induced-type I IFN production. (A, B) PK-15 cells were co-transfected with HA-PDCoV-N (0 ng, 200 ng, 400 ng), Flag-poIRF7 or empty vector expression plasmids, along with pGL3-pIFN-β or pGL3-pIFN-α, pRL-TK (as a normalizing control). (C, D) LLC-PK1 cells were uninfected or infected with PDCoV at a MOI of 0.5 for 6 h, and then the cells were co-transfected with the pIFN-α (C) or pIFN-β (D) promoter luciferase reporter and the Flag-tagged poIRF7 or an empty vector plasmids for 24 h. The cell lysates were harvested and subjected to a dual-luciferase assay. (E, F) HEK293 T cells were co-transfected with HA-PDCoV-N (0 ng, 200 ng, 400 ng), Flag-hIRF7, or empty vector, along with pGL3-hIFN-β or pGL3-hIFN-α, pRL-TK. (G) DF1 cells were co-transfected with HA-PDCoV-N (0 ng, 200 ng, 400 ng), V5-chIRF7 or empty vector expression plasmids, along with pGL3-chIFN-β, pRL-TK. Dual-luciferase assays were performed at 24 h post-transfection. The relative firefly luciferase activity was relative to that of an empty vector control. Western blot was used to detect the protein expression of PDCoV N with HA antibody. The β-actin was the loading control. (H-M) PK-15 cells cultured in 12-well plates were transfected with HA-PDCoV-N or empty expression plasmids together with the Flag-poIRF7 for 24 h. The cells were lysed with TRIZOL to extract the total RNA. The qRT-PCR or PCR was used to detect the relative expression of PDCoV-N (H), poIRF7 (I), porcine type I IFN genes (including IFNA1 and IFNB1) (J, K) and ISGs (L, M) mRNA. All data are presented as means ± SD of three independent experiments (*p < 0.05 and **p < 0.01).