Fig. 1.

PDCoV nsp15 antagonizes SEV-induced IFN-β production. (A, C) LLC-PK1 cells (A) or HEK-293T cells (C) cultured in 24-well plates were co-transfected with IFN-β-Luc and pRL-TK together with increasing amounts (0.1, 0.5, and 1.0 μg) of pCAGGS-HA-nsp15 or empty vector. After 24 h, the cells were mock-infected or infected with SEV (10 hemagglutinating activity units/well) for 12 h and subjected to a dual-luciferase reporter assay. The relative firefly luciferase activity was normalized to the Renilla luciferase activity with the untreated empty vector control value set to 1. (B, D) LLC-PK1 cells (B) or HEK-293T cells (D) cultured in 24-well plates were co-transfected with 1.0 μg of pCAGGS-HA-nsp15 or empty vector for 24 h, and then left untreated or infected with SEV (10 hemagglutinating activity units/well). At 8 h after infection, the cells were collected, and total RNA was extracted to detect the expression levels of IFN-β and GAPDH by SYBR Green PCR assay. (E, F) LLC-PK1 (E) or HEK-293T (F) cells were transfected with 1.0 μg of pCAGGS-HA-nsp15 or empty vector. At 24 h after transfection, both cell lines were infected with SEV for 12 h and the cell supernatants were collected. The UV-irradiated cell supernatants were overlaid onto fresh LLC-PK1 or HEK-293T cells in 24-well plates. After 24 h of incubation, cells were infected with VSV-GFP for 12 h. The replication of VSV-GFP was detected via fluorescence microscopy. Data are representative of three independent experiments. ***, p <  0.001; **, p <  0.01; ns, not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)