Porcine epidemic diarrhea virus inhibits dsRNA-induced interferon-ß production in porcine intestinal epithelial cells by blockade of the RIG-I-mediated pathway

Cao, Liyan; Ge, Xuying; Gao, Yu; Herrler, Georg; Ren, Yudong; Ren, Xiaofeng; Li, Guangxing

We then used luciferase reporter assay system to determine whether IRF-3 and NF-κB are linked with the inhibition of IFN-β production after PEDV infection. As shown in Fig. 2b , IRF-3 luciferase activity was sharply decreased in PEDV-infected cells, and poly (I:C)-induced IRF-3 activation was also inhibited by PEDV in comparison to a remarkably signal in poly (I:C)-transfected cells. However, in Fig. 2c , compared with mock-infected To detect the activation of IRF-3 and p65 after PEDV infection, the cell extracts were prepared at the indicated times and subjected to western blot analysis with antibodies specific for IRF-3, p65, p-IRF-3, p-p65 and PEDV N McAb. Anti-β-actin was included as a control for sample loading. These experiments were performed in duplicate. b and c IECs were infected or mock-infected with PEDV at an MOI of 1 for 12 h, and then cells were cotransfected with (PRDIII-I) 4-Luc (b) or pNF-κB-Luc (c) and phRL-TK for additional 24 h. Cells were retransfected with poly (I:C) for 12 h, harvested, and then subjected to a dual-luciferase assay. All data are expressed as means ± SD of 3 independent experiments. **p < 0.01 as compared with poly (I:C). d IRF3-GFP fusion protein transfected with IECs and then infected with PEDV at an MOI of 1 and mock-infected cells served as negative controls. 24 h later, cells were retransfected with poly (I:C) (positive control) (c and d) or untransfected (a and b) for 12 h. Cells were fixed with 4 % paraformaldehyde, permeabilized with 0.1 % Triton X-100, and stained by DAPI (blue). Cells were incubated with anti-PEDV rabbit polyclonal antibody (red) and TRITC-labeled goat anti-rabbit secondary antibody, then analyzed for fluorescence by confocal microscopy. Magnification, ×20 (Leica, Wetzlar, Germany) (See figure on previous page.) Fig. 1 PEDV does not induce IFN-β production and inhibits poly (I:C)-mediated IFN-β induction. a IECs were cotransfected with IFN-β-Luc and phRL-TK, then infected with PEDV at an MOI of 1 and 0.1 for 24 h. Cells were retransfected with poly (I:C) as a positive control. After 12 h, the cells were harvested and subjected to a dual-luciferase assay. b IECs were infected with PEDV at an MOI of 1, mock-infected as a negative control, or transfected with poly (I:C) as a positive control. At the indicated time points, total RNA was extracted and IFN-β and β-actin mRNA were subjected to real-time PCR. RNA expression levels were normalized to β-actin. c In contrast to a, IECs were first mock-infected or infected with PEDV at an MOI of 1 for 12 h and then cotransfected with IFN-β-Luc and phRL-TK for 24 h. Cells were retransfected with or without poly (I:C) for an addition 12 h, harvested, and then subjected to a dual-luciferase assay. All data are expressed as means ± SD of 3 independent experiments. *p < 0.05; **p < 0.01 as compared with poly (I:C) cells, NF-κB luciferase activity significantly enhanced both in PEDV-infected and poly (I:C)-transfected cells. In addition, we found that poly (I:C)-induced activation of NF-κB was not blocked by PEDV. To further identify PEDV-inhibited poly (I:C)-mediated activation of IRF-3, confocal microscopy assay was used. As a result, IRF3-GFP remained in the cytoplasm of both mockinfected ( Fig. 2d. a) and PEDV-infected (Fig. 2d . b) IECs compared with poly (I:C) controls, in which clear translocation to the nucleus was observed (Fig. 2d. d) . Furthermore, PEDV could block poly (I:C)-mediated IRF-3 nucleus migration (Fig. 2d. c) . Take