FIG 1.
Inhibition of IFN-β signaling by BoHV-1 VP8. (A and B) Vero cells were transfected with pISREluc and pRL-TK together with pCMV4.1K, pFLAG-EYFP, or pFLAG-VP8. (A) At 24 h posttransfection, cells were stimulated with 2,500 units of human IFN-β in 0.5 ml or left untreated. After 6 h of incubation, cell lysates were made and reporter gene activity was measured. (B) Expression of VP8 and EYFP was confirmed by Western blotting with monoclonal anti-FLAG antibody. (C) EBTr cells were transfected with pISREluc and pRL-TK. At 24 h posttransfection cells were mock infected or infected with BoHV-1 or BoHV1-ΔUL47, and another 24 h later cells were treated with 400 ng of bovine IFN-β in 0.5 ml or left untreated. After 6 h of incubation, cell lysates were made and reporter gene activity was measured. (D to G) EBTr cells were transfected with pISREluc and pRL-TK, and at 20 h posttransfection cells were either left untreated (D and E) or were pretreated with ActD (F and G) before mock infection or infection with BoHV-1, BoHV1-ΔUL47, or BoHV1-UL47R. (D and F) At 1 h postinfection, cells were stimulated with IFN-β for 1 h, and at 2 h postinfection cell lysates were collected and reporter gene activity was measured. ActD was maintained in the medium throughout the infection. (E and G) Expression of VP8, bICP0, and bICP4 in untreated cells (E) but not in Act-treated cells (G) was confirmed by Western blotting with monoclonal anti-FLAG antibody and bICP0- and bICP4-specific rabbit antibodies, respectively. IFN-β-induced firefly luciferase reporter values were normalized to the expression of Renilla luciferase. The values are presented as percentages of IFN-stimulated controls and are expressed as means ± standard deviations (SD) for six samples. Statistical significance is indicated by asterisks (***, P < 0.001).