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. 2009 Sep 18;5(9):e1000587. doi: 10.1371/journal.ppat.1000587

Figure 2. Enhanced activation of MAPK/ERK pathway and inhibition of IFN-α/β signaling by hPIV3-C.

Figure 2

(A) HEK-293T cells were transfected with pFA2-Elk1 to express Elk1 transcription factor fused to the DNA binding domain of Gal4, pGal4-UAS-Luc that contains the firefly luciferase reporter gene downstream of a promoter sequence containing Gal4 binding site, and pRL-CMV that drives Renilla luciferase expression constitutively. In addition to these three plasmids, cells were co-transfected with an expression vector encoding 3×FLAG-tagged MV-C, hPIV3-C or Nipah-C or the corresponding empty vector pCI-neo-3×FLAG. 12 h after transfection, cells were starved and 6 h later EGF was added at a final concentration of 100 ng/ml. After 24 h, relative luciferase activity was determined. (B) HEK-293T cells were transfected with pISRE-Luc, a plasmid containing a luciferase gene of which expression is controlled by five ISREs, and pRL-CMV. In addition to these two plasmids, cells were co-transfected with an expression plasmid encoding 3×FLAG-tagged MV-C, hPIV3-C or Nipah-C or the corresponding empty vector pCI-neo-3×FLAG. 24 h after transfection, 1000 IU/ml of recombinant IFN-β were added. After 24 h, relative luciferase activity was determined. (C) HEK-293T cells were infected with hPIV3 (MOI = 3) and then transfected with pFA2-Elk1, pGal4-UAS-Luc, pRL-CMV vectors. 12 h later, cells were starved during 6 h and stimulated with EGF at a final concentration of 100 ng/ml. After 24 h, relative luciferase activity was determined. (D) Same experiment as (A) but 20 µM of MEK1/2 specific inhibitor U0126 was added as indicated. (A–D) All experiments were achieved in triplicate, and data represent means±SD. (E) HEK-293T cells were infected as in (C), and hPIV3-HN expression determined by immunostaining and flow cytometry analysis. (F) HEK-293T cells were transfected to express 3×FLAG-tagged MV-C, hPIV3-C or Nipah-C as described in (A) and (B), and relative expression levels were determined 36 h later by western blot analysis (left panel). In a parallel experiment, HEK-293T cells were transfected to express hPIV3-C and were cultured with or without EGF in the presence or absence of U0126 as described in (D). hPIV3-C expression level was determined by western blot analysis (right panel).