Phosphorylation of ERK1/2, eIF4E and small ribosomal subunit S6 protein are stimulated by hPIV3-C expression or hPIV3 infection To further document hPIV3-C impact on MAPK/ERK signaling pathway, we compared the kinetic of ERK1/2 phosphorylation in HEK-293T cells expressing hPIV3-C or not. Cells were transfected hPIV3-C Impact on IFN-a/b and EGF Pathways In addition to these three plasmids, cells were co-transfected with an expression vector encoding 36FLAG-tagged MV-C, hPIV3-C or Nipah-C or the corresponding empty vector pCI-neo-36FLAG. 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 36FLAG-tagged MV-C, hPIV3-C or Nipah-C or the corresponding empty vector pCI-neo-36FLAG. 24 h after transfection, 1000 IU/ml of recombinant IFN-b 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 mM of MEK1/2 specific inhibitor U0126 was added as indicated. (A-D) All experiments were achieved in triplicate, and data represent means6SD. (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 36FLAG-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). doi:10.1371/journal.ppat.1000587.g002 with 36FLAG-tagged hPIV3-C or a control plasmid and 24 h post transfection, they were starved before stimulation with EGF. ERK1/ 2 phosphorylation was determined at 10, 30 and 120 min after stimulation. As illustrated by one representative experiment in Figure 3A , EGF stimulation induced ERK1/2 phosphorylation in control cells but signal was markedly and reproducibly increased by hPIV3-C expression at maximum phosphorylation time point (1.4 to 2.8 fold increase; p = 0.005; n = 4). We then determined the phosphorylation level of two downstream targets of this pathway that are involved in the control of mRNA translation, the translation initiation factor eIF4E and the ribosomal protein S6 ( Figure 3B ). Before EGF stimulation, low levels of phosphorylated eIF4E and S6 were detectable in mock-treated cells ( Figure 3B and 3D). hPIV3-C expression had virtually no effects on this background. Thus, eIF4E and S6 phosphorylation levels were determined at different time-points after EGF stimulation. Because ERK1/2 activation precedes eIF4E and S6 phosphorylation, maximal phosphorylation occurs at later time points and was determined at 30 min, 2 h, 6 h and 24 h after stimulation. As observed for ERK1/2, phosphorylation levels of eIF4E and S6 were enhanced by hPIV3-C expression when stimulating the cells with EGF.