The C-terminal region of hPIV3-C required to bind STAT1 and GRB2 in the yeast two-hybrid system is highly conserved among Respiroviruses ( Figure 7A ) and suspected to fold into a structured coiled-coil domain [18] . Furthermore, virtually the same C-terminal region of Sendai virus C protein (AA 85-204) was previously reported to mediate the interaction with mouse STAT1 [32] . To further validate our observations performed in the yeast two-hybrid system, we retested by co-affinity purification the ability of hPIV3-C fragment encompassing AA 90-195 (hPIV3-C 90-195 ) to interact with STAT1 and GRB2 in HEK-293T cells. GST-tagged hPIV3-C 90-195 was expressed together with 36-FLAG-tagged STAT1 or GRB2, and purified with glutathionsepharose beads. Full-length hPIV3-C and the N-terminal region encompassing AA 1-89 (hPIV3-C 1-89 ) were used as positive and negative controls, respectively. As shown in Figure 7B and 7C, hPIV3-C 90-195 interacted with STAT1 and GRB2, whereas hPIV3-C 1-89 did not. Although hPIV3-C 90-195 interacted with GRB2 as efficiently as full-length hPIV3-C ( Figure 7C ), interaction with STAT1 was weaker suggesting that more residues contribute to the stabilization of this interaction ( Figure 7B ). Altogether, these results confirm that AA 90-195 of hPIV-3 include both STAT1 and GRB2 binding sites. As described in Figure 2 , HEK-293T, Hela, Vero, A549 or BEAS-2B cells were transfected with pFA2-Elk1, pGal4-UAS-Luc, pRL-CMV to measure the activation level of MAPK/ERK signaling pathway. Cells were co-transfected with plasmids 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 stimulated 6 h later with 100 ng/ml of EGF. After 24 h, expression of luciferase was quantified. Results were normalized so that reporter activity in cells transfected with a control vector equals 1. Experiments were performed in triplicates and data represent means6SD. doi:10.1371/journal.ppat.1000587.t001