Fig 3

PIV5-V inhibits IFN induction by RIG-I ligands. (A) HEK293 cells were transfected with pIFΔ(−116)lucter, pJatlacZ, and pEFplink2, pEF.PIV5-V, or pEF.PIV5-P. At 30 h after transfection, cells were induced with poly(dA-dT), RNA purified from influenza virus-infected cells, or RIG-Ipan RNA for 16 h prior to harvesting for luciferase and β-galactosidase assays. (B) Expression of PIV5-V and -P was confirmed by immunoblotting with an anti-V/P antibody. (C) A549/pr(IFN-β).GFP cells were mock infected or infected with the W3A strain of PIV5 at an MOI of 5 for 24 h. Cells were then induced with poly(I · C), poly(dA-dT), RNA from uninfected cells (mock RNA), or RNA from influenza virus-infected cells (flu RNA) for a further 24 to 48 h. Expression of GFP was visualized by fluorescence microscopy. To verify that the majority of cells had been infected, PIV5 P/V protein expression was visualized by immunofluorescence using the anti-Pk antibody. (D) HEK293 cells were transfected with pIFΔ(−116)lucter and pJatlacZ. At 24 h after transfection, cells were either mock infected or infected with PIV5 W3A at an MOI = 5 and then induced 6 h later with either poly(I · C) or influenza virus RNA for 16 h prior to harvesting for luciferase and β-galactosidase assays.