hMPV G protein modulates viral-induced NF-κB activation.

(A) A549 cells were transfected with a luciferase reporter plasmid containing the human IL-8 promoter and infected with rhMPV-WT or -ΔG, at MOI of 2. Cells were harvested at 15 h p.i. to measure luciferase activity. Uninfected plates served as controls. For each plate luciferase was normalized to the β-galactosidase reporter activity. Data are representative of two independent experiments and are expressed as mean±standard deviation of normalized luciferase activity. *, P<0.05 relative to rhMPV-WT. (B) A549 cells were transfected with a luciferase reporter plasmid containing multimers of the IL-8 NF-κB site together with G or F protein expression plasmid or thes empty vector and infected with rhMPV-WT or -ΔG, at MOI of 2. Cells were harvested at 15 h p.i. to measure luciferase activity. Uninfected plates served as controls. For each plate luciferase was normalized to the β-galactosidase reporter activity. Data are representative of two independent experiments and are expressed as mean±standard deviation of normalized luciferase activity. *, P<0.05 relative to rhMPV-ΔG-infected-CV transfected A549 cells. (C) A549 cells were infected with rhMPV-WT or rhMPV-ΔG, at MOI of 2, for various lengths of time and harvested to prepare nuclear extracts. Equal amounts of protein from uninfected and infected cells were analyzed by Western blot using either an anti-p50 or anti-p65 antibody. Membranes were stripped and reprobed for lamin b, as control for equal loading of the samples. Densitometric analysis of NF-κB band intensity, performed using the histogram function of Adobe Photoshop, is shown after normalization to lamin b.