IRF3-NF-kB Pathway Utilization Figure 4. IKKcD couples to the canonical NF-kB activation pathway. (A–C) Confocal immunofluoresence microscopy. IKKc2/2 MEFs were transfected with the indicated expression vectors encoding EGFP alone (EV), EGFP-IKKc-WT or EGFP-IKK–cD. Cells were untreated, TNF stimulated (30 ng/ml, 1 h) or RSV infected (MOI 1, 24 h). Cells were fixed, stained with anti-RelA Ab and DAPI. Shown are relevant channels for EGFP, RelA and DAPI. (D) Stably transfected IKKc-WT or IKKcD-expressing cells were RSV infected, and a time series of cytoplasmic and nuclear extracts prepared. Top Panel, Western blot for Flag (top), IkBa in cytoplasmic extracts. Bottom panel, EMSA using radiolabeled NF-kB probe. (E), IKKc2/2 MEFs transfected with empty vector, IKKc-WT or IKKcD were RSV infected for 16 h (MOI = 1). Total RNA was extracted and QRT-PCR was conducted using primers for RIG-I. **, P,0.01. doi:10.1371/journal.pone.0008079.g004 with empty vector or in those reconstituted with IKKcD (Figure 5D). However, RSV induced a 4-fold increase of IRF3-dependent reporter gene activity in the cells transfected with IKKc-WT; by contrast empty vector-and IKKcD transfected cells did not induce IRF3 dependent transcription (Figure 5D). These data suggested IKKc-WT comple- mented the IRF3 signaling defect in Hela S3 cells, cells selectively defective in IRF3 signaling but having an otherwise have an intact canonical NF-kB pathway. Varying Ratios of IKKcD (Keeping IKKc Constant) Affects Viral Induced Type I IFN Production Our findings in HeLa S3 cells suggested that the endogenous ratio of IKKc-WT:IKKcD is one determinant of type I IFN production in response to ssRNA virus infection. To more fully explore this hypothesis, we conducted an experiment varying the ratios of IKKc-WT:IKKcD in the IKKc2/2 MEF background. In this experiment, we fixed the total amount of IKKc to a constant level while only changing the IKKcWT:IKKcD ratio (Figure 6A). Type I IFN production was then quantified in response to RSV infection using QRT-PCR. In cells expressing only IKKc-WT, a 3,000-fold induction of IFNb transcripts were observed in response to RSV infection, a response that was reduced in a dose-dependent manner upon the expression of IKKcD (Figure 6B). Under expression conditions where IKKcD was the predominant isoform, type I IFN production was significantly blunted (Figure 6B). Similar findings were observed PLoS ONE | www.plosone.org 8 November 2009 | Volume 4 | Issue 11 | e8079