IKKc-WT and IKKcD, were investigated [18]. These cells have been previously shown to have intact NF-kB signaling in response to TNFa stimulation and IKKa/b expression [18]. In response to RSV infection, we found that these cells also had defective type I IFN expression (Supplementary Figure S2A online). Similar findings were produced in stable transfectants in response to SeV infection, both in terms of defective type I IFN production as well as impaired ISG expression (Supplementary Figures S2B, S2C online). A biological action of epithelial type I IFN production is to induce paracrine activation of the Jak-STAT pathway in neighboring cells to produce a mucosal anti-viral state [27]. In this process, STAT1 is tyrosine phosphorylated and its expression upregulated via a positive feedback loop [28]. We therefore analyzed RSV-induced inducible STAT1 tyrosine phosphoryla- tion and expression in IKKc2/2 MEFs transfected with either empty-, IKKc-WT and IKKcD expression vectors. The induction of phospho-Tyr701 STAT1 and upregulation of STAT1 protein were only observed in IKKc-WT reconstituted cells (Figure 2C). Similar results were observed in response to SeV infection (Figure 2D). Collectively, these data suggest that, in contrast to IKKc-WT, IKKcD does not effectively differently couple to type I IFN induction resulting in a deficient ISG response after ssRNA virus infection. IRF3-NF-kB Pathway Utilization IKKcD Is Deficient in Viral Induced IRF3 Activation Previous studies have demonstrated that IKKc is an essential adapter for IRF3 activation downstream of RIG-I?MAVS [15]. Because IRF3 is a major regulator of type I IFN production, we therefore tested whether IKKc and IKKcD differentially affected viral induced IRF3 or NF-kB transcription. Myc epitope-tagged IKKc and IKKcD were co-transfected in the absence or presence of MAVS along with NF-kB-selective (IFNb PRDII domain) or IRF3- selective (PRDIII) luciferase reporter genes into IKKc2/2 MEFs. MAVS expression was determined by anti-Flag Ab in Western Immunoblot (Figure 3A, left, top panel) and that of IKKc and IKKcD by anti-Myc Ab in in Western immunoblot (Figure 3A, left, middle panel). We noted that IKKc isoform expression did not affect MAVS expression. As expected, MAVS was unable to activate NF-kB-driven luciferase reporter activity in IKKc2/2 MEFs, and mediated a 4-fold increase in IKKc-WT transfectants (Figure 3B). Important- ly, MAVS activated NF-kB-driven luciferase reporter activity to a slightly greater degree (5-fold) in cells expressing IKKc2D (Figure 3B, left, top panel). Conversely, although MAVS induced 3.5-fold increase in IRF3-driven luciferase reporter activity in cells expressing IKKc-WT, no detectable induction of IRF3-driven luciferase activity was seen in cells expressing IKKcD (Figure 3B, right, top panel). Figure 3. IKKcD is defective in mediating IRF3 signaling. (A) Expression vectors encoding Myc-tagged IKKc or IKKcD were co-transfected into IKKc2/2MEFs, in the presence of MAVS or NH2 terminal RIG-I (RIG-N). 48 h later, the expression of Flag-MAVS (top left panel), Flag-RIG-N (top right panel), Myc-IKKc and Myc-IKKcD (middle panel) were detected by Western immunoblot. (B) IFN-b PRDII or PRDIII driven luciferase reporter genes were co-transfected with MAVS or RIG-N in the absence or presence of IKKc and IKKcD as in (A). 36 h later, reporter activity was measured. Shown is normalized luciferase activity. (C) Nuclear extracts were prepared using sucrose-cushion from SeV infected IKKc-WT and IKKcD stably transfected MEFs. The nuclei were denatured in 1% SDS PAGE loading buffer and IRF3 abundance measured by Western immunoblot. b-actin was used as loading control. (D) EMSA using an ISRE probe was conducted using nuclear extracts from a similar experiment as described in (C). (E,F) IKKc2/2, IKKc reconstituted and IKKcD reconstituted MEFs were infected by SeV (E) or RSV (F) for times indicated. 50 mg WCEs were native gel-fractionated and Western immunoblot conducted using anti-IRF3 Ab. The location of monomer and dimers are indicated. doi:10.1371/journal.pone.0008079.g003 PLoS ONE | www.plosone.org 6 November 2009 | Volume 4 | Issue 11 | e8079