IRF3-NF-kB Pathway Utilization adjacent graph (Figure 1E, right). Together these data suggested that IKKcD reconstituted MEFs were defective in restricting viral expression relative to those expressing the IKKc-WT isoform. IKKcD Transfectants Are Defective in Type I IFN Production To understand the mechanism for the enhanced viral replication rate in IKKcD-reconstituted MEFs, viral induced expression of type I IFN (IFN-b, -a1 and -a4), were quantified by QRT-PCR. In IKKc2/2 MEFs, RSV infection did not induce a detectable change in expression of any type I IFN. Conversely, in cells reconstituted with IKKc-WT, RSV induced a 150-fold increase in IFN-b, a 10-fold increase in IFN-a1 and a 350-fold increase in IFN-a4 (Figure 2A). Strikingly, in IKKcD-reconstituted cells, the expression of all three type I IFNs was significantly less, and for IFNa1, indistinguishable from that of IKKc2/2 MEFs (Figure 2A). A major mechanism for IFN induced antiviral activity involves the expression of downstream IFN-stimulated genes (ISGs). To confirm that the attenuated type I IFN production in IKKcD expressing cells was biologically relevant, we next measured ISG expression. In RSV infected cells reconstituted with IKKc-WT, robust 1,200-fold induction of the IFN response factor-7 (IRF7), a 550-fold induction of IFN inducible gene -10 (IP10), and a 350-fold induction of RANTES were observed (Figure 2B). Conversely, in both IKKc2/2 MEFs and those reconstituted with IKKcD, ISG expression was significantly reduced (Figure 2B). To exclude the possibility that IKKc reconstitution by transient transfection affects cellular signaling in response to ssRNA virus infection, the response of IKKc2/2-deficient cells stably expressing Figure 2. IKKcD does not mediate IFN or ISG gene expression in response to RNA virus infection. (A) IKKc2/2 MEFs were reconstituted by empty vector, IKKc-WT or IKKcD as indicated, and RSV infected for 16 h. Total RNA was extracted and QRT-PCR was conducted using probes for IFN-b, -a1, -a4. (B) Experiment as in (A) where QRT-PCR was was performed with probes for IRF7, IP10 and RANTES. (C) Experiment as in (A) where WCEs were extracted 0, 12 or 24 h after RSV infection and Western blot performed for phospho-Tyr701 STAT1 (pSTAT1, top panel) and total STAT1 (middle panel). b2actin was loading control (bottom panel). (D) Experiment as in (C) except cells were SeV infected for 0, 12 and 24 h. doi:10.1371/journal.pone.0008079.g002 PLoS ONE | www.plosone.org 5 November 2009 | Volume 4 | Issue 11 | e8079