IRF3-NF-kB Pathway Utilization Figure 7. IKKcD is a dominant negative inhibitor of type I IFN expression. IKKc2/2 MEFs were transfected with increasing amounts of IKKcD eukaryotic expression vectors in the presence of a constant amount of IKKc-WT as indicated. 24 h later, transfectants were RSV infected for 16 h. Total cellular RNA was assayed by QRT-PCR for the expression of IFNb, IFNa1, IRF7 or IFNa4 as indicated. Shown is X6SD of the fold mRNA induction relative to uninfected IKKc-WT transfected cells. Results were repeated three times with similar results. doi:10.1371/journal.pone.0008079.g007 unable to recruit TANK-IKKi to the activated RIG-I?MAVS complex. The Dominant Negative Effect of IKKcD Is Mediated by Displacement of IKKc-WT from MAVS To determine the mechanism for the inhibitory effect of IKKcD (Figure 7), we sought to determine whether IKKcD could displace the IRF3 signaling-competent IKKc-WT isoform from the activated MAVS complex. For this experiment, Myc epitope tagged IKKc-WT was co-transfected with FLAG-tagged MAVS in the absence or presence of IKKcD. Lysates were then subjected to nondenaturing coimmunoprecipitation using anti-FLAG, and IKKc isoform association was determined by Western blot using anti-Myc Ab. In the absence of IKKcD, IKKc-WT was associated with MAVS, however, the expression of IKKcD completely displaced IKKc-WT from the MAVS complex (Figure 9). Discussion IKKc was identified as an essential regulatory subunit of the canonical IKK complex because IKKc deficient cells were unable to activate NF-kB in response to most known stimuli [19,32]. IKKc plays multiple adapter roles in IKK activation through its ability to organize the assembly of IKKs into the activated high molecular weight complex [33,34], bind ubiquitylated upstream signaling adapters [32,35,35–37], and recruit the IkBa inhibitor into the activated IKK complex [33]. Through these activities, IKKc forms a molecular bridge between IKK, its upstream activators, and its substrate. In the innate immune response pathway, IKKc recruits IKK2a and -b catalytic complexes to RIG-I?MAVS, resulting in IkBa proteolysis and canonical NF-kB activation. Similarly IKKc is a binding target for TANK, an adapter that links TBK1 and IKKe, two key kinases controlling IRF3 activation [16,38]. In this manner, IKKc is the final common shared signaling adapter upstream of the divergent IRF3 and the canonical NF-kB pathways. Both IRF3 and NF-kB signaling play important, yet distinct, roles in anti-viral and inflammatory signaling in response to ssRNA viral infection. For example, IRF3 is a major mediator of type I IFN production, important in mucosal anti-viral response; in IKKc2/2 cells, the replication of RNA viruses is significantly increased due to the inability to produce type I IFNs [15]. Similarly, NF-kB signaling is important in initiating mucosal inflammation and the adaptive immune response. The coordination and timing of these two arms of innate immune signaling response may affect the resolution of viral infection, yet the mechanisms for selection of these two pathways are not yet fully elucidated. In this study, we have extended our previous work describing the signaling properties of a ubiquitously expressed IKKc alternative splice product. Previously we reported that IKKcD is defective in mediating HTLV-Tax recruitment, but more efficiently mediates NF-kB activation by IKKa/b and MAP3Ks, NIK and TAK/TAB [18]. These earlier studies showed that IKKcD efficiently binds to IKKa/b isoforms in coimmunopreci- pitation experiments, and induces IKK kinase activity to a greater degree than does IKKc-WT. Here we find that IKKcD is primarily defective in IRF3 signaling, reducing type I IFN production and ISG signaling by displacing IKKc-WT from PLoS ONE | www.plosone.org 11 November 2009 | Volume 4 | Issue 11 | e8079