Figure 10. IKKc-WT-IKKcD affects IRF3-NF-kB pathway utiliza- tion in ssRNA viral infection. Schematic diagram of two distinct signaling complexes produced by IKKc splice variants. RIG-I recognition of ssRNA intermediates induces the activated RIG-IMAVS signaling complex. Depending on the relative abundance of IKKc-WT and IKKcD isoforms, distinct signaling pathways are activated downstream. Mito, mitochondrion. doi:10.1371/journal.pone.0008079.g010 role in anti-viral response. In this regard, we note that others have suggested that NF-kB signaling mediates anti-viral activity [42]. Inhibition of NF-kB signaling in response to hPIV or RSV infection resulted in enhanced viral replication in an IFN-independent manner. The selective deficiency in IRF3 signaling in IKKcD expressing cells may allow the isolated study or identification of this potential anti-viral pathway. The data in this study and that of our previous work have indicated that IKKcD is competent to mediate signals through the canonical NF-kB pathway. This conclusion is supported by multiple lines of evidence: 1. the ability of ectopic RIG-I or MAVS to activate NF-kB dependent gene expression in IKKcD expressing MEFs (Figure 3); 2. the ability of RSV infection to induce IkBa proteolysis in IKKcD expressing MEFs (Figure 3); 3. the ability of TNF to induce IkBa proteolysis in Hela S3 cells predominately expressing the IKKcD isoform (Figure 4); and 4. the ability of catalytic IKKa/b isoforms to bind IKKcD in nondenaturing coimmunoprecipitation assays (Figure 8). Previous protein interaction mapping studies that show the IKKa/b interaction motif lies in a 119 aa region in the far NH2 terminus of IKKc [37,43,44], upstream of residues encoded by exon 5. Despite the ability to mediate productive IKKa/b binding and interaction, we note the qualitative difference in the kinetics of IkBa proteolysis in IKKcD expressing MEFs induced by RSV infection (Figure 4). Our data suggest that the slower kinetic IRF3-NF-kB Pathway Utilization response of NF-kB activation in IKKcD expressing cells is due to reduction in IRF3-IFN-RIG-I cross talk pathway. Viral activation of RIG-I?MAVS signaling is thought to occur in two sequential phases. The first phase is mediated by low ambient concentrations of RIG-I early in the course of viral infection, where viral RNA is in low abundance [45]. Here, initial activation of IRF3 is mediated by the TRIM25 or Riplet/RNF135 ubiquitin ligases, inducing RIG-I ubiquitylation vis Lys 63- linked ubiquitin polymers, a modification that promotes its association with MAVS, initiating downstream IFN production [46]. The subse- quent potent upregulation of RIG-I expression induced by this first wave of IFN [46] produces amplification of the signaling pathway. We observed that RSV-induced RIG-I upregulation is attenuated in IKKcD expressing cells, nearly to that seen in IKKc2/2 MEFS (Figure 3). Taken in context with our earlier work showing that IFN-induced RIG-I upregulation is required for RSV-induced NF-kB activation [47], these data may suggest a mechanism for cross-talk between the IRF3 and NF-kB pathway in viral induced inflammation. In the absence of IRF3-IFN signaling, seen in IKKcD expressing cells, reduced RIG-I upregulation may result in delayed and or attenuated NF-kB activation. Secondary structure predictions of IKKcD reveals that the occlusion of exon 5 affects the COOH terminus of an extended NH2 terminal coiled-coil motif at aa 174–224, a motif important in protein-protein interaction [18]. This alternative splice variant exhibits differential signaling by binding distinct signaling molecules. In this regard, IKKc is essential for TNF signaling via its ability to recruit IKK to activated TNF receptors, mediate interaction with upstream MAP3Ks, and directly and/or recruit IkBa into the activated IKK for stimulus-induced phosphoryla- tion. Recent work has shown that this scaffolding function of IKKc may be due to its inducible post-translational modification via a unique chemistry of head-to-tail ubiquitin polymers catalyzed by the LUBAC ubiquitin ligase complex. These inducible ubiquitin polymers enhance the binding of upstream MAP3Ks, that phosphorylate associated catalytic IKKa/b subunits, resulting in their activation [36]. Interestingly, the LUBAC-mediated ubiquitin polymerization is involved in TNF inducible NF-kB signaling, but not by the related cytokine, IL-1. The role of post- translational modifications of IKKc in response to ssRNA viral infection is not known. We note from co-immunoprecipitation experiments, that IKKcD associates with MAVS in the absence of ssRNA infection (Figure 8A), suggesting to us, that IKKc-MAVS complex may be a stable, pre-formed complex whose activity is initiated by binding activated RIG-I. Our findings that IKKcD displaces IKKc-WT from the MAVS complex explains the dominant negative effect of IKKcD to reduce IRF3 activation. IKKcD is defective in TANK binding and consequently recruiting downstream TBK1?IKKi, a kinase complex essential in IRF3 phosphorylation. Like IKKc, TANK is itself a scaffolding protein responsible for recruiting IKKe and TBK1 into an activated complex [38]. Structure-function studies of TANK have revealed a C2H2-type zinc finger in the TANK COOH terminus essential for IKKc association [48]. Conversely, sequential mutagenesis and mapping studies on IKKc have identified aa 150–250 aa as the TANK binding domain [15,38]. Our study is consistent with these findings where IKKcD, lacking exon5-encoded 174–224 aa, is unable to bind TANK. In the absence of TANK binding, in IKKcD expressing cells, RIG?I MAVS is unable to signal to the IRF3 pathway, induce type I IFN expression or activate ISG signaling. In summary, our findings reveal ubiquitously expressed IKKc splice variant differentially couples IKK signaling to the IRF3 PLoS ONE | www.plosone.org 13 November 2009 | Volume 4 | Issue 11 | e8079