A similar experiment was performed with activated form of RIG-I, encoding the NH2 terminal CARD domain (RIG-N). Expression of RIG-N was determined by Western immunoblot (Figure 3A, top right), and that of co-transfected IKKc isoforms (Figure 3A, middle right). Consistently with the findings of MAVS, RIG-N was unable to activate NF-kB-dependent reporter activity in IKKc2/2 MEFs (Figure 3B, left, bottom panel) but did so in cells expressing either IKKc-WT or IKKcD isoforms, where a 3-fold induction of PRDII was observed. Strikingly, and in a manner consistent with the expression of MAVS, RIG-N activated IRF3- dependent transcription only in the presence of IKKc-WT, but was unable to activate IRF3 in IKKcD expressing cells (Figure 3B, right, bottom panel). Together these data indicated that IKKc-WT mediates both NF-kB and IRF3 pathways, whereas IKKcD is unable to support IRF3 signaling. To further define this mechanism, we examined whether IRF3 was induced to translocate into the nucleus in SeV-infected IKKc2/2 deficient MEFs stably expressing IKKc-WT or IKKcD. Sucrose cushion-purified nuclear extracts, free of cytoplasmic markers (tubulin [29]), were assayed by Western immunoblot using an anti-IRF3 Ab. In cells expressing IKKc-WT, nuclear IRF3 was undetectable at 0- and 6 h, but appeared in the nuclear compartment after 12- and 24 h of SeV infection (Figure 3C). By contrast, in IKKcD expressing cells, no IRF3 was detected in the nucleus (Figure 3C), despite effective viral replication (Figure 1E). Next, nuclear extracts from SeV-infected MEFs were assayed for IRF3 DNA binding activity in EMSA using a radiolabeled ISRE site (taken from the ISG15 promoter). SeV induced a specific DNA binding activity 12- and 24 h after infection only in IKKc-WT expressing cells; no DNA binding activity was seen in IKKcD expressing cells (Figure 3D; this band was previously shown to be DNA sequence specific and contain IRF3 [1]). To further confirm defective IRF3 activation, IRF3 dimer formation was quantified by Western immunoblot of native gel-fractionated whole cell extracts prepared from IKKc2/2, IKKc-WT-reconstituted and IKKcD- reconstituted MEFs infected for various times by either SeV or RSV. IRF3 dimer formation was detected only in IKKc-WT- expressing cells in response to either type of viral infection (Figures 3E,F). We conclude that, in contrast to IKKc-WT, IKKcD is unable to mediate IRF3 nuclear translocation, DNA binding, dimerization or transcriptional activation. We next investigated whether IKKcD was coupled to the NF-kB pathway. First, confocal immunofluorescence experiments were performed for RelA nuclear translocation in IKKc2/2 MEFs complemented with either EGFP-IKKc or EGFP-IKKcD. Trans- fectants were then either treated with TNF (30 ng/ml, 1 h) or infected with RSV (MOI = 1, 24 h). Cells were fixed, RelA stained using anti-RelA Ab and transfectants imaged using confocal microscopy. Nuclei were counterstained with DAPI, and the presence of RelA examined in EGFP-expressing cells. In untreated controls, RelA was cytoplasmic in empty vector, IKKc-WT or IKKcD expressing cells (Figure 4A). In response to TNF stimulation, a strong nuclear concentration of RelA was observed in either IKKc-WT or IKKcD expressing cells but not those transfected with empty vector (Figure 4B). Similarly, RSV induced RelA nuclear translocation only in either IKKc-WT and IKKcD expressing cells (Figure 4C). A hallmark of the activated canonical NF-kB pathway involves cytoplasmic IkBa proteolysis via a ubiquitin proteasome-indepen- dent pathway [10], a phenomenon that is IKKc-dependent [13]. To confirm that RSV-induced RelA nuclear translocation was mediated by canonical NF-kB pathway activation, IkBa proteol- ysis was measured in cytoplasmic extracts using Western immunoblot. In IKKc-WT expressing cells, cytoplasmic IkBa IRF3-NF-kB Pathway Utilization proteolysis is clearly evident 6 h after RSV infection (Figure 4D), and is resynthesized 24 h after viral exposure via the RelA-IkBa positive feedback loop [30]. In IKKcD expressing cells, cytoplas- mic IkBa proteolysis is also observed, although with slower kinetics. Conversely, in nuclear extracts, NF-kB DNA binding increases in IKKc-WT-complemented cells 6 h after RSV infection, at times when cytoplasmic IkBa is degraded, and declines as IkBa is resynthesized, trapping NF-kB back in its cytoplasmic location (Figure 4D; supershifting experiments in RSV infected IKKc2/2 MEFs have previously demonstrated this complex to be composed of RelA?p50 complexes[13]). Consistent with the qualitative differences in kinetics of IkBa proteolysis, NF-kB DNA binding increases in IKKcD-expressing cells, peaking at later times and persisting 24 h after infection (Figure 4D). We sought to further understand the mechanism for qualitative difference in NF-kB activation in the IKKcD background. Previously we showed that RIG-I is strongly induced in response to RSV infection, and its expression is required for RSV inducible NF-kB activation [1]. Because RIG-I is type I IFN dependent, we examined whether RSV-induced RIG-I upregulation was attenuat- ed in IKKcD-expressing cells by QRT-PCR. Although RIG-I mRNA is induced by over 100-fold in IKKc-WT expressing cells, in both IKKc2/2 and IKKcD expressing cells, viral inducible RIG-I expression was significantly reduced, accounting, in part, for the reduced NF-kB activation (Figure 4E). Together, these data indicate that the primary defect in IKKcD signaling is the IRF3 pathway, and the attenuated NF-kB activation is because of secondarily reduced RIG-I expression ( note ectopic RIG-I or MAVS expression can activate the canonical NF-kB pathway, seen in Figures 3A,B). Defective IRF3 Activation in Cells Expressing Increased IKKcD:IKKc-WT Ratios Previous work from our lab demonstrated that IKKcD was universally expressed in various ratios in different tissue- and cell types with IKKc-WT [18]. To illustrate, the expression of IKKc and IKKcD was surveyed in 7 different cell types by Western immunoblot, where the 43 kDa IKKcD and 50 kDa IKKc-WT isoforms could be resolved. Both bands are specific as demonstrated by peptide competition experiments (Supplementary Figure S3 online). Both IKKc and IKKcD isoforms are expressed from ,2:1 IKKc:IKKcD ratios in HepG2 or HEK293 cells, to 1:2 IKKc:IKKcD ratios in WT MEF cells, to 1:4 ratios in HeLa S3 cells (Figure 5A). We postulated that cells natively expressing high amounts of IKKcD could be defective in IRF3 activation. We therefore assayed IRF3 dimer formation in Hela S3 cells by Western immunoblot after native gel fractionation. Despite the finding that RSV replicated in Hela S3 better than that of Hela CCL2 cells (Supplementary Figure S4 online), HeLa S3 cells showed no evidence of IRF3 dimer formation. By contrast, efficient IRF3 dimer formation was observed in HeLa CCL2 cells (Figure 5B). To confirm HeLa cells could efficiently couple to the canonical NF-kB pathway, the response to TNF was measured. We observed that Hela S3 cells have an intact canonical NF-kB activation pathway indicated by rapid cytoplasmic IkBa proteolysis and appearance of nuclear NF-kB DNA binding activity in EMSA (Figure 5C these complexes have been extensively characterized by competition and supershift as containing RelA?p50 heterodimers [11,31]). To confirm that HeLa S3 cells had an otherwise intact RIG?MAVS- IRF3 pathway, HeLa S3 were complemented with IKKc-WT. For this experiment, HeLa S3 cells were co-transfected with emp- ty(pcDNA), IKKc-WT, or IKKcD expression vectors and the IRF3- driven IFNb PRDIII luciferase reporter gene. Cells were then RSV infected, and luciferase activity measured 24 h later. RSV was unable to activate PRDIII luciferase reporter activity in the cells transfected PLoS ONE | www.plosone.org 7 November 2009 | Volume 4 | Issue 11 | e8079