IRF3-NF-kB Pathway Utilization Figure 5. Defective IRF3 activation in cells expressing high endogenous levels of IKKcD. (A) 100 mg whole cell extracts (WCE) from HepG2, HEK293, Hela CCL3, Hela S3, A549, IKKc2/2 MEFs and wild type MEFs were fractionated by SDS-PAGE and Western immunoblot was conducted using anti-IKKc Ab. Location of IKKc isoform is indicated. (B) Hela S3 cells were infected with RSV for 0, 12 or 24 h. WCEs were assayed for IRF3 dimerization using native gel electrophoresis. Shown is Western blot probed with anti-IRF3 Ab. Hela CCL2 cells were used as control. (C). Time course of TNF stimulation. Hela S3 cells were stimulated with TNF (30 ng/ml) for indicated times prior to cytoplasmic and nuclear protein extraction. Top, Western immunoblot using anti-IkBa Ab. b-actin staining is loading control. Bottom, NE were used in EMSA. Shown is a region of the autoradiogram with the specific NF-kB binding complex. (D) HelaS3 cells were co-transfected with the IRF3-dependent PRDIII luciferase reporter gene along with an empty vector, IKKc-WT or IKKcD. 24 h after transfection, cells were infected with RSV for another 24 h. WCEs were collected and the luciferase assay was conducted. Shown is normalized luciferase reporter activity in triplicate independent plates. Data was reproduced in two independent experiments. n.s., not significant. doi:10.1371/journal.pone.0008079.g005 for the ,90-fold induction of IFNa1, 350-fold induction of IRF7 and 500-fold induction of IFNa4 mRNAs (Figure 6B). As expected from the blunted RIG-I induction in cells expressing IKKcD (see Figure 4E), the RSV inducible expression of NF-kB dependent Grob and A20 genes were also blunted. Together we conclude that the relative IKKc-WT:IKKcD ratio, independent of changes in total IKKc abundance, controls cellular IRF3-IFN responsive- ness to ssRNA virus infection. IKKcD Overexpression Is a Dominant Negative Inhibitor of IKKc-WT Mediated IFN Production Earlier we showed that IKKcD is a dominant negative inhibitor of HLTV-I Tax induced NF-kB activation, despite the ability of both isoforms to bind HTLV-I Tax protein [18]. To explore whether IKKcD functioned as a dominant negative inhibitor of type I IFN production, we performed an alternative experiment where IKKcD was expressed in increasing amounts in the presence of a constant amount of IKKc-WT in IKKc2/2 MEFs (Figure 7). Type I IFN production was quantified in RSV using QRT-PCR. Co-transfection of IKKcD at 2.5 mg reduced type I IFN expression by ,50% that was further reduced in a dose- dependent manner up to 7.5 mg expression plasmid, where the response was almost completely abolished. Together we conclude that the relative IKKc-WT:IKKcD ratio primarily mediates IRF3-IFN responsiveness in response to RNA virus infection through its dominant negative effect. IKKcD Is Defective in Recruiting the TBK1 Adapter, TANK Previous work has demonstrated that an interaction between the TBK1 adapter, TANK, and IKKc is required for coupling RIG-I?MAVS complex to IRF3 activation [15]. Because IKKcD fails to activate IRF3, we first tested whether IKKcD associates with MAVS. For this purpose, Myc epitope-tagged IKKc-WT or IKKcD expression vectors were co-transfected with Flag-tagged MAVS and subjected to nondenaturing coimmunoprecipitation using anti-Myc Ab. MAVS association was detected by immuno- blot using anti-Flag Ab. We observed that both IKKc and IKKcD effectively bound to MAVS (Figure 8A). We next asked whether IKKcD was able to bind TANK. In this experiment, Myc epitope tagged IKKc-WT or IKKcD expression vectors were co-transfected with V5-epitope tagged TANK. A nondenaturing co-immunoprecipitation assay experiment was then performed using anti-Myc Ab as the primary immunopre- cipitating Ab, followed by Western immunoblot of the immuno- precipitates using anti-V5 Ab. V5-TANK was only observed in immunoprecipitates in cells expressing IKKc-WT, but not IKKcD (Figure 8B, top panel). Equivalent amounts of IKKc-WT and IKKcD were seen in the immunoprecipitates (Figure 8B, bottom panel). TANK is an external adaptor that mediates the recruitment of the atypical IKK, IKKe, to IKKc. Because IKKcD is unable to bind TANK, we investigated whether IKKcD was defective in IKKe recruitment. For this purpose, either Flag epitope tagged PLoS ONE | www.plosone.org 9 November 2009 | Volume 4 | Issue 11 | e8079