rich domain [7]. This complex, serves as a scaffold for recruitment of signaling adapters mediating activation of the divergent NF-kB and IRF3 pathways. Downstream activation of the IRF3 pathway results in dramatic upregulation of RIG-I expression and signal amplification. RIG-I?MAVS activates two distinct pathways controlling NF-kB, termed the canonical and cross-talk pathways. The canonical pathway is mediated by activating the IKK complex, a signaling complex containing the two closely related kinase subunits, IKKa and IKKb, and a third regulatory subunit, IKKc [8]. In the process of IKK activation, IKKc is required for recruiting the catalytic IKK-a and b subunits to activated RIG-I?MAVS, where they are serine phosphorylated in their activation loops. IKK activation effects the phosphorylation and inducible degradation of the IkB inhibitor, resulting in nuclear translocation of the NF-kB/RelA transcriptional activator [9,10]. Here, activated nuclear NF-kB induces expression of inflammatory cytokines such as Grob, IL-6, IL-8 and others [11,12]. By contrast, the cross-talk pathway is mediated by RIG-I?MAVS direct interacting with the IKKa-NF- kB inducing kinase (NIK) complex, in an IKKc -independent manner [13]. This pathway, time-delayed relative to the canonical pathway, results in RelA and RelB release from cytoplasmic- sequestered p100. In this way RIG-I?MAVS induces two effector arms converging on NF-kB, producing mucosal inflammation. RIG-I?MAVS also induces the IRF3 pathway, a pathway controlled by a complex of two IKK-related kinases, TANK- binding kinase 1 (TBK1) and an inducible subunit, IKKe [14]. Here, the TRAF-associated NF-kB activator (TANK) links TBK1and IKKe with upstream TRAF molecules [15,16]. Importantly, IRF3 activation also requires the IKKc signaling adapter; in IKKc-deficient cells, IRF3 activation is also abolished in response to different RNA viruses [15]. As a result of IRF3 activation, the expression of type I IFNs results in a potent upregulation of RIG-I and its ubiquitin ligases, thereby potenti- ating coordinate signaling by the NF-kB and IRF3 innate signaling responses [17]. In this way, IKKc serves as the final adaptor molecule in RIG-I?MAVS signaling that is shared between the canonical NF-kB and the IRF3 pathways. In previous work, we identified an alternatively spliced IKKc isoform, termed IKKcD. IKKcD is missing a crucial region in the NH2 terminal coiled coil domain whose functional effect is to couple IKK to distinct upstream signals. Interestingly, IKKcD efficiently mediates cytokine-induced canonical NF-kB activation by associating with the IKKa/b kinases, and mediates TAK/TAB and NIK inducible NF-kB activation, but is resistant to HTLV Tax [18]. Here we investigate its signaling role in response to ssRNA infection. In response to RSV infection, we find that IKKc WT isoform is potently upregulated relative to the IKKcD splice form. In cells only expressing IKKcD, enhanced viral replication and cytopathic effect were seen due to deficient IRF3 signaling and type I IFN production. IKKcD functions as a dominant- negative inhibitor of IRF3 signaling being unable to couple to the TANK-IKKe complex and displaces IKKc from activated RIG-I?MAVS. These data suggest that endogenous expression of IKKcD is involved in balancing inflammatory and anti-viral signaling response to ssRNA infection. Materials and Methods Cell Cultures Human A549 pulmonary type II epithelial cells (American Type Culture Collection [ATCC]) were grown in F12K medium (Gibco) with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 g/ml) at 37uC in a 5% CO2 incubator. Wild type IRF3-NF-kB Pathway Utilization and IKKc2/2 [19] MEFs were cultured in Eagle’s minimum essential medium (Gibco) with 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, and 10% FBS. IKKc and IKKcD reconstituated stable MEFs were described previously [18]. HEK293 cells were cultured in Eagle’s minimum essential medium (Gibco) with 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, and 10% FBS. Virus Preparation and Infection The human RSV A2 strain was propogated in Hep2 cells and purified on sucrose cushion gradient [1]. Cells were infected at an MOI of 1.0 for indicated times. Sendai virus was purchased from Charles River Laboratory. Cells were infected with 100 hemag- glutinin units/ml [20]. Plasmid Construction Expression vectors encoding Flag epitope-tagged RIG-I?NH2 terminus and Flag eiptope-tagged MAVS were described [21,22]. pEF6-Flag-IKKa and pEF6-Flag-IKKb were described in [18]. Myc-epitope tagged IKKc and IKKcD were constructed by cloning PCR generated IKKc cDNA into BamH1/HindIII sites of pcDNA3Myc. The PCR primers used were: 59-ATCAATG- GATCC ATGGAACAGAAGTTGATTTC CGAAGAAGAG CTCGGATCCATGAATTAGGCA CCT-39 (upstream, Bam site underlined) and 59-AGTATCAAGCTTCTACTC AATG- CACTCC ATGACAT-39 (downstream). pEGFP IKKc and IKKcD were constructed by cloning the same cDNAs amplified using the same upstream primer and the downstream primer 59- AGTATCAAGCTTCTC AATGCACTCC ATGACAT-39 (to remove the stop codon) into Bam H1/HindIII digested pcDNA3EGFP, encoding a EGFP fusion on the COOH terminus of the IKKc isoform. Plasmids were purified on Qiagen columns and sequenced for authenticity. Transfection Two million freshly isolated cells were transfected in suspension with indicated plasmids according to the manufacturers recom- mendation (Amaxa). After transfection, cells were immediately transferred to DMEM and cultured for 24 h before treatment. Luciferase reporter assays were performed as previously described [23]. Data represents mean6SD of triplicate plates of normalized luciferase reporter activity. Quantitative Real-Time PCR (QRT-PCR) Total RNA was extracted using acid guanidium phenol extraction (Tri Reagent; Sigma). For IFN and ISG analyses, 1 mgm of RNA was reversely transcribed using Super Script III in a 20 ml reaction mixture. One ml of cDNA product was diluted 1:2, and 2 mL was amplified in a 20 mL reaction mixture containing 12.5 mL of SYBR Green Supermix (Bio-Rad) and 0.4 mM each of forward and reverse gene-specific primers (Supplementary data, Table S1), aliquoted into 96-well, 0.2-mm thin-wall PCR plates, and covered with optical-quality sealing tape. The plates were denatured for 90 s at 95uC and then subjected to 40 cycles of 15 s at 94uC, 60 s at 60uC, and 1 min at 72uC in iCycler (BioRAD). The IKKc isoform specific QRT-PCR assays were performed in triplicate in an ABI Prism 7000 Sequence Detection System using the SYBR Green PCR Master Mix (ABI #4364344) as specified by the manufacturer. The final primer concentration was 900 nM (Supplementary data, Table S1). The PCR assays were denatured for 10 min at 95uC, followed by 40 cycles of 15 s at 95uC and 60 s at 60uC. After PCR was performed, PCR products were subjected to melting curve analysis to assure a single PLoS ONE | www.plosone.org 2 November 2009 | Volume 4 | Issue 11 | e8079