Expression of an IKKc Splice Variant Determines IRF3 and Canonical NF-kB Pathway Utilization in ssRNA Virus Infection Ping Liu1., Muping Lu1., Bing Tian 1 , Kui Li2, Roberto P. Garofalo3,4, Deborah Prusak4, Thomas G. Wood4,5, Allan R. Brasier1,4* 1 Department of Medicine, University of Texas Medical Branch (UTMB), Galveston, Texas, United States of America, 2 Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America, 3 Department of Pediatrics, University of Texas Medical Branch (UTMB), Galveston, Texas, United States of America, 4 Sealy Center for Molecular Medicine, University of Texas Medical Branch (UTMB), Galveston, Texas, United States of America, 5 Department of Biochemistry and Molecular Biology, University of Texas Medical Branch (UTMB), Galveston, Texas, United States of America Abstract Single stranded RNA (ssRNA) virus infection activates the retinoic acid inducible gene I (RIG-I)- mitochondrial antiviral signaling (MAVS) complex, a complex that coordinates the host innate immune response via the NF-kB and IRF3 pathways. Recent work has shown that the IkB kinase (IKK)c scaffolding protein is the final common adapter protein required by RIG-I?MAVS to activate divergent rate-limiting kinases downstream controlling the NF-kB and IRF3 pathways. Previously we discovered a ubiquitous IKKc splice-variant, IKKcD, that exhibits distinct signaling properties. Methodology/Principal Findings: We examined the regulation and function of IKKc splice forms in response to ssRNA virus infection, a condition that preferentially induces full length IKKc-WT mRNA expression. In IKKcD-expressing cells, we found increased viral translation and cytopathic effect compared to those expressing full length IKKc-WT. IKKcD fails to support viral-induced IRF3 activation in response to ssRNA infections; consequently type I IFN production and the induction of anti- viral interferon stimulated genes (ISGs) are significantly attenuated. By contrast, ectopic RIG-I?MAVS or TNFa-induced canonical NF-kB activation is preserved in IKKcD expressing cells. Increasing relative levels of IKKc-WT to IKKcD (while keeping total IKKc constant) results in increased type I IFN expression. Conversely, overexpressing IKKcD (in a background of constant IKKc-WT expression) shows IKKcD functions as a dominant-negative IRF3 signaling inhibitor. IKKcD binds both IKK-a and b, but not TANK and IKKe, indicating that exon 5 encodes an essential TANK binding domain. Finally, IKKcD displaces IKKcWT from MAVS explaining its domainant negative effect. Conclusions/Significance: Relative endogenous IKKcD expression affects cellular selection of inflammatory/anti-viral pathway responses to ssRNA viral infection. Citation: Liu P, Lu M, Tian B, Li K, Garofalo RP, et al. (2009) Expression of an IKKc Splice Variant Determines IRF3 and Canonical NF-kB Pathway Utilization in ssRNA Virus Infection. PLoS ONE 4(11): e8079. doi:10.1371/journal.pone.0008079 Editor: Dong-Yan Jin, University of Hong Kong, Hong Kong Received May 21, 2009; Accepted November 5, 2009; Published November 26, 2009 Copyright: 2009 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was supported by P01 AI062885 (to A.R.B.), RO1 GM086885 and NHLBI Proteomics Technologies in Airway Inflammation (N01-HV-28184). Core Laboratory support was from NIEHS grant P30 ES06676. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: arbrasie@utmb.edu . These authors contributed equally to this work. Introduction Activation of the mucosal innate immune response in sentinel epithelial cells is vital to the resolution of mucosal viral infection. Here, viral replication intermediates are sensed by cytoplasmic pattern recognition receptors, an event that activates two important signaling pathways, one mediated by the NF-kB transcription factor controlling inflammatory cytokine expression, and the second mediated by IRF3 controlling anti-viral type I IFN-a and -b expression. The coordinated expression of these two pathways is responsible for limiting viral replication and activating the adaptive immune response. Significant advances have been made in identifying the structure of these two pathways and their mechanism of control. Cytoplasmic RNA virus infections, including Sendai (SeV)-, influenza-, Japanese encephalitis-, respiratory syncytial (RSV)- and others, produce 5’triphosphate modified- or ds-RNA products during their replication cycle. These ‘‘non-self’’ RNA species are bound by RIG-I, a cytoplasmic DExD/H box RNA helicase [1–3]. RNA-bound RIG-I is rapidly polyubiquitylated by E3 ligases (TRIM25 and Riplet/RNF13) that catalyze addition of Lys 63- linked ubiquitin polymers into the RIG-I?NH2 terminus [4,5]. Lys 63-ubiquitinated RIG-I, in turn, associates with the mitochondrial antiviral signaling (MAVS) protein via its NH2 terminal caspase recruitment domain (CARD), producing an activated dimeric complex [6]. The assembled RIG-I?MAVS complex, in turn, recruits the TNF Receptor-associated factors (TRAFs)- 2, -3 and -6 to multiple TRAF-interaction motifs located in the MAVS proline PLoS ONE | www.plosone.org 1 November 2009 | Volume 4 | Issue 11 | e8079