pathway and the induction of type I IFNs. Our studies further indicate that the relative level of expression of IKKc splice forms affects IFN-mediated antiviral signaling in the host cell and may affect viral tissue tropism. Manipulation of the expression of these two isoforms may provide a mechanism to modulate the two arms of the innate immune pathway where preferential expression of NF-kB inflammatory signaling or IRF3 induced anti-viral signaling would be desired. Supporting Information Table S1 Primers used for QRT-PCR Found at: doi:10.1371/journal.pone.0008079.s001 (5.05 MB TIF) Figure S1 Enhanced cytopathic effect in IKKc expressing cells. Wild type, empty vector, IKKc-WT and IKKcD reconstituted IKKc2/2-deficient MEFs were infected by RSV (M.O.I. = 1) for 24 h. Cells were also 4% paraformaldehyde fixed, stained with SYTOX (Molecular Probes) and imaged by fluorescence micros- copy (magnification of 10X). Representative multinucleated cells are indicated by white arrows. In IKKc2/2 or IKKcD,expressing cells, 13 and 15 multinucleated cells/high power field were detected respectively, while in wild type- and IKKc-WT- reconstituted MEFs, only 1 and 2 fusion cells were observed. Found at: doi:10.1371/journal.pone.0008079.s002 (4.49 MB TIF) Figure S2 Defective IFN response in cells stably transfected with IKKcD response to RNA virus infection. (a) WT MEFs, or References 1. Liu P, Jamaluddin M, Li K, Garofalo RP, Casola A, et al. (2007) Retinoic acid- inducible gene I mediates early antiviral response and toll-like receptor 3 expression in respiratory syncytial virus-infected airway epithelial cells. J Virol 81: 1401–1411. 2. Kato H, Sato S, Yoneyama M, Yamamoto M, Uematsu S, et al. (2005) Cell Type-Specific Involvement of RIG-I in Antiviral Response. Immunity 23: 19–28. 3. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, et al. (2006) Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441: 101–105. 4. Oshiumi H, Matsumoto M, Hatakeyama S, Seya T (2008) Riplet/RNF135, a RING-finger protein, ubiquitinates RIG-I to promote interferon-beta induction during the early phase of viral infection. J Biol Chem M804259200. 5. Gack MU, Shin YC, Joo CH, Urano T, Liang C, et al. (2007) TRIM25 RING- finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 446: 916–920. 6. Baril M, Racine ME, Penin F, Lamarre D (2009) MAVS Dimer Is a Crucial Signaling Component of Innate Immunity and the Target of Hepatitis C Virus NS3/4A Protease. J Virol 83: 1299–1311. 7. Kumar H, Kawai T, Kato H, Sato S, Takahashi K, et al. (2006) Essential role of IPS-1 in innate immune responses against RNA viruses. J Exp Med 203: 1795–1803. 8. Zandi E, Karin M (1999) Bridging the gap: composition, regulation, and physiological function of the IkappaB kinase complex. Mol Cell Biol 19: 4547–4551. 9. Garofalo R, Sabry M, Jamaluddin M, Yu RK, Casola A, et al. (1996) Transcriptional activation of the interleukin-8 gene by RSV infection in alveolar epithelial cells: Nuclear translocation of the Rel A transcription factor as a mechanism producing airway mucosal inflammation. J Virol 70: 8773–8781. 10. Jamaluddin M, Casola A, Garofalo RP, Han Y, Elliott T, et al. (1998) The major component of IkBa proteolysis occurs independently of the proteasome pathway in Respiratory Syncytial Virus-infected pulmonary epithelial cells. J Virol 72: 4849–4857. 11. Tian B, Nowak D, Brasier AR (2005) A TNF Induced Gene Expression Program Under Oscillatory NF-kB Control. BMC Genomics 6: 137. 12. Tian B, Brasier AR (2005) The Nuclear Factor-kB (NF-kB) Gene Regulatory Network. In: Microarrays and Transcription Factor Networks. Landes Bioscience. 13. Liu P, Li K, Garofalo RP, Brasier AR (2008) Respiratory syncytial virus induces RelA release from cytoplasmic 100-kDa NF-kappa B2 complexes via a novel retinoic acid-inducible gene-I{middle dot}NF- kappa B-inducing kinase signaling pathway. J Biol Chem 283: 23169–23178. 14. Hiscott J, Pitha P, Genin P, Nguyen H, Heylbroeck C, et al. (1999) Triggering the interferon response: the role of IRF-3 transcription factor. J Interferon Cytokine Res 19: 1–13. IRF3-NF-kB Pathway Utilization IKKc2/2 MEFs stably reconstituted with IKKc-WT or IKKcD were RSV infected for 16 h (MOI = 1). Total RNA was extracted and QRT-PCR was conducted using probes for IFN-b, -a1, -a4. (b) WT MEFs, or IKKc2/2 MEFs stably reconstituted with IKKc-WT or IKKcD were Sendai virus infected for 16 h. Total RNA was extracted and QRT-PCR was conducted using probes for IFN-b, and -a4. (c) Same experiment as in (b) where QRT- PCR was performed with probes for IRF7, IP10 and RANTES. Found at: doi:10.1371/journal.pone.0008079.s003 (5.61 MB TIF) Figure S3 Antibody specificity. The staining specificity of anti- IKKc Ab was evaluated using peptide preadsorption. Anti-IKKc Ab was preadsorbed with nothing or 10-fold molar excess of recombinant purified GST-IKKcD-WT, and used as primary Ab in Western immunoblot. Both bands are reduced by 50%. Found at: doi:10.1371/journal.pone.0008079.s004 (0.19 MB TIF) Figure S4 Effective RSV replication in HeLa S3 cells. Western immunoblot of HeLa S3 cells infected with RSV for indicated times. Found at: doi:10.1371/journal.pone.0008079.s005 (0.50 MB TIF) Author Contributions Conceived and designed the experiments: PL ML TGW ARB. Performed the experiments: PL ML BT DP. Analyzed the data: PL ML BT KL RPG DP TGW ARB. Contributed reagents/materials/analysis tools: PL BT KL ARB. Wrote the paper: PL ML BT KL RPG DP TGW ARB. 15. Zhao T, Yang L, Sun Q, Arguello M, Ballard DW, et al. (2007) The NEMO adaptor bridges the nuclear factor-kappaB and interferon regulatory factor signaling pathways. Nat Immunol 8: 592–600. 16. Guo B, Cheng G (2007) Modulation of the interferon antiviral response by the TBK1/IKKi adaptor protein TANK. J Biol Chem 282: 11817–11826. 17. Yount J, Moran TM, Lopez CB (2007) Cytokine-Independent Upregulation of MDA5 in Viral Infection. J Virol 81: 7316–7319. 18. Hai T, Yeung ML, Wood TG, Wei Y, Yamaoaka S, et al. (2006) An Alternative Splice Product of IkB Kinase (IKK)-c, IKKc2D, Differentially Mediates Cytokine And HTLV-I Tax Induced NF-kB Activation. J Virol 80: 4227–4241. 19. Yamaoka S, Courtois G, Bessia C, Whiteside ST, Weil R, et al. (1998) Complementation cloning of NEMO, a component of the IkB kinase complex essential for NF-kB activation. Cell 93: 1231–1240. 20. Foy E, Li K, Wang C, Sumpter R Jr, Ikeda M, et al. (2003) Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science 300: 1145–1148. 21. Foy E, Li K, Sumpter R Jr, Loo YM, Johnson CL, et al. (2005) Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-I signaling. Proc Natl Acad Sci U S A 102: 2986–2991. 22. Seth RB, Sun L, Ea CK, Chen ZJ (2005) Identification and Characterization of MAVS, a Mitochondrial Antiviral Signaling Protein that Activates NF-[kappa]B and IRF3. Cell 122: 669–682. 23. Brasier AR (1990) Reporter system using firefly luciferase. In: Ausubel FM, ed. Current Protocols in Molecular Biology. New York: John Wiley & Sons. pp 9.6.10–9.6.13. 24. Tian B, Zhang Y, Luxon BA, Garofalo RP, Casola A, et al. (2002) Identification Of NF-kB Dependent Gene Networks In Respiratory Syncytial Virus-Infected Cells. J Virol 76: 6800–6814. 25. Li K (2009) Regulation of interferon regulatory factor 3-dependent innate immunity by the HCV NS3/4A protease. In: Methods in Molecular Biology. Clifton, NJ: Springer. pp 211–226. 26. Jamaluddin M, Wang S, Garofalo R, Elliott T, Casola A, et al. (2001) INFb mediates coordinate expression of antigen-processing genes in RSV infected pulmonary epithelial cells. Am J Physiol Lung Cell Mol Physiol 280: L248–L257. 27. Smieja J, Jamaluddin M, Brasier AR, Kimmel M (2008) Model-based analysis of interferon-{beta} induced signaling pathway. Bioinformatics 24: 2363–2369. 28. Lehtonen A, Matikainen S, Julkunen I (1997) Interferons up-regulate STAT1, STAT2, and IRF family transcription factor gene expression in human peripheral blood mononuclear cells and macrophages. The Journal of Immunology 159: 794–803. 29. Forbus J, Spratt H, Wiktorowicz J, Wu Z, Boldogh I, et al. (2006) Functional Analysis Of The Nuclear Proteome Of Human A549 Alveolar Epithelial Cells By HPLC- High Resolution 2D Gel Electrophoresis. Proteomics 6: 2656–2672. 30. Lipniacki T, Paszek P, Brasier AR, Luxon B, Kimmel M (2004) Mathematical model of NF-kB regulatory module. J Theor Biol 228: 195–215. PLoS ONE | www.plosone.org 14 November 2009 | Volume 4 | Issue 11 | e8079