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Involvement of the IB Kinase (IKK)-Related Kinases Tank-Binding Kinase 1/IKKi and Cullin-Based Ubiquitin Ligases in IFN Regulatory Factor-3 Degradation¹

Annie Bibeau-Poirier^*, Simon-Pierre Gravel^*, Jean-François Clément^*, Sébastien Rolland^*, Geneviève Rodier, Philippe Coulombe, John Hiscott, Nathalie Grandvaux, Sylvain Meloche and Marc J. Servant^2,*

^* Faculty of Pharmacy, Institute of Research in Immunology and Cancer, Centre Hospitalier de l’Université de Montréal and Department of Biochemistry, University of Montreal, Montreal, Canada; and Lady Davis Institute for Medical Research, McGill University, Montreal, Canada

Activation of the innate arm of the immune system following pathogen infection relies on the recruitment of latent transcription factors involved in the induction of a subset of genes responsible for viral clearance. One of these transcription factors, IFN regulatory factor 3 (IRF-3), is targeted for proteosomal degradation following virus infection. However, the molecular mechanisms involved in this process are still unknown. In this study, we show that polyubiquitination of IRF-3 increases in response to Sendai virus infection. Using an E1 temperature-sensitive cell line, we demonstrate that polyubiquitination is required for the observed degradation of IRF-3. Inactivation of NEDD8-activating E1 enzyme also results in stabilization of IRF-3 suggesting the NEDDylation also plays a role in IRF-3 degradation following Sendai virus infection. In agreement with this observation, IRF-3 is recruited to Cullin1 following virus infection and overexpression of a dominant-negative mutant of Cullin1 significantly inhibits the degradation of IRF-3 observed in infected cells. We also asked whether the C-terminal cluster of phosphoacceptor sites of IRF-3 could serve as a destabilization signal and we therefore measured the half-life of C-terminal phosphomimetic IRF-3 mutants. Interestingly, we found them to be short-lived in contrast to wild-type IRF-3. In addition, no degradation of IRF-3 was observed in TBK1^–/– mouse embryonic fibroblasts. All together, these data demonstrate that virus infection stimulates a host cell signaling pathway that modulates the expression level of IRF-3 through its C-terminal phosphorylation by the IB kinase-related kinases followed by its polyubiquitination, which is mediated in part by a Cullin-based ubiquitin ligase.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by research grants from the Canadian Institutes of Health Research (CIHR) to M.J.S. (MOP-53282) and S.M. (MOP-14168). M.S. is a recipient of a Rx&D/CIHR Health Research Foundation Career Award in Health Sciences. S.M. and N.G. are both recipients of Canada Research Chairs. J.H. is supported by a CIHR Senior Scientist award. P.C. is a recipient of a studentship from the CIHR. J.-F.C and S.-P.G are both recipients of a studentship from the Fonds de la Recherche en Santé du Québec. G.R. holds a fellowship from the American Association for Cancer Research.

² Address correspondence and reprint requests to Dr. Marc J. Servant, Faculté de Pharmacie, Université de Montréal, C.P. 6128, succursale Centre-Ville, Montréal, Québec, Canada H3C 3J7. E-mail address: marc.servant{at}umontreal.ca

³ Abbreviations used in this paper: IRF-3, IFN regulatory factor-3; IKK, IB kinase; TBK1, Tank-binding kinase 1; RING, really interesting new gene; Cul1, Cullin1; SCF, Skp1-Cul1-F-box; TPEN, N,N,N',N',-tetrakis(2-pyridylmethyl)ethylenediamine; MEF, murine embryonic fibroblast; SeV, Sendai virus; HA, hemagglutinin; HCMV, human CMV; RIPA, radioimmunoprecipitation assay; APP-BPI, amyloid percursor protein-binding protein 1; NEDD8, neural precursor cell expressed developmentally down-regulated protein 8.

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