Involvement of the IkappaB kinase (IKK)-related kinases tank-binding kinase 1/IKKi and cullin-based ubiquitin ligases in IFN regulatory factor-3 degradation.
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 IkappaB kinase-related kinases followed by its polyubiquitination, which is mediated in part by a Cullin-based ubiquitin ligase.
Bibeau-Poirier
Annie
A
Faculty of Pharmacy, University of Montreal, Montreal, Canada.
Gravel
Simon-Pierre
SP
Clément
Jean-François
JF
Rolland
Sébastien
S
Rodier
Geneviève
G
Coulombe
Philippe
P
Hiscott
John
J
Grandvaux
Nathalie
N
Meloche
Sylvain
S
Servant
Marc J
MJ
D000818:Animals; D002460:Cell Line; D044842:Cullin Proteins / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D029082:Sendai virus; D015398:Signal Transduction; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D014777:Virus Diseases / Q000378:metabolism*
D044842:Cullin Proteins; C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D017346:Protein Serine-Threonine Kinases; C403191:TBK1 protein, human; D051550:I-kappa B Kinase