Identification of the minimal phosphoacceptor site required for in vivo activation of interferon regulatory factor 3 in response to virus and double-stranded RNA

Marc J. Servant, Nathalie Grandvaux, Benjamin R. TenOever, Delphine Duguay, Rongtuan Lin, John Hiscott

Research output: Contribution to journalArticlepeer-review

183 Scopus citations

Abstract

The ubiquitously expressed latent interferon regulatory factor (IRF) 3 transcription factor is activated in response to virus infection by phosphorylation events that target a cluster of Ser/Thr residues, 382GGASSLEN-TVDLHISNSHPLSLTSDQY408 at the C-terminal end of the protein. To delineate the minimal phosphoacceptor sites required for IRF-3 activation, several point mutations were generated and tested for transactivation potential and cAMP-response element-binding protein-binding protein/p300 coactivator association. Expression of the IRF-3 S396D mutant alone was sufficient to induce type I IFN β, IFNα1, RANTES, and the interferon-stimulated gene 561 promoters. Using SDS-PAGE and immunoblotting with a novel phosphospecific antibody, we show for the first time that, in vivo, IRF-3 is phosphorylated on Ser396 following Sendai virus infection, expression of viral nucleocapsid, and double-stranded RNA treatment. These results demonstrate that Ser396 within the C-terminal Ser/Thr cluster is targeted in vivo for phosphorylation following virus infection and plays an essential role in IRF-3 activation. The inability of the phosphospecific antibody to detect Ser396 phosphorylation in lipopolysaccharide-treated cells suggests that other major pathways may be involved in IRF-3 activation following Toll-like receptor 4 stimulation.

Original languageEnglish
Pages (from-to)9441-9447
Number of pages7
JournalJournal of Biological Chemistry
Volume278
Issue number11
DOIs
StatePublished - 14 Mar 2003
Externally publishedYes

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