Phosphorylation of interferon regulatory factor 9 (IRF9)

Area of Science:

• Immunology
• Molecular Biology
• Virology

Background:

• IRF9 (Interferon regulatory factor 9) is a key transcription factor in the Janus kinase-Signal transducer and activator of transcription (JAK-STAT) pathway, mediating interferon-stimulated gene (ISG) expression.
• While the JAK-STAT pathway is well-characterized for phosphorylation events, IRF9 has remained an enigma, with its own phosphorylation status and regulatory mechanisms unexplored.
• Understanding IRF9 phosphorylation is crucial given its significant role in establishing host antiviral states.

Purpose of the Study:

• To investigate the phosphorylation status of IRF9.
• To identify specific phosphorylation sites on IRF9.
• To elucidate the functional consequences of IRF9 phosphorylation on the expression of interferon-stimulated genes (ISGs).

Main Methods:

• Utilized phosphoprotein enrichment and Phos-tag™ assays to confirm IRF9 phosphorylation.
• Employed tandem mass spectrometry on immunoprecipitated IRF9 to identify phosphorylated amino acid residues.
• Performed site-directed mutagenesis of identified phosphorylation sites and evaluated the impact on USP18 and Mx1 gene expression via qPCR.

Main Results:

• IRF9 was found to be phosphorylated at serine 252 (S252) and serine 253 (S253) under interferon-beta (IFNβ) induced conditions, and at arginine 242 (R242) under non-induced conditions.
• Mutagenesis of S252 and S253 to alanine or aspartic acid showed a modest effect on the upregulation of USP18, a negative regulator of the type I interferon response.
• The phosphorylation status of IRF9 did not significantly affect Mx1 gene expression.

Conclusions:

• This preliminary study demonstrates that IRF9 is indeed phosphorylated.
• IRF9 phosphorylation may play a role in regulating USP18 gene expression.
• Further in vivo investigations are necessary to fully ascertain the biological significance of IRF9 phosphorylation in the context of antiviral immunity.