Tyrosine phosphorylation of IRF3 by BLK facilitates its sufficient activation and innate antiviral response

Viral infection triggers the activation of transcription factor IRF3, and its activity is precisely regulated for robust antiviral immune response and effective pathogen clearance. However, how full activation of IRF3 is achieved has not been well defined. Herein, we identified BLK as a key kinase that positively modulates IRF3-dependent signaling cascades and executes a pre-eminent antiviral effect. BLK deficiency attenuates RNA or DNA virus-induced ISRE activation, interferon production and the cellular antiviral response in human and murine cells, whereas overexpression of BLK has the opposite effects. BLK-deficient mice exhibit lower serum cytokine levels and higher lethality after VSV infection. Moreover, BLK deficiency impairs the secretion of downstream antiviral cytokines and promotes Senecavirus A (SVA) proliferation, thereby supporting SVA-induced oncolysis in an in vivo xenograft tumor model. Mechanistically, viral infection triggers BLK autophosphorylation at tyrosine 309. Subsequently, activated BLK directly binds and phosphorylates IRF3 at tyrosine 107, which further promotes TBK1-induced IRF3 S386 and S396 phosphorylation, facilitating sufficient IRF3 activation and downstream antiviral response. Collectively, our findings suggest that targeting BLK enhances viral clearance via specifically regulating IRF3 phosphorylation by a previously undefined mechanism. Transcription factor IRF3-mediated type I interferon production is essential for antiviral innate immunity. Understanding how full activation of IRF3 is regulated is important to decipher the detailed mechanisms of IRF3-dependent signal transduction. Here, we report that BLK promotes IRF3 activation, leading to elevated antiviral cytokine production against multiple invading viruses both in vivo and in vitro. Upon viral infection, BLK undergoes Y309 autophosphorylation and directly phosphorylates IRF3 at Y107, together with TBK1-induced IRF3 S386 and S396 phosphorylation, to achieve sufficient IRF3 activation and elicit robust downstream antiviral responses. Overall, these data suggest that BLK is a critical effector of IRF3 phosphorylation. This study advances the understanding of the precise regulatory mechanisms of IRF3 activation. Manipulation of these mechanisms may help to develop novel therapeutics for infectious and inflammatory diseases.