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.

The African swine fever virus I10L protein inhibits the NF-κB signaling pathway by targeting IKKß.

Proinflammatory factors play important roles in the pathogenesis of African swine fever virus (ASFV), which is the causative agent of African swine fever (ASF), a highly contagious and severe hemorrhagic disease. Efforts in the prevention and treatment of ASF have been severely hindered by knowledge gaps in viral proteins responsible for modulating host antiviral responses. In this study, we identified the I10L protein (pI10L) of ASFV as a potential inhibitor of the TNF-α- and IL-1ß-triggered NF-κB signaling pathway, the most canonical and important part of host inflammatory responses. The ectopically expressed pI10L remarkably suppressed the activation of NF-κB signaling in HEK293T and PK-15 cells. The ASFV mutant lacking the I10L gene (ASFVΔI10L) induced higher levels of proinflammatory cytokines production in primary porcine alveolar macrophages (PAMs) compared with its parental ASFV HLJ/2018 strain (ASFVWT). Mechanistic studies suggest that pI10L inhibits IKKß phosphorylation by reducing the K63-linked ubiquitination of NEMO, which is necessary for the activation of IKKß. Morever, pI10L interacts with the kinase domain of IKKß through its N-terminus, and consequently blocks the association of IKKß with its substrates IκBα and p65, leading to reduced phosphorylation. In addition, the nuclear translocation efficiency of p65 was also altered by pI10L. Further biochemical evidence supported that the amino acids 1-102 on pI10L were essential for the pI10L-mediated suppression of the NF-κB signaling pathway. The present study clarifies the immunosuppressive activity of pI10L, and provides novel insights into the understanding of ASFV pathobiology and the development of vaccines against ASF. IMPORTANCE African swine fever (ASF), caused by the African swine fever virus (ASFV), is now widespread in many countries and severely affects the commercial rearing of swine. To date, few safe and effective vaccines or antiviral strategies have been marketed due to large gaps in knowledge regarding ASFV pathobiology and immune evasion mechanisms. In this study, we deciphered the important role of the ASFV-encoded I10L protein in the TNF-α-/IL-1ß-triggered NF-κB signaling pathway. This study provides novel insights into the pathogenesis of ASFV and thus contributes to the development of vaccines against ASF.

BLK positively regulates TLR/IL-1R signaling by catalyzing TOLLIP phosphorylation.

TLR/IL-1R signaling plays a critical role in sensing various harmful foreign pathogens and mounting efficient innate and adaptive immune responses, and it is tightly controlled by intracellular regulators at multiple levels. In particular, TOLLIP forms a constitutive complex with IRAK1 and sequesters it in the cytosol to maintain the kinase in an inactive conformation under unstimulated conditions. However, the underlying mechanisms by which IRAK1 dissociates from TOLLIP to activate TLR/IL-1R signaling remain obscure. Herein, we show that BLK positively regulates TLR/IL-1R-mediated inflammatory response. BLK-deficient mice produce less inflammatory cytokines and are more resistant to death upon IL-1ß challenge. Mechanistically, BLK is preassociated with IL1R1 and IL1RAcP in resting cells. IL-1ß stimulation induces heterodimerization of IL1R1 and IL1RAcP, which further triggers BLK autophosphorylation at Y309. Activated BLK directly phosphorylates TOLLIP at Y76/86/152 and further promotes TOLLIP dissociation from IRAK1, thereby facilitating TLR/IL-1R-mediated signal transduction. Overall, these findings highlight the importance of BLK as an active regulatory component in TLR/IL-1R signaling.