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. 2023 Oct 23;19(10):e1011742.
doi: 10.1371/journal.ppat.1011742. eCollection 2023 Oct.

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

Wei-Wei Li  1   2   3 , Xu-Xu Fan  1   3 , Zi-Xiang Zhu  1   3 , Xue-Jing Cao  1   3 , Zhao-Yu Zhu  1   3 , Dan-Shi Pei  1   3 , Yi-Zhuo Wang  1   3 , Ji-Yan Zhang  1   3 , Yan-Yi Wang  2 , Hai-Xue Zheng  1   3
Affiliations

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

Wei-Wei Li et al. PLoS Pathog. .

Abstract

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Identification of BLK as a positive regulator of RNA or DNA virus-triggered signaling.
(A) Effects of BLK on SeV-induced activation of IFN-β and ISRE. U87MG cells (1 × 105) were co-transfected with the indicated reporter (IFN-β (0.05 μg), ISRE (0.05 μg)), pRL-TK (Renilla luciferase) reporter (0.01 μg) and increased amounts of BLK expression plasmids (0.01, 0.05 μg) for 24 h. Cells were then uninfected or infected with SeV (MOI, 1) for 10 h before luciferase assays. (B) Effects of BLK on SeV-, HSV-1- or EMCV-induced transcription of downstream antiviral genes. Raji cells were transduced with vector (Vec) or BLK expression plasmids by lentivirus-mediated gene transfer to establish the stable cell lines. Cells (2 × 105) were then uninfected or infected with viruses (MOI, 1) for 8 h before qPCR analysis. (C and D) Effects of BLK deficiency on SeV-, VSV- or EMCV-induced transcription of downstream antiviral genes. Jurkat (C) or Raji (D) cells were transduced with control (Con) or the indicated gRNA plasmids targeting BLK by the CRISPR/Cas9 method to establish the stable cell lines. BLK-deficient and control cells (2 × 105) were then uninfected or infected with viruses (MOI, 1) for the indicated times before qPCR analysis. (E) Effects of BLK knockdown on VSV- or EMCV-induced transcription of downstream antiviral genes. U87MG cells (2 × 105) were transfected with the indicated siRNA (final concentration, 40 nM). Forty-eight hours later, cells were uninfected or infected with viruses (MOI, 1) for the indicated times before qPCR analysis. (F and G) Effects of BLK deficiency on SeV- or HSV-1-induced phosphorylation of TBK1 and IRF3. BLK-deficient and control Jurkat (F) or Raji (G) cells (2 × 105) were infected with viruses (MOI, 1) for the indicated times before immunoblot analysis. (H) Effects of BLK knockdown on VSV-induced phosphorylation of TBK1 and IRF3. U87MG cells (2 × 105) were transfected with the indicated siRNA (final concentration, 40 nM). Forty-eight hours later, cells were uninfected or infected with VSV-GFP (MOI, 1) for the indicated times before immunoblot analysis. Graphs show mean ± SD (n = 2 biological replicates in A, n = 2 technical replicates in B-E) from one representative experiment. Data are representative of at least three independent experiments with similar results. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired, two-tailed Student’s t-test).
Fig 2
Fig 2. Blk is required for RNA or DNA virus-induced innate antiviral response in murine cells.
(A-C) Effects of Blk deficiency on SeV-, VSV- or EMCV-induced transcription of downstream genes and phosphorylation of TBK1 and IRF3. A20 cells were transduced with control (Con) or the indicated gRNA plasmids targeting Blk by the CRISPR/Cas9 method to establish the stable cell lines. Blk-deficient and control cells (2 × 105) were then uninfected or infected with viruses (MOI, 1) for the indicated times before qPCR (A) and immunoblot (B and C) analyses. (D and E) Effects of Blk deficiency on VSV- or HSV-1-induced transcription of downstream genes and phosphorylation of TBK1 and IRF3. pDCs derived from the spleens of Blk+/+ and Blk-/- mice (1 × 105) were infected with VSV or HSV-1 (MOI, 0.5) for 8 h before qPCR analysis (D) or infected with VSV (MOI, 0.5) for the indicated times before immunoblot analysis (E). (F and G) Effects of Blk deficiency on the replication of VSV. (F) pDCs derived from the spleens of Blk+/+ and Blk-/- mice (1 × 105) were infected with VSV-GFP (MOI, 0.2) for 8 h before flow cytometry analysis. (G) Statistical analysis in the form of a bar graph for the data in F. Graphs show mean ± SD (n = 2 technical replicates in A and D, n = 3 biological replicates in G) from one representative experiment. Data are representative of at least three independent experiments with similar results. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired, two-tailed Student’s t-test).
Fig 3
Fig 3. BLK is critical for host defense against VSV infection in mice.
(A) Effects of Blk deficiency on VSV-induced death in mice. Sex- and age-matched Blk+/+ and Blk-/- mice (n = 10 for each group) were infected intraperitoneally with VSV (1 × 108 PFU per mouse), and mouse survival was recorded daily for 16 days. (B) Effects of Blk deficiency on VSV-induced serum levels of IFN-α and IFN-β. Sex- and age-matched Blk+/+ and Blk-/- mice (n = 6 for each group) were infected intraperitoneally with VSV (1 × 108 PFU per mouse) for 6 h before serum cytokines were measured by ELISA. (C and D) Effects of Blk deficiency on viral replication in the spleens of VSV-infected mice. Sex- and age-matched Blk+/+ and Blk-/- mice (n = 4 for each group) were infected intraperitoneally with VSV (1 × 108 PFU per mouse) for 4 days. Viral titers and genomic copy numbers in the spleens of VSV-infected mice were quantified by qPCR (C) and plaque (D) assays, respectively. Graphs show mean ± SD from one representative experiment. Data are representative of at least two independent experiments with similar results. **P < 0.01, ***P < 0.001 (unpaired, two-tailed Student’s t-test). For the mouse survival study in A, Kaplan-Meier survival curves were generated and analyzed by the log-rank test.
Fig 4
Fig 4. BLK is essential for host defense against SVA infection in the U87MG xenograft tumor model.
(A) Schematic diagram of the SVA efficacy experiment in vivo. (B) Effects of BLK deficiency on SVA-induced cytokine levels in U87MG xenograft tumors. Athymic female mice (BALB/c-nu) bearing wild-type or BLK-deficient U87MG tumors (n = 7 for each group) were challenged with SVA (1 × 108 PFU per mouse) for 24 h via a single intravenous delivery. Subsequently, tumors were excised and subjected to ELISA measurement. (C and D) Effects of BLK deficiency on SVA replication kinetics in U87MG xenograft tumors. Athymic female mice (BALB/c-nu) bearing wild-type or BLK-deficient U87MG tumors (n = 4 for each group) were challenged with SVA (1 × 108 PFU per mouse) for 3 days via a single intravenous delivery. Subsequently, viral titers and genomic copy numbers in tumor tissues were quantified by plaque (C) and qPCR (D) assays, respectively. 5’UTR is the N-terminal noncoding region of the SVA genome. (E) Effects of BLK deficiency on SVA-induced in vivo antitumor efficacy. Athymic mice bearing pre-established U87MG xenograft tumors within each cohort were randomly distributed (n = 3 for each group) and challenged with SVA or PBS at a dose of 1 × 108 PFU per mouse into the lateral tail vein. Antitumor efficacy was determined by measuring tumor volumes every 2 days following SVA administration. Graphs show mean ± SD from one representative experiment. Data are representative of at least two independent experiments with similar results. *P < 0.05, **P < 0.01 (unpaired, two-tailed Student’s t-test).
Fig 5
Fig 5. BLK associates with IRF3.
(A) Identification of the BLK interactome by silver staining and mass spectrometry methods. Silver staining showing GST-associated factors (Control) and GST-BLK-associated factors. IRF3 was one of the convincing interacting factors. (B) BLK interacts with IRF3 in the mammalian overexpression system. HEK293 cells (2 × 106) were transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. (C) BLK interacts with IRF3 and IRF9. Coimmunoprecipitation and immunoblot analysis were similarly performed as in B. (D) BLK associates with IRF3. Prokaryotically expressed and purified GST-BLK protein coupled to glutathione sepharose beads was incubated with purified TBK1-Flag or IRF3-Flag protein for 3 h at 4°C and then subjected to in vitro GST pull-down assays. GST: glutathione S-transferase. (E) BLK directly binds to IRF3. Prokaryotically expressed and purified His-IRF3 and GST-BLK proteins were subjected to in vitro GST pull-down assays. (F) BLK associates with endogenous IRF3 upon SeV infection. Jurkat cells stably expressing BLK-Flag (2 × 107) were uninfected or infected with SeV (MOI, 1) for the indicated times. Coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. Data are representative of at least three independent experiments with similar results.
Fig 6
Fig 6. Phosphorylation of IRF3 at Y107 mediated by BLK is essential for virus-triggered innate antiviral response.
(A) Effects of BLK and BLK(K269A) on SeV- or EMCV-induced transcription of downstream antiviral genes. U87MG cells (2 × 105) were transfected with BLK or BLK(K269A) plasmids for 24 h. Cells were then uninfected or infected with viruses (MOI, 1) for 8 h before qPCR analysis. (B) BLK mediates tyrosine phosphorylation of IRF3. HEK293 cells (2 × 106) were transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. (C) BLK phosphorylates IRF3 at Y107. HEK293 cells (2 × 106) were transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. (D) BLK phosphorylates IRF3 but not IRF3-Y107F. Purified Flag-tagged BLK and BLK(K269A) coupled with Protein G sepharose beads were subjected to in vitro kinase assays with purified GST-tagged IRF3 or IRF3-Y107F. “Vector” here means the control Protein G sepharose beads. (E) Effects of BLK deficiency on SeV-triggered IRF3 Y107 phosphorylation. BLK-deficient and control U87MG cells (2 × 107) were uninfected or infected with SeV (MOI, 1) for the indicated times. Coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. (F) Effects of IRF3 or its mutant recovery on SeV-induced ISRE activation. U87MG cells were transduced with gRNA plasmids targeting IRF3 by the CRISPR/Cas9 method to establish the stable cell lines with puromycin (1 μg/mL) selection. IRF3-deficient and control U87MG cells (1 × 105) were co-transfected with ISRE reporter (0.05 μg), pRL-TK reporter (0.01 μg), and either IRF3 or its mutant (0.05 μg) plasmids for 24 h and then uninfected or infected with SeV (MOI, 1) for 10 h before luciferase assays. The lower blots show the expression levels of IRF3 and its mutants as detected by anti-Flag or anti-IRF3 antibodies, respectively. (G) Effects of IRF3 or IRF3(Y107F) recovery on SeV-induced IRF3 nuclear translocation. IRF3-deficient U87MG cells were transduced with vector, IRF3, or IRF3(Y107F) plasmids by lentivirus-mediated gene transfer to establish the stable cell lines with blasticidin S (10 μg/mL) selection. Subsequently, the indicated cell lines (1 × 105) were infected with SeV (MOI, 1) for 4 h, then fixed with 4% paraformaldehyde and stained with anti-IRF3 antibody before confocal microscopy. Scale bars, 50 μm. Graphs show mean ± SD (n = 2 technical replicates in A, n = 2 biological replicates in F) from one representative experiment. Data are representative of at least three independent experiments with similar results. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant (unpaired, two-tailed Student’s t-test).
Fig 7
Fig 7. Autophosphorylation of BLK at Y309 is critical for its regulation of IRF3-dependent antiviral signaling.
(A) BLK undergoes tyrosine phosphorylation upon SeV infection. U87MG cells stably expressing BLK-Flag (2 × 107) were uninfected or infected with SeV (MOI, 1) for the indicated times. Coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. (B) BLK catalyzes autophosphorylation in a kinase activity-dependent manner. Purified Flag-tagged BLK and BLK(K269A) coupled with Protein G sepharose beads were subjected to in vitro BLK autophosphorylation assays. The detailed procedures are shown in the Materials and Methods. “Vector” here means the control Protein G sepharose beads. (C) Identification of potential autophosphorylation sites of BLK by mass spectrometry. The in vitro phosphorylated BLK in Fig 7B was subjected to mass spectrometry. The list shows the phosphorylated peptide sequences and phosphorylation (STY) probabilities of BLK analyzed by mass spectrometry. (D) BLK catalyzes autophosphorylation mainly at Y309. Purified Flag-tagged BLK and its mutants coupled with Protein G sepharose beads were subjected to in vitro BLK autophosphorylation assays. (E) BLK(Y309F) fails to phosphorylate IRF3. HEK293 cells (2 × 106) were transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. (F) Effects of BLK or its mutant recovery on SeV-induced transcription of downstream antiviral genes. BLK-deficient and control U87MG cells (2 × 105) were transfected with the same amount of BLK or its mutants for 24 h. Cells were then uninfected or infected with SeV (MOI, 1) for 8 h before qPCR analysis. The lower blot shows the expression levels of BLK and its mutants as detected by anti-Flag antibody. (G) Sequence alignment of BLK from the indicated species. The sequences correspond to aa293-312 of human BLK. The conserved tyrosine residues are highlighted in black. (H) Effects of Blk or Blk(Y303F) recovery on VSV-induced transcription of downstream antiviral genes. Wild-type and Blk-deficient A20 cells were transduced with empty vector (EV), Blk or Blk(Y303F) plasmids by lentivirus-mediated gene transfer to establish the stable cell lines with blasticidin S (10 μg/mL) selection. The indicated cell lines (2 × 105) were then uninfected or infected with VSV (MOI, 1) for 8 h before qPCR analysis. The lower blot shows the expression levels of Blk and Blk(Y303F) in the indicated cell lines as detected by anti-Flag antibody. Graphs show mean ± SD (n = 2 technical replicates in F and H) from one representative experiment. Data are representative of at least three independent experiments with similar results. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant (unpaired, two-tailed Student’s t-test).
Fig 8
Fig 8. BLK promotes sufficient activation of IRF3 to facilitate cellular antiviral response.
(A and B) BLK promotes the recruitment of IRF3 to TBK1 in a kinase activity-dependent manner. HEK293 cells (2 × 106) were transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. (C) Effects of BLK deficiency on SeV-induced recruitment of IRF3 to TBK1. BLK-deficient and control U87MG cells (2 × 107) were uninfected or infected with SeV (MOI, 1) for the indicated times before coimmunoprecipitation and immunoblot analysis. (D) Effects of BLK on TBK1-mediated phosphorylation and dimerization of IRF3. IRF3- or IRF3(Y107F)-reconstituted U87MG cells (5 × 105) were co-transfected with the same amount of TBK1 or BLK plasmids for 24 h before native PAGE and SDS PAGE analyses with the indicated antibodies. (E) Effects of BLK deficiency on SeV-triggered dimerization of IRF3. BLK-deficient and control U87MG cells (1 × 106) were uninfected or infected with SeV (MOI, 1) for the indicated times before native PAGE and SDS PAGE analyses. (F) Effects of BLK deficiency on SeV-triggered nuclear translocation of IRF3. BLK-deficient and control U87MG cells (1 × 106) were uninfected or infected with SeV (MOI, 1) for the indicated times before the nuclear and cytoplasmic extraction assays. (G and H) Effects of BLK or BLK(Y309F) recovery on SeV-triggered nuclear translocation of IRF3. (G) BLK-deficient U87MG cells were transduced with BLK or BLK(Y309F) plasmids by lentivirus-mediated gene transfer to establish the stable cell lines with blasticidin S (10 μg/mL) selection. Subsequently, the indicated cell lines (1 × 105) were infected with SeV (MOI, 1) for 4 h, then fixed with 4% paraformaldehyde and stained with anti-Flag and anti-IRF3 antibodies before confocal microscopy. Scale bars, 50 μm. (H) Quantitative analysis of IRF3 fluorescence intensity in the cytoplasm and nucleus in Fig 8G. Statistical analyses were based on colocalization images (covering dozens of cells) using ImageJ software. (I) Effects of BLK or BLK(Y309F) on IRF3(S386/396D)-induced transcription of downstream antiviral genes. IRF3-deficient U87MG cells (2 × 105) were co-transfected with the indicated plasmids (IRF3(S386/396D) 0.5 μg, BLK 1.5 μg, BLK(Y309F) 1.5 μg) for 24 h before qPCR analysis. The lower blots show the expression levels of the indicated plasmids. Graphs show mean ± SD (n = 12 cells from three individual images in H, n = 2 technical replicates in I) from one representative experiment. Data are representative of at least three independent experiments with similar results. *P < 0.05, **P < 0.01, ns, not significant (unpaired, two-tailed Student’s t-test).
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Grants and funding

This work was supported by the grants from the National Key R&D Program of China (2021YFD1801300, awarded to H.X.Z.), the National Natural Science Foundation of China (32202781, awarded to W.W.L.), the Fundamental Research Funds for the Central Universities (awarded to H.X.Z.), the Key Technologies R&D Program of Gansu Province (21ZD3NA001, awarded to H.X.Z., 20ZD7NA006, awarded to H.X.Z., and 19ZD2NA001, awarded to H.X.Z.), the Natural Science Foundation of Gansu Province (22JR5RA034, awarded to W.W.L.), the Open Competition Program of Top Ten Critical Priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (2023SDZG02, awarded to H.X.Z.), the Earmarked Fund for CARS-35 (awarded to H.X.Z.) and CARS-39-13 (awarded to H.X.Z.), the Chinese Academy of Agricultural Science and Technology Innovation Project (CAAS-ZDRW202006, awarded to H.X.Z., and CAAS-ASTIP-2022-LVRI, awarded to H.X.Z.), and the Basic Scientific Research Fund of LVRI (1610312021009, awarded to W.W.L.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.