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 P P t-test).