Fig 2: CSNK2B interacts directly with IRF1 and modulates the binding of IRF1 to chromatin, thereby promoting expression of antiviral genes.

Fig 3: CSNK2B interacts with IRF1 and regulates its antiviral activity independently of phosphorylation. (A) 293FT cell lysates expressing IRF1-FLAG or empty vector was immunoprecipitated with anti-FLAG M2 antibody. Proteins eluted from the precipitates were subjected to an SDS-PAGE followed by western blotting with specific antibodies against CK2 components. (B) Pull-down analysis showing direct interaction between purified IRF1-FLAG and recombinant human CSNK2B (rCSNK2B) proteins. (C) Phos-tag SDS-PAGE of FLAG-tagged IRF1. PH5CH8 cell lysates were subjected to immunoblotting before (−) and after (+) digestion with lambda protein phosphatase (λ PPase). An arrowhead shows phospho-IRF1. (D, E) Phos-tag gel analysis of ectopically expressed IRF1-FLAG in 293FT cells (D) or endogenously expressed IRF1 in PH5CH8 cells (E). Means ± S.D. of values for abundance of phospho-IRF1 relative to non-phosphorylated IRF1 are shown on right (n = 3). (F) PH5CH8 cells were transfected with siRNAs targeting CK components and infected 48 h later with HAV at an m.o.i. of 10. Percentage of HAV RNA levels relative to non-target control siRNA was determined 4 d.p.i. by RT-qPCR. Immunoblots showing depletion of catalytic CK2 subunits are shown on right. **P < 0.01 versus control (n = 3 or 4, one-way ANOVA with Dunnett's multiple comparisons test). (G) Immunoblots showing CSNK2B protein abundance in Huh-7.5 cells treated with 10 μM CX-4945 for 24 h. (H) Effects of CX-4945 on replication of HAV/NLuc (18f/NLuc) in Huh-7.5 cells and cell viability. **P < 0.01, ***P < 0.0001 versus control (n = 3, one-way ANOVA with Bonferroni's multiple comparisons test). (I) Effects of CX-4945 on HAV replication and PLAAT4 expression in IRF1-depleted and control Huh-7.5 cells. **P < 0.01, ***P < 0.0001 (n = 3, two-way ANOVA with Sidak's multiple comparisons test). (J) NLuc reporter analysis of Huh-7.5 cells expressing NLuc reporter (pNL-4×IRF1). Cells were transfected with indicated siRNAs for 48 h, and relative NLuc values secreted at 48–72 h post-transfection are shown. Immunoblots showing depletion of each siRNA target are shown on right. *P < 0.05, **P < 0.01 versus control (n = 4, one-way ANOVA with Dunnett's multiple comparisons test).

Fig 4: CSNK2B regulates transcription and phosphorylation of AFAP1 that lowers permissiveness to flavivirus replication. (A) Immunoblots of AFAP1 and GAPDH as loading control in lysates of PH5CH8 cells transfected with indicated siRNAs, with and without IFNγ stimulation. (B) Immunoblots of AFAP1, IRF1 and GAPDH in lysates of PH5CH8 cells depleted of either (or both) CSNK2B and IRF1. Quantitation of AFAP1 protein abundance is shown on the right. **P < 0.01, ***P < 0.0001 (n = 3, one-way ANOVA with Dunnett's multiple comparisons test). (C) Immunoblots of AFAP1 and GAPDH in lysates of PH5CH8 cells treated with either lambda protein phosphatase (λ PPase, left panels) or indicated concentrations of CX-4945 (right panels). (D) Immunoblots of AFAP1 and Src in lysates of PH5CH8 cells treated with 10 μM CX-4945. (E) Immunoblots of PH5CH8 cell lysates transfected with indicated siRNAs targeting AFAP1 (left panels) or CSNK2B (right panels). (F) Scheme of CSNK2B-regulated AFAP1 signaling cascades that lead to Src activation. (G) Immunoblots of PH5CH8 cell lysates transfected with indicated siRNAs (top). DENV RNA levels were determined at 48 h p.i. (bottom). *P < 0.05 (n = 3, two-tailed Student's t-test). (H) Immunoblots of AFAP1 in lysates of PH5CH8 versus Huh-7.5 cells stably transduced with AFAP1 (top). Huh-7.5 cells stably expressing AFAP1 or vector control were challenged with DENV/NLuc (bottom). NLuc activities at the indicated time points post-infection are shown. **P < 0.01 (n = 3, two-way ANOVA with Sidak's multiple comparisons test). (I) PH5CH8 cells were transfected with indicated siRNAs and infected 48 h later with dengue virus (DENV) at an m.o.i. of 0.1. CX-4945 (5 μM) was added 2 h post-infection. Infectious titers were determined 48 h p.i. by focus formation assays. FFU, focus forming units. **P < 0.01, ***P < 0.0001 versus control (n = 3, one-way ANOVA with Dunnett's multiple comparisons test). (J) PH5CH8 cells were infected with DENV or Zika virus (ZIKV) as in (I) and viral RNA levels determined by RT-qPCR. **P < 0.01 (n = 3, two-tailed Student's t-test).

Fig 5: Genome-wide mapping of CSNK2B-regulated IRF1 binding sites by CUT&RUN. (A) In vitro pull-down experiments using biotinylated DNA probe containing IRF1 binding motifs derived from the PLAAT4 promoter in lysates of CSNK2B-depleted PH5CH8 cells versus control (top). Means ± S.D. of relative values for abundance of DNA-bound IRF1 in CSNK2B-depleted cell lysates versus control (bottom). **P < 0.01 (n = 3, two-tailed Student's t-test). (B) Stacked bar plots showing peak annotation of IRF1-bound sites in PH5CH8 cells transfected with CSNK2B siRNA versus control. The number of peaks is shown on each of the stacked bar. (C) Volcano plots showing changes in relative abundance of the peaks identified in CUT&RUN between PH5CH8 cells transfected with CSNK2B versus control siRNAs. Each symbol represents the log2 fold-change in CSNK2B siRNA-transfected cells over control from three biological replicates. Combined P-value for the peak was determined using Sime's method. The P-values were adjusted using false discovery rate (FDR) correction. (D) Gene ontology analysis and functional annotation of genes differentially bound by IRF1 in CSNK2B-depleted cells. (E) Logo depiction of HOMER de novo motif analysis of all peaks (top) and peaks significantly decreased in CSNK2B-depleted cells (bottom). (F) Example genomic regions showing binding peaks of IRF1 in control versus CSNK2B-depleted PH5CH8 cells for all three biological replicates. (G) Bar graphs showing RT-qPCR measurements of transcripts from representative CSNK2B-responsive genes in PH5CH8 (upper panels) and A549 (lower panels) cell lines. Cells were stimulated with IFNγ (100 U/ml, + IFNγ) or vehicle control (− IFNγ) for 24 h. *P < 0.05, **P < 0.01, ***P < 0.0001 (n = 3, two-way ANOVA with Sidak's multiple comparisons test). (H) Heatmaps showing the relative abundance of CSNK2B-regulated gene transcripts linked to antiviral defense and innate immune responses as determined by RT-qPCR, with clustering according to CSNK2B regulation of basal expression.