Fig 1.
PRV and HSV-1 employ US3 to decrease Bclaf1 in a proteasome-dependent manner.
(A) IB analysis of Bclaf1, TK and US3 in PK15 cells infected with PRV (MOI = 1) for the indicated hours. α-Tubulin was used as loading control. (B) IB analysis of Bclaf1, VP5 and US3 in HEp-2 cells infected with HSV-1 (MOI = 5) for the indicated hours. (C) IB analysis of Bclaf1, UL42 and US3 in PK15 cells infected with PRV (MOI = 1) followed by untreatment (U) or treatment with DMSO or MG132. (D) IB analysis of Bclaf1, VP5 and US3 in HEp-2 cells infected with HSV-1 (MOI = 5) followed by untreatment (U) or treatment with DMSO or MG132. (E) IB analysis of Bclaf1, UL50, EP0 and US3 in PK15 cells infected with indicated PRV strains (MOI = 1) at 8 hpi. (F) IB analysis of Bclaf1, TK and US3 in PK15 cells infected with PRV WT or PRV ΔUS3 (MOI = 1) for the indicated hours. (G) IB analysis of Bclaf1, VP5 and US3 in HEp-2 cells infected with HSV-1 WT or HSV-1 ΔUS3 (MOI = 5) for the indicated hours. (H) IB analysis of endogenous Bclaf1 in HEK293T cells transfected with Flag-tagged PRV/HSV-1 US3 expression plasmids followed by treatment with DMSO or MG132.
Fig 2.
Bclaf1 contributes to the inhibition of IFNα to PRV and HSV-1.
(A) PK15 cells were treated with PBS or porcine IFNα (500U/ml) for 12 hours followed infected with PRV WT or PRV ΔUS3 (MOI = 0.5) for 24 hours. IB analyzed TK, UL42, US3 and Bclaf1 expression. (B) Plaque assay analyzed virus titers in supernatants as described in (A). (C) HEp-2 cells were treated with PBS or human IFNα (500U/mL) for 12 hours followed infected with HSV-1 WT or HSV-1 ΔUS3 (MOI = 1) for 24 hours. IB analyzed VP5, US3, ISG15 and Bclaf1 expression. (D) Plaque assay analyzed virus titers in supernatants as described in (C). (E) IB analysis of TK, UL42 and Bclaf1 in PK15 cells transfected with si-control or si-Bclaf1 followed by PBS or porcine IFNα (500U/mL) treatment for 12h and then infected with PRV ΔUS3 (MOI = 1) for 24h. (F) Plaque assay analyzed virus titers in supernatants as described in (E). (G) IB analysis of VP5, ISG15 and Bclaf1 in control and Bclaf1-KO HeLa cells pre-treated with PBS or human IFNα (500U/mL) for 12h followed by HSV-1 ΔUS3 infection (MOI = 5) for 24h. (H) Plaque assay analyzed virus titers in supernatants as described in (G). Data are shown as mean ± SD of three independent experiments. Statistical analysis was performed by the two-way ANOVA test. *p<0.05; **p<0.01; ***p<0.001.
Fig 3.
Bclaf1 facilitates IFNα-induced ISG expression.
(A) ISRE-luciferase assay in HeLa WT and HeLa Bclaf1-KO cells treated with human IFNα (500U/mL) for 10h. (B) qRT-PCR analysis of ISG15, IFIT1, IFIT2 and OAS1 mRNA levels in HeLa WT and HeLa Bclaf1-KO cells treated with human IFNα (500U/mL) for the indicated time. (C) IB analysis of ISG15 and PKR in HeLa WT and HeLa Bclaf1-KO cells treated with human IFNα for 12h. (D) qRT-PCR analysis of ISG15, IFIT1 and OAS1 mRNA levels in HEp-2 cells transfected with si-control or si-Bclaf1 followed by human IFNα (500U/mL) treatment for 4h. IB analyzed the knocking down efficiency. (E) qRT-PCR analysis of OAS1 and IFIT1 mRNA levels in indicated HeLa cells transfected Flag-tagged EV or Bclaf1 expression plasmids followed by PBS or human IFNα (500U/mL) treatment for 4h. IB analyzed the expression of Bclaf1. Data are shown as mean ± SD of three independent experiments. Statistical analysis was performed by the two-way ANOVA test (A and B) and one-way ANOVA test (D and E). **p<0.01; ***p<0.001.
Fig 4.
Loss of Bclaf1 attenuates IFNα-mediated STAT1/STAT2 phosphorylation.
(A) IB analysis of phosphorylated(P)-STAT1, P-STAT2, STAT1, STAT2 and Bclaf1 in HeLa WT and HeLa Bclaf1-KO cells treated with human IFNα (500U/mL) for the indicated time. Data were quantified and shown as the ratio of P-STAT1 to STAT1 and P-STAT2 to STAT2. (B) IB analysis of P-STAT1, P-STAT2, STAT1, STAT2 and Bclaf1 in HEp-2 cells transfected with si-control or si-Bclaf1 followed by PBS or human IFNα (500U/mL) treatment for the indicated time. Data were quantified and shown as the ratio of P-STAT1 to STAT1 and P-STAT2 to STAT2. (C) IB analysis of P-STAT1, P-STAT2, STAT1, STAT2 and Bclaf1 in cytoplasmic and nuclear extracts of HEp-2 cells transfected with si-control or si-Bclaf1 followed by PBS or human IFNα (500U/mL) treatment for the indicated time. α-Tubulin and Histone H3 were used as the cytoplasmic and nuclear controls, respectively.
Fig 5.
Bclaf1 binds with ISRE and promotes the association of ISGF3 with DNA.
(A) ChIP analysis of STAT1/STAT2/IRF9 DNA-binding in promoters of IFIT1 and IFIT2 in HeLa WT and HeLa Bclaf1-KO cells simulated with PBS or human IFNα (500U/mL) for 1h. (B) IB analysis of Bio-ISRE pull-down STAT1, STAT2, IRF9 and Bclaf1. Unlabeled ISRE was used for control. (C) IB analysis of ISRE-binding Bclaf1. Unlabeled ISRE and Bio-GFP were used for control. (D) ChIP analysis of Bclaf1 DNA-binding in promoters of ISG15, IFIT1 and IFIT2 in HeLa cells simulated with PBS or human IFNα (500U/mL) for 1h. An amplicon located in IFIT1 exon2 was also tested for control. (E) IB analysis of WT or mutated (1–3) Bio-ISRE pull-down Bclaf1.
Fig 6.
Bclaf1 interacts with STAT1/STAT2/IRF9.
(A) IB analysis of STAT1, STAT2, P-STAT1, P-STAT2 and Flag-Bclaf1 in cytoplasmic or nuclear immunoprecipitates of a HEp-2-Flag-Bclaf1 cell line treated with PBS or human IFNα (500U/mL) for 2h. IgG was used for control immunoprecipitation. (B) IB analysis of IRF9 and Flag-Bclaf1 in cytoplasmic or nuclear immunoprecipitates of a HEp-2-Flag-Bclaf1 cell line treated with PBS or human IFNα (500U/mL) for 4h. (C) IB analysis of immunoprecipitates of HEK293T cells co-transfected with Flag-tagged Bclaf1 truncations and Ha-tagged STAT1/STAT2IRF9 expression plasmids. (D) qRT-PCR analysis of IFIT1 mRNA levels in HEp-2 cells transfected with Flag-tagged EV, full-length Bclaf1 or its truncations expression plasmids followed by PBS or human IFNα (500U/mL) treatment for 3h. IB analyzed the expression of Bclaf1. Data are shown as mean ± SD of three independent experiments. Statistical analysis was performed by the one-way ANOVA test. ***p<0.001.
Fig 7.
Bclaf1 interacts with ISGF3 mainly through STAT2.
(A) GST pulldown analysis of the interaction between His-STAT1/STAT2/IRF9 and GST-Bclaf1 F2. (B) IB analysis of immunoprecipitates of HEK293T cells co-transfected with Flag-tagged Bclaf1, Ha-tagged STAT1 or STAT2/IRF9 expression plasmids. (C) IB analysis of immunoprecipitates of HEK293T cells co-transfected with Flag-tagged Bclaf1, Ha-tagged IRF9 or STAT2/STAT1 expression plasmids. (D) IB analysis of STAT1, STAT2, IRF9 and Flag-Bclaf1 in nuclear immunoprecipitates of a HEp-2-Flag-Bclaf1 cell line transfected with si-control or si-STAT2 followed by PBS or human IFNα (500U/mL) treatment for 3h. (E) IB analysis of Bio-ISRE pull-down STAT1, STAT2, IRF9 and Bclaf1.
Fig 8.
Bclaf1-knockdown mice are more sensitive to PRV ΔUS3 infection.
(A) IB analysis of Bclaf1 and UL42 in PRV-infected mice lungs and brains at 6 dpi. (B) Control and Bclaf1-knockdown mice were infected with PRV ΔUS3. UL42 and Bclaf1 in lungs were detected by western blot. (C) Plaque assay analyzed virus titers in lungs as described in (B). (D) Hematoxylin and eosin staining of lung tissue section from mice as described in (B). Scale bar: 50 μm. (E) A working model of how Bclaf1 regulates IFN response pathway. Data are shown as mean ± SD of three independent experiments. Statistical analysis was performed by the two-way ANOVA test. **p<0.01; ***p<0.001.