Porcine Circovirus Type 3 Cap Inhibits Type I Interferon Induction Through Interaction With G3BP1

Figures

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  Figure 1. PCV3 Cap inhibits DNA-induced IFN-β production. (A) qPCR analysis of IFN-β mRNA in PK15 cells transfected with FLAG empty vector (EV) or the FLAG-Cap expression vector and stimulated with 2 μg/ml of ISD for 6 h at 24 h post-transfection. (B–D) Cap inhibits DNA-induced IFN promoter activation. Cells were co-transfected with 100 ng IFN-luc, 50 ng TK-luc, and 500 ng FLAG-Cap or the EV. After 24 h, the cells were stimulated by transfection with 2 μg/ml ISD (B) or 1 μg Poly (dA:dT) (C) for another 12 h and then lysed with cell lysis buffer for a dual-luciferase assay. (D) PK15 cells were transfected with the indicated amount of the FLAG-Cap expression vector. The results represent data from three independent experiments, *p < 0.01; ***p < 0.001.

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  Figure 2. PCV3 Cap inhibits cGAS binding to DNA. (A) PCV3 Cap did not affect IFN promoter induction by cGAS and STING. HEK 293T cells were co-transfected with IFN-Luc, TK-Luc, FLAG-Cap, empty vector (EV), FLAG-cGAS, and FLAG-STING. At 24 hpt, the cell lysates were prepared for luciferase analysis. (B) Cap inhibits ISD binding of cGAS. HEK 293T cells were co-transfected with FLAG-cGAS, FLAG-Cap, or the empty vector (EV). At 24 hpt, the cells were transfected with biotin-ISD for another 2 h, and the cell lysates were prepared from them for biotin-ISD pull-down and immunoblotting analysis.

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  Figure 3. Identifying Cap-interacting proteins. (A) SDS-PAGE analysis of Cap pull-down samples. PK15 cells were transfected with FLAG-Cap or FLAG vector. The cells were lysed for immunoprecipitation (IP) with anti-FLAG antibody. The precipitates were analyzed by SDS-PAGE. (B–D) Interaction between Cap and G3BP1. HEK 293T cells were co-transfected with FLAG-Cap and HA-G3BP1 (B) or various HA-G3BP1 mutants (D). The cells were lysed at 24 h post-transfection for IP using an anti-HA antibody, and the IP products were analyzed by western blotting. (C) Mapping the interacting regions between G3BP1 and Cap.

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  Figure 4. G3BP1 is involved in DNA-induced IFN promoter activation. (A) Western blotting analysis of G3BP1 expression after siRNA transfection. (B) Depletion of G3BP1 inhibits DNA-induced IFN promoter activation. PK15 cells were transfected with the negative control (NC) or G3BP1 #1 siRNA and the indicated reporter plasmids 24 h after siRNA transfection. The cells were then treated with ISD (2 μg/ml) for 12 h, and their luciferase activities were determined. G3BP1 and β-actin expression were detected by western blotting. (C) Depletion of G3BP1 inhibits DNA-induced TBK1 activation. PK15 cells transfected with NC or siRNA were treated with ISD, and 24 h after siRNA transfection, the cell lysates were analyzed by western blotting and the intensities of the pTBK1 and β-actin bands were quantified and normalized to β-actin. The results represent data from three independent experiments, **p < 0.01; ***p < 0.001.

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  Figure 5. Cap inhibits the interaction between cGAS and G3BP1. (A) Cap reduced the amount of cGAS co-precipitated by G3BP1. HEK 293T cells were co-transfected with FLAG-cGAS, FLAG-cap and HA-G3BP1, or the empty vector (EV). Cell lysates were used for immunoprecipitation (IP) with the anti-HA antibody. IP samples were analyzed by western blotting. (B) Cap inhibited the interaction of endogenous cGAS and G3BP1. PK15 cells were transfected with FLAG-cap or the empty vector. The cell lysates were used for IP with the anti-cGAS antibody, and the IP products were western blotted. The results represent data from three independent experiments, ***p < 0.001.

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  Figure 6. G3BP1 reduces the inhibition activity of Cap on cGAS-STING signaling. (A) Cap inhibited ISD binding to cGAS and overexpression of G3BP1 reversed this effect. HEK 293T cells co-transfected with indicted vector and transfected with biotin-ISD (2 μg/well) 24 h after the vector transfection. The cell lysates were used for DNA pull-down assays and western blotting. (B) Cap inhibits TBK1 and IRF3 phosphorylation, whereas G3BP1 has the opposite effect. PK15 cells were transfected with the expression vector indicated and then transfected with ISD (2 μg/ml) 24 h after the vector transfection. The cells were lysed and used for western blotting analysis. The results represent data from three independent experiments, *p < 0.05; **p < 0.01; ***p < 0.001.