Enterovirus D68 VP3 Targets the Interferon Regulatory Factor 7 To Inhibit Type I Interferon Response

Figures

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  FIG 1. EV-D68 VP3 suppresses virus-induced IFN-β activation. (A) The UV-inactivated EV-D68 virus dose-dependently inhibited the IFN-β promoter induced by SeV infection. RD cells were treated with an increasing dose of UV-EV-D68 (MOI = 0.5, 1, 2, 3) for 24 h. The positive control group was cells infected with active EV-D68 (MOI = 0.5). HEK293T cells were transfected with 200 ng of the reporter plasmid IFN-β-Luc and 2 ng of pRLSV40 for 24 h. After transfection, the cells were treated with SeV (20 HA/mL) or UV-EV D68 (MOI = 0.5, 1, 2, 3) for 24 h, and the cell lysates were subjected to a luciferase assay. (B) Detection of anti-IFN-I responses by viral structural proteins. HEK293T cells were transfected with 200 ng of the reporter plasmid IFN-β-Luc, 2 ng of pRL-SV40, and 600 ng of VP1, VP2/4, or VP3. Twenty-four hours later, the cells were treated with medium or SeV (20 HA/mL) for 16 h, the cell lysates were subjected to a luciferase assay, and the expression of plasmid was confirmed by Western blotting. (C) The treatment of EV-D68 VP3 inhibits IFN-β mRNA synthesis. HEK293T cells were transfected with 600 ng VP3-HA or empty vector. Twenty-four hours after transfection, cells were infected…

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  FIG 2. EV-D68 VP3 interacts with IRF7 and inhibits IRF7-mediated activation of the IFN-I pathway. (A) VP3 functions specifically at or downstream of IRF7. HEK293T cells were transfected with 200 ng of ISRE-Luc, 2 ng of pRL-SV40, 600 ng of VP3-Myc, or 600 ng of empty vector (MOCK) and the indicated plasmids, including 200 ng of RIG-I, MDA5, MAVS, TBK1, IKKε, IRF3, or IRF7. Twenty-four hours after transfection, the cell lysates were subjected to a luciferase assay. (B and C) Coexpression of VP3 and IRF7 could inhibit IRF7-induced IFN-I activation. HEK293T cells were transfected with 600 ng VP3-Myc, 300 ng IRF7-Flag, and 200 ng IFN-β-Luc (B) or IFN-α4-Luc (C); 2 ng of pRL-SV40 was used as an internal control. Twenty-four hours after transfection, the cell lysates were subjected to a luciferase assay. (D) VP3 interacts with IRF7 in HEK293T cells. HEK293T cells were transfected with 3 μg VP3-Myc and empty vector or 3 μg IRF7-Flag or IRF3-Flag. The cell lysates were immunoprecipitated (IP) with anti-Flag beads and immunoblotted (IB) with anti-Myc or anti-Flag antibody. (E) VP3 interacts with IRF7 in RD-IRF7-Flag cells. Lane 1 RD cells were transfected with 3 μg VP3-Myc. Lane 1 RD-IRF7-Flag…

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  FIG 3. VP3 inhibits SeV-induced IRF7 phosphorylation and nuclear import. (A) p-IRF7 is suppressed by VP3 plasmid treatment or EV-D68 (MOI = 0.1) infection in RD-IRF7 cells. RD-IRF7 cells were transfected with 1 μg VP3-Myc. Twenty-four hours later, cells were infected with SeV (20 HA/mL) for 16 h. Then, the cell lysates were subjected to Western blotting with p-IRF7, IRF7, EVD68-VP3, and GAPDH antibodies. (B) Quantification of the relative expression of P-IRF7/IRF7 in panel A. (C) The endogenous IRF7 expression level was detected. A significant decrease in IRF7 is observed in the nucleus. Lane 3 HEK293T cells were transfected with 1 μg VP3-Myc. Lane 4 HEK293T cells were infected with EV-D68 (MOI = 0.1). Twenty-four hours after transfection, cells were infected with SeV (20 HA/mL) for 16 h. The nuclear and cytoplasmic proteins were separated. Cell lysates were subjected to Western blotting with the following antibodies, anti-IRF7, anti-IRF3, anti-Myc, anti-EV-D68 VP1 and GAPDH. (D) Quantification of the relative expression of IRF7/histone in panel C. (E) EV-D68 VP3 inhibits the nuclear translocation of IRF7 after SeV stimulation in HeLa cells. HeLa cells were seeded on glass coverslips…

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  FIG 4. VP3 inhibits the TRAF6-induced ubiquitination level and the transactivation ability of IRF7. (A) EV-D68 VP3 disrupts the interaction of TRAF6 and IRF7 in a dose-dependent way. HEK293T cells were transfected with 2 μg IRF7-Flag, 2 μg Ub-K63-Myc, 2 μg TRAF6-HA or empty vector, and 2 μg VP3-HA or empty vector. Cell lysates were immunoprecipitated with anti-Flag beads followed by immunoblots using HA, Flag, Myc, and GAPDH antibodies. (B) Quantification of the relative expression of K63/GAPDH in panel A. (C) The TRAF6-induced IRF7-dependent transcription level of IFN-α4-Luc was suppressed. HEK293T cells were transfected with VP3-HA (200 ng, 400 ng, 800 ng), 150 ng of TRAF6-V5, and 200 ng of IFN-α4-Luc; 2 ng of pRL-SV40 was used as an internal control. Twenty-four hours after transfection, the cell lysates were subjected to a luciferase assay. (D) Expression of VP3 protein dose-dependently inhibits TRAF6 induced ubiquitination of IRF7. HEK293T cells were transfected with 1.5 μg IRF7-Flag, 2.5 μg VP3-HA, and 600 ng TRAF6-V5. Cell lysates were IP with anti-Flag beads. Immunoprecipitates and aliquots of cell lysates were subjected to Western blotting with HA, Flag, V5, and GAPDH…

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  FIG 5. Identification of VP3 and IRF7 interacting region. (A) Four Flag-tagged IRF7-truncated mutants were constructed. (B) Removing 305 to 468 amino acids resulted in a constitutively active form of IRF7. HEK293T cells were transfected with 600 ng of IRF7-truncated mutants, 200 ng of IFN-β-Luc, and 2 ng of pRL-SV40. The cell lysates were subjected to a luciferase assay, and the expression of plasmid was confirmed by Western blotting. (C) IRF7Δ305–503 could not be coimmunoprecipitated with VP3. HEK293T cells were cotransfected with 3 μg of VP3-Myc and 3 μg of IRF7-truncated mutants. The cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-HA or anti-Flag. (D) TRAF6 binds to the C terminus of IRF7. The 305 to 503 amino acid region of IRF7 is the major binding site of TRAF6 to IRF7 protein to facilitate K63-linked polyubiquitination. HEK293T cells were cotransfected with 2 μg IRF7-Flag or IRF7Δ305–503 or empty vector, 2 μg Ub-K63-Myc, and 2 μg TRAF6-Myc or empty vector. The cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-Myc or anti-Flag. (E) Three VP3-truncated mutants were constructed. (F) HEK293T cells were transfected with 600…

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  FIG 6. The VP3 proteins of CV-A16 and EV-A71 interact with IRF7. (A) The exogenous expression of VP3 protein from EV-A71 and CV-A16 inhibited SeV-induced IRF7-dependent transcriptional activation. HEK293T cells were transfected with 600 ng VP3-HA, 600 ng EV-A71 VP3-HA, 600 ng CV-A16 VP3-HA, and 200 ng IFN-β-Luc; 2 ng pRL-SV40 was used as an internal control. Twenty-four h after transfection, cell lysates were assayed for promoter-luciferase activities. Cell lysates were subjected to Western blotting with HA and GAPDH antibodies. (B) EV-A71 and CV-A16 VP3 inhibit the SeV-induced production of IFN-β mRNA. HEK293T was transfected with 4 μg IRF7-Flag and 4 μg EV-A71 VP3-HA or CVA16 VP3-há; 48 h after transfection, cell lysates were IP with anti-Flag beads. Immunoprecipitates and aliquots of cell lysates were subjected to Western blotting with HA, Flag, and GAPDH antibodies. (C and D) The exogenous expression of VP3 protein from EV-A71 and CV-A16 inhibits SeV-induced IRF7-dependent transcriptional activation. HEK293T cells were transfected with 600 ng VP3-HA, 600 ng EV-A71 VP3-HA, 600 ng CV-A16 VP3-HA, and 200 ng ISRE-Luc (C) or IFN-α4-Luc (D); 2 ng pRL-SV40 was used as an internal control.…

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  FIG 7. The molecular model of IRF 7 targeting by EV-D68 VP3 to inhibit type I interferon response. The EV-D68 structural protein VP3 interacts with IRF7, resulting in the inhibition of virus-induced phosphorylation and nuclear translocation of IRF7 and suppression of downstream IFN production. EV-D68 VP3 also disturbs the TRAF6-triggered K63-ubiquitination of IRF7, thus inhibiting the phosphorylation of IRF7 and the following IFN promoter activation and IFN expression.