Foot-and-Mouth Disease Virus Structural Protein VP1 Destroys the Stability of the TPL2 Trimer by Degradation of TPL2 To Evade Host Antiviral Immunity

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  FIG 1. TPL2 played an antiviral role during FMDV replication. (A) PK-15 cells were transfected with increasing amounts of Myc-tagged TPL2-expressing plasmid (0, 1, 2, or 3 μg), and the empty Myc vector (3, 2, 1, or 0 μg) was used in the transfection process to ensure that the cells received the same amounts of total DNA plasmids. At 24 hpt, the cells were infected with equal amounts of FMDV (MOI of 1) for 12 h. The replication levels of FMDV were measured by RT-qPCR, and the expression levels of Myc-TPL2 and viral proteins were detected by Western blotting. A rabbit anti-FMDV polyclonal antibody was used to confirm the expression of viral proteins. Western blotting showed three protein bands of VP0 (40 kDa), VP1 (25 kDa), and VP3 (26 kDa). The viral titers were measured by 50% tissue culture infective dose (TCID50) assay. (B) PK-15 cells were transfected with 100 pM NC siRNA or TPL2 siRNA for 48 h. The knockdown efficiency was determined by Western blotting. Relative fold change in abundance of TPL2 protein was determined by densitometric analysis. (C) PK-15 cells were transfected with 100 pM NC siRNA or TPL2 siRNA for 48 h, followed by infection with equal amounts of FMDV (MOI of 1)…

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  FIG 2. TPL2 enhanced antiviral effectors induced by FMDV infection in PK-15 cells. PK-15-TPL2 and PK-15-TPL2−/− cells were transfected with Myc-TPL2-expressing plasmid (3 μg) or empty Myc vector (3 μg) for 24 h and then mock infected or infected with equal amounts of FMDV (MOI of 1) for 12 h. The mRNA expression levels of IFN-α, IFN-β, IFN-γ, ISG15, ISG56, IL-6, IL-8, and TNF-α were measured by RT-qPCR assay.

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  FIG 3. TPL2 was essential for host against FMDV infection in mice. (A) Knockout (KO) schematic representation position of the sequence for TPL2 in TPL2−/− mice. The mouse genome of TPL2 contained eight exons. CRISPR/Cas9 technology was used to design a targeted vector to delete exon 3 and exon 4. (B) Protein levels of TPL2 in TPL2+/+ and TPL2−/− mice were detected by Western blotting to determine TPL2 knockout efficiency. (C) Three-day-old TPL2+/+ (n = 6 in each group) and TPL2−/− (n = 4 in each group) suckling mice were used and divided into three groups. One group was subcutaneously injected with equal amounts of PBS, and the other two groups were injected subcutaneously with two concentrations of FMDV (7 × 103 and 7 × 104 PFU). The survival rate was monitored every 12 h. (D) Three-day-old TPL2+/+ (n = 4 in each group) and TPL2−/− (n = 4 in each group) mice were used and divided into two groups in total. Two groups were subcutaneously injected with equal amounts of PBS and FMDV (7 × 104 PFU) and euthanized at 72 h after FMDV infection. Samples from FMDV-infected mouse were detected by RT-qPCR individually. Samples from mock-infected mice were mixed, and samples from infected mice were…

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  FIG 4. TPL2, ABIN2, and P105 levels were decreased after FMDV infection in vivo and in vitro. (A) PK-15 cells were infected with equal amounts of FMDV (MOI of 1) for 0, 4, 8, or 12 h. The transcriptional levels of FMDV and TPL2 were measured by RT-qPCR. (B) PK-15 cells were infected with equal amounts of FMDV (MOI of 1) for 0, 2, 4, 8, 12, 16, and 20 h. The expression levels of endogenous TPL2 and viral proteins were detected by Western blotting with anti-TPL2 and anti-FMDV antibodies. (C) The mRNA expression levels of p105 and ABIN2 were measured by RT-qPCR for the samples from panel A. (D) The expression levels of endogenous p105, ABIN2, and viral proteins were detected by Western blotting for the samples from panel B with anti-p105, anti-ABIN2, and anti-FMDV antibodies. (E) PK-15 cells with or without actinomycin D (10 μg/ml) treatment were infected with equal amounts of FMDV (MOI of 1) for 0, 4, 8, 12, and 16 h. The expression levels of endogenous TPL2 and viral proteins were detected by Western blotting. (F and G) Three-day-old wild-type mice (n = 4 in each group) were divided into two groups. Two groups were subcutaneously injected with equal amounts of FMDV (7 × 104 PFU) and PBS…

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  FIG 5. FMDV VP1 protein was responsible for reducing the expression of TPL2, p105, and ABIN2 at protein level but not transcription level. (A) HEK293T cells were cotransfected with Myc-TPL2 (1 μg), along with increasing amounts of Flag-L-, Flag-VP1-, or Flag-VP2-expressing plasmid (0, 1, 3, and 5 μg), and the empty Flag vector (5, 4, 2, and 0 μg) was used in the transfection process. At 24 hpt, the expression levels of Myc-TPL2 and Flag-tagged viral proteins (L, VP1, and VP2) were analyzed by Western blotting with anti-Myc and anti-Flag antibodies. (B) PK-15 cells were transfected with increasing amounts of Flag-L-, Flag-VP1-, or Flag-VP2-expressing plasmid (0, 1, 3, and 5 μg), and the empty Flag vector (5, 4, 2, and 0 μg) was used in the transfection process. At 24 hpt, the expression levels of endogenous TPL2 and Flag-tagged viral proteins were analyzed by Western blotting with anti-TPL2 and anti-Flag antibodies. (C) HEK293T cells were cotransfected with HA-p105 or Flag-ABIN2 (1 μg), along with increasing amounts of Flag-VP1- or HA-VP1-expressing plasmid (0, 1, 3, and 5 μg), and the empty Flag vector or empty HA vector (5, 4, 2, and 0 μg) was used in the transfection process. At 24…

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  FIG 6. VP1 interacted with host TPL2 but not with p105 and ABIN2. (A) HEK293T cells were cotransfected with 8 μg Myc-TPL2- or HA-p105- and Flag-VP1-expressing plasmid. The cells were lysed at 24 hpt, and the lysates were immunoprecipitated with mouse IgG antibody or mouse anti-Flag antibody and subjected to Western blotting. Lysate and immunoprecipitation (IP) antibody-antigen complexes were analyzed by immunoblotting (IB) using anti-HA, anti-Myc, anti-Flag, and anti-β-actin antibodies. (B) The method is the same as that for panel A. The lysates were immunoprecipitated with mouse IgG antibody or mouse anti-HA antibody and subjected to Western blotting. (C and D) PK-15 cells were transfected with Flag-VP1-expressing plasmid (8 μg) or empty Flag vector (Vec; 8 μg) for 24 h. The cells were then lysed and immunoprecipitated with normal IgG antibody, anti-Flag antibody (C), or anti-TPL2 antibody (D). The lysate and IP antibody-antigen complexes were analyzed by IB using anti-Flag, anti-TPL2, or anti-β-actin antibodies. (E and F) PK-15 cells were mock infected or infected with FMDV (MOI of 1) for 6 or 12 h. MG132 (20 μM) was added to prevent the degradation of endogenous TPL2. The cells were…

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  FIG 7. Degradation pathways of TPL2, ABIN2, and p105 induced by VP1 in HEK293T and PK-15 cells. (A) PK-15 cells treated with MG132 (20 μM), CQ (100 μM), NH4Cl (20 μM), or Z-VAD-FMK (50 μM) for 1 h and then with or without FMDV infection (MOI of 1) for 12 h. The expression levels of endogenous TPL2 and viral proteins were detected by Western blotting. DMSO, dimethyl sulfoxide. (B) HEK293T cells were cotransfected with Flag-VP1-expressing plasmid (2 μg) or empty Flag vector (2 μg) and Myc-TPL2-expressing plasmid (1 μg) and empty Myc vector (1 μg) and maintained in the presence or absence of MG132 (2 or 20 μM), CQ (50 or 100 μM), NH4Cl (10 or 20 μM), or Z-VAD-FMK (10 or 50 μM) for 36 h. The expression levels of Myc-TPL2 and Flag-VP1 proteins were detected by Western blotting. (C) PK-15 cells were transfected with HA-VP1-expressing plasmid (2 μg) or empty HA vector (2 μg) and maintained in the presence or absence of MG132 (20 μM), CQ (100 μM), NH4Cl (20 μM), or Z-VAD-FMK (50 μM) for 36 h. The expression levels of endogenous TPL2 and HA-VP1 proteins were detected by Western blotting. (D) HEK293T cells were cotransfected with HA-VP1 (5 μg) or empty HA vector (5 μg) and Myc-tagged TPL2…

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  FIG 8. FMDV and FMDV VP1 destroy the stability of the TPL2-p105-ABIN2 trimer complex. (A) PK-15 cells were cotransfected in pairs with plasmid expressing Myc-TPL2 (5 μg), HA-p105 (5 μg), or Flag-ABIN (5 μg) for 24 h, with or without FMDV infection (MOI of 1) for 12 h. The cells were lysed, and the lysates were immunoprecipitated with anti-Myc antibody, mouse anti-Flag antibody, anti-HA antibody, and IgG antibody and subjected to Western blotting. (B) PK-15 cells with or without FMDV infection (MOI of 1) for 12 h. The cells were lysed and immunoprecipitated with mouse anti-TPL2 antibody. The lysate and IP antibody-antigen complexes were analyzed by IB using anti-TPL2, anti-p105, anti-ABIN2, anti-FMDV, and anti-β-actin antibodies to measure the expression of endogenous TPL2, p105, and ABIN2 separately. (C) HEK293T cells were cotransfected in pairs with plasmid expressing Myc-TPL2 (5 μg), HA-p105 (5 μg), and V5-ABIN2 (5 μg). Flag-VP1-expressing plasmid (8 μg) and empty Flag vector (8 μg) were transfected at the same time. At 24 hpt, the cells were lysed and the lysates were immunoprecipitated with anti-Myc antibody, anti-V5 antibody, anti-HA antibody, and mouse normal IgG antibody. (D)…

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  FIG 9. Schematic representation showing the model of TPL2 involvement in FMDV replication. TPL2 inhibits FMDV replication by promoting FMDV-induced interferons and other antiviral factors. FMDV VP1 protein destroys the stability of TPL2 trimer by targeting TPL2 degradation, leading to the reduction of TPL2, which can induce the production of antiviral cytokines, thereby facilitating FMDV replication.