Foot-and-Mouth Disease Virus VP3 Protein Acts as a Critical Proinflammatory Factor by Promoting Toll-Like Receptor 4-Mediated Signaling

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  FIG 1. Foot-and-mouth virus (FMDV) potentiates the lipopolysaccharide (LPS)-induced signaling pathway. (A) PAMs are susceptible to FMDV. PK-15 and PAMs were seeded in a 12-well plate for 12 h. The cells then were infected with FMDV (MOI, 0.5 or 1.0) for the indicated times before RT-PCR experiments. (B) Effects of FMDV on LPS-induced increases in Ifnb, Isg56, Rantes, and Cxcl-10 in porcine alveolar macrophages (PAMs). PAMs were seeded in a 12-well plate for 12 h. The cells then were uninfected or infected with FMDV (MOI, 1.0) for the indicated times. Cells were treated or left untreated with LPS (1 μg/ml) for 4 h before RT-PCR experiments. (C) Inactivated FMDV did not induce the transcription of Ifnb in PAMs. After PAMs were seeded in a 12-well plate for 12 h, cells were uninfected or infected with FMDV or inactive-FMDV (obtained by UV treatment for 30 min) (MOI, 1.0) for the indicated time. The cells were treated or left untreated with LPS (1 μg/ml) for 4 h before RT-PCR. (D) FMDV increases the LPS-induced phosphorylation of TBK1, IRF3, and IκBα. PAMs were seeded in a 12-well plate for 24 h. Cells then were uninfected or infected with FMDV (MOI, 1.0) for the indicated times. Cells were…

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  FIG 2. FMDV VP3 regulates the TLR4-mediated signaling pathway. (A) Effect of the overexpression of FMDV proteins on LPS-triggered activation of the Nifty promoter. 293-TLR4 cells (1 × 105) were transfected with the Nifty reporter (0.1 μg) and the indicated expression plasmids. Twenty hours after transfection, cells were treated or left untreated with LPS for 12 h before luciferase assays. Rel. Luc. Act., relative luciferase activity. (B) Dose-dependent effects of FMDV VP3 on LPS-triggered activation of the Nifty promoter. The experiments were performed following similar methods to those in panel A. (C) Effects of FMDV VP3 on the LPS-induced increases in Ifnb, Isg56, Rantes, and Cxcl-10. VP3-overexpessing PAMs (2 × 105) were left untreated or treated with LPS (1 μg/ml) for 12 h before RT-PCR experiments were performed with the indicated primers. (D) FMDV VP3 increases LPS-induced phosphorylation of TBK1, IRF3, and IκBα. FMDV VP3-overexpressed PAM cells were left untreated (2 × 105) or were treated with LPS (1 μg/ml) for the indicated times. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Data are shown as means ± SD. Data are representative of at least three…

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  FIG 3. FMDV VP3 regulates the TLR4 signaling pathway at the TLR4 level. 293-TLR4 cells (1 × 105) were transfected with the Nifty reporter (0.1 μg) and the indicated expression (0.1 μg) plasmids for 24 h before luciferase assays. Data are shown as means ± SD. Data are representative of at least three independent experiments. SD, standard deviation; Luc, luciferase. EV, empty vector; Con, control.

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  FIG 4. FMDV VP3 interacts with TLR4. (A) FMDV VP3 interacted with TLR4 and TRAF6 but not with Myd88, TRIF, TBK1, or IRF3 in the overexpression system. 293T cells (2 × 106) were transfected with the indicated plasmids (5 μg each). Coimmunoprecipitation and immunoblot analyses were performed with the indicated antibodies. The expression of transfected proteins was analyzed by immunoblotting with anti-HA or anti-Flag antibodies. (B) Endogenous interactions between VP3 and TLR4. PAMs were left uninfected or were infected with FMDV for the indicated times before coimmunoprecipitation and immunoblotting. (C) Colocalization of VP3 with endogenous TLR4. PAMs were mock infected or infected with FMDV. Cells were fixed at 12 h postinfection and subjected to indirect immunofluorescence to detect VP3 (green) and TLR4 (red) with mouse anti-VP3 and rabbit anti-TLR4 antibodies. Nuclei are indicated by 4′,6-diamidino-2-phenylindole (DAPI) (blue) staining in the merged image.

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  FIG 5. TLR4 inhibits FMDV replication. (A) Effects of TLR4 overexpression in PAMs on FMDV replication. TLR4-overexpressing PAMs were infected with FMDV (MOI, 1.0) for the indicated times. FMDV genome replication was evaluated by RT-PCR. The results are representative of three independent experiments, and means ± SD from triplicate assays are shown. (B) Effects of TLR4 overexpression on FMDV titers in PAM cells. TLR4-overexpressing PAMs were infected with FMDV (MOI, 1.0) for the indicated times. The virus titers in the supernatants were determined by immunofluorescence and are expressed as 50% tissue culture infective dose (TCID50)/0.1 ml. The results are representative of three independent experiments, and means ± SD are shown. (C) Effects of TLR4 overexpression on FMDV protein expression in PAMs. TLR4-overexpressing PAMs were infected with FMDV (MOI, 1.0) for the indicated times. FMDV VP3 was detected by immunoblotting with the indicated antibodies. (D) Effects of TLR4 knockdown in PAMs on FMDV replication. The experiments were performed by following methods similar to those for panel A. (E) Effects of TLR4 knockdown on FMDV titers in PAMs. The experiments were performed by following…

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  FIG 6. FMDV potentiates TLR4 via the lysozyme pathway. (A) Effects of FMDV on endogenous TLR4 in PAMs. Briefly, PAMs were uninfected or infected with FMDV for the indicated times and then analyzed by immunoblotting with the indicated antibodies. (B) Effects of inhibitors on the FMDV-mediated stability of TLR4. PAMs were treated with the indicated inhibitors for 1 h. The cells then were left uninfected or infected with FMDV for 6 h and were analyzed by immunoblotting with the indicated antibodies. DMSO, dimethyl sulfoxide.

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  FIG 7. FMDV VP3 potentiated TLR4 expression by downregulating Rab7b expression. (A) FMDV VP3 interacted with Rab7b in the overexpression system. 293T cells (2 × 106) were transfected with the indicated plasmids (5 μg each). Coimmunoprecipitation and immunoblot analyses were performed with the indicated antibodies. The expression of transfected proteins was analyzed by immunoblotting with anti-Myc or anti-Flag antibodies. (B) VP3 inhibits the expression of Rab7b protein. 293T cells (2 × 105) were transfected with Myc-Rab7b (1 μg) and Flag-VP3 (0 μg, 0.25 μg, 0.5 μg, and 1.0 μg) for 24 h. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (C) Rab7b increases the expression of VP3. The methods were similar to those for panel B. (D) Rab7b inhibits the expression of TLR4. The methods were similar to those for panel B. (E) Effects of inhibitors on the VP3-mediated stabilization of Rab7b. 293T cells (4 × 105) were transfected with the indicated plasmids. Eighteen hours after transfection, the cells were treated with the indicated inhibitors for 6 h before immunoblot analyses. (F) Effects of FMDV VP3 on the interaction between TLR4 and Rab7b. The methods were similar to…

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  FIG 8. Rab7b overexpression promotes FMDV replication. (A) Effects of Rab7b overexpression in PAMs on FMDV replication. Rab7b-overexpressed PAMs were infected with FMDV (MOI, 1.0) for the indicated times. FMDV genome replication was evaluated by RT-PCR. The results are representative of three independent experiments, and means ± SD are shown. (B) Effect of Rab7b overexpression on FMDV titers in PAMs. Rab7b-overexpressed PAMs were infected with FMDV (MOI, 1.0) for the indicated times. The virus titers in the supernatants were determined by immunofluorescence and are expressed as TCID50/0.1 ml. The results are representative of three independent experiments and are presented as means ± SD from three technical replicates. (C) Effect of Rab7b overexpression on FMDV protein expression in PAMs. Rab7b-overexpressing PAMs were infected with FMDV (MOI, 1.0) for the indicated times. FMDV VP3 protein expression was detected by immunoblotting with the indicated antibodies. (D) Effect of Rab7b knockdown in PAMs on FMDV replication. The methods were similar to those for panel A. (E) Effect of Rab7b knockdown on FMDV titers in PAMs. The methods were similar to those for panel B. (F) Effect of Rab7b…