Poly (rC) binding protein 2 interacts with VP0 and increases the replication of the foot-and-mouth disease virus

Figures

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  Fig. 1. PCBP2 potentiates FMDV replication. a PCBP2 increased FMDV replication in PCBP2-overexpressing PK-15 cells. PCBP2-overexpressing PK-15 cells were infected with FMDV (MOI = 0.1) for 4 and 8 h, and the copy number of FMDV RNA was evaluated by RT-PCR. b Western immunoblotting analysis of FMDV VP3 protein levels in PCBP2-overexpressing PK-15 cells. The experiments were similar to those described in a. Western immunoblotting analysis with the antibodies against flag, VP3, and β-actin proteins. c Western immunoblotting analysis of PCBP2 protein levels in PCBP2-knockdown PK-15 cells. Western immunoblotting analysis with the antibodies against PCBP2 and β-actin proteins. Degree of PCBP2-knockdown was calculated by Image J software. d PCBP2 reduced FMDV replication in PCBP2-knockdown PK-15 cells. The experiments were similar to those described in a. e Effects of FMDV on the transcription of FMDV VP0 and PCBP2 genes. PK-15 cells were infected with FMDV (MOI = 0.1) for 0, 4, and 8 h, and the mRNA levels of VP0 and PCBP2 were analyzed by RT–PCR. The data represent the mean ± SD of the three independent experiments (*p < 0.05; **p < 0.01; NS, not significant, p> 0.05). EV empty vector, Coni…

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  Fig. 2. PCBP2 interacts with FMDV proteins. a, b The interaction between PCBP2 and FMDV proteins in mammalian overexpression systems was identified by coimmunoprecipitation experiment. The HEK293T cells (2 × 106) were transfected with HA-PCBP2 plasmid or expressing Flag-tagged VP2, 2B, 3A, L, VP0, 2C, 3C, and 3D proteins (5 μg each) for 24 h. Afterward, PCBP2 and FMDV proteins were analyzed by coimmunoprecipitation and immunoblotting analyses using the antibodies against flag and HA. c, d GST pull-down assay. Glutathione beads conjugated with GST or with GST-PCBP2 fusion protein were incubated with recombinant FMDV proteins. After washing three times, the proteins were eluted from the beads, and SDS-PAGE was performed. The presence of FMDV proteins was detected via immunoblotting utilizing the anti-flag monoclonal antibody. GST and GST-PCBP2 protein expression was confirmed via immunoblotting using the rabbit anti-GST polyclonal antibody. F flag tag, H HA tag, LC light chain

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  Fig. 3. PCBP2 inhibits IFN-β signaling pathway. a, b The effects of PCBP2 overexpression on SeV-triggered IFN-β promoter and ISRE activation were evaluated. HEK293T cells (1 × 105) were transfected with IFN-β reporter or ISRE (0.1 μg) and with PCBP2 expression (0.1 μg) plasmids for 24 h. The cells were then infected with SeV for 12 h or were uninfected (left) before luciferase assays were performed. EV empty vector. c, d Dose-dependent effects of PCBP2 on VISA-triggered activation of IFN-β promoter and ISRE were assessed. The experiments were performed similar to those described in a. EV empty vector. e Panel shows schematic representations of PCBP2 proteins. f, h The sites or domains of PCBP2 inhibit IFN-β signaling pathway. The experiments were performed similar to those described in a. EV, empty GFP vector. The data represent the mean ± SD of the three independent experiments (*p < 0.05; **p < 0.01). Luc luciferase

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  Fig. 4. Effects of FMDV proteins synergized with PCBP2 on VISA-triggered signaling. a–g Effects of L a, 2C b, 2B c, 3D d, 3A e, VP0 f, and VP2 g proteins of FMDV synergized with PCBP2 on VISA-triggered activation of IFN-β promoter. HEK293T cells (1 × 105) were transfected with IFN-β reporter (0.1 μg) and the indicated expression (0.1 μg) plasmids for 24 h before luciferase assays were performed. h Effects of 2B, 2C, 3D, and VP0 proteins of FMDV synergized with PCBP2 on VISA-triggered activation of IFN-β promoter. The experiments were performed similar to those described in a. The data represent the mean ± SD of the three independent experiments (*p < 0.05; **p < 0.01). EV empty vector, Luc luciferase, Con control

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  Fig. 5. Effects of overexpression of FMDV proteins on the interaction between PCBP2 and VISA. a–d HEK293T cells (2 × 106) were transfected with the indicated plasmids (5 μg each) for 24 h. Coimmunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. e Dose-dependent effects of FMDV VP0 protein on the interaction between VISA and PCBP2. The experiments were performed similar to those described in a. f Effects of FMDV on the interaction between VISA and PCBP2. PK-15 cells (5 × 107) were infected with FMDV (MOI = 0.1) for 0, 6, and 12 h. Coimmunoprecipitation experiments were performed with antibody against VISA, and the immunoprecipitates were analyzed by immunoblotting using the antibodies against PCBP2 and VISA protein. The lysates were analyzed by immunoblotting with the indicated antibodies. g Effects of RNase A/T1 treatment on PCBP2–VP0 interaction. Extractions of HEK293T cells overexpressing different proteins were treated or not treated with RNases A/T1 for 1 h before coimmunoprecipitation and immunoblotting with the indicated antibodies. Ethidium bromide-stained agarose gels showed the total amount of RNA present. EV empty vector

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  Fig. 6. Effects of overexpression of PCBP2 and FMDV proteins on the expression level of VISA. a–d Effects of overexpression of PCBP2 and FMDV VP0, 2B, 2C, and 3D on the expression level of VISA. HEK293T cells (5 × 105) were transfected with the indicated expression plasmids (2 μg each) for 24 h. Immunoblotting analyses were performed using the indicated antibodies. e–f Effects of overexpression of PCBP2 and FMDV VP0 and 3D on the expression level of endogenous VISA. The experiments were performed similar to those described in a. g RT-PCR was used to detect the transcription of VISA in VISA-deficient MEF cells. h Deficiency of VISA increases FMDV replication in MEF cells. VISA-deficient MEF cells were infected with FMDV (MOI = 0.1) for the indicated time. RT-PCR analysis was carried out to evaluate FMDV replication. The data represent the mean ± SD of the three independent experiments (*p < 0.05; **p < 0.01)

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  Fig. 7. FMDV VP0 coexpresses with PCBP2-mediated degradation of VISA via the apoptotic pathway. a–d Coexpression of PCBP2 and FMDV VP0 degraded VISA via the apoptotic pathway. HEK293T cells (5 × 105) were transfected with the indicated expression plasmids (2 μg each) for 18 h and then treated with dimethyl sulfoxide (DMSO), MG-132, 3-MA, or NH4Cl for 6 h. Immunoblotting analyses were performed using the indicated antibodies. e Coexpression of PCBP2 and FMDV VP0 degraded endogens VISA via the apoptotic pathway. The PK-15 cells (5 × 105) were transfected with the indicated expression plasmids (4 μg each) for 18 h and then treated with dimethyl sulfoxide (DMSO), and MG-132 for 6 h. Immunoblotting analyses were performed using the indicated antibodies. f FMDV VP0 increases the interaction between endogenous PCBP2 and VISA. The PK-15 cells (2 × 106) were transfected with the indicated plasmids (8 μg each) for 24 h. Coimmunoprecipitation and immunoblotting analyses were performed using the indicated antibodies. g Effects of VP0 and PCBP2 on the apoptosis of HEK293T cells. HEK293T cells were transfected with the indicated plasmids (1 μg) for 24 h. The cellular apoptosis rate was measured by…

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  Fig. 8. Effect of endogenous PCBP2 and FMDV VP0 on the expression of VISA. a Effects of overexpression of FMDV VP0 on the expression of VISA in PCBP2-knockdown PK-15 cells. The PCBP2-knockdown PK-15 cells (5 × 105) were transfected with the indicated plasmids (2 μg each) for 24 h. Then, the immunoblotting analyses were performed utilizing the indicated antibodies. b Effects of VP0-RNAi plasmids on the transcription of FMDV VP0. The VP0-knockdown PK-15 cells were infected with FMDV (MOI = 0.1) for 12 h before real-time RT–PCR experiments were performed. c Effects of knockdown of FMDV VP0 on the expression of FMDV VP3 protein. The experiments were performed similar to those described in b. c Effects of knockdown of FMDV VP0 on the expression level of VISA. The VP0- knockdown PK-15 cells were infected with FMDV (MOI = 0.1) for 12 h or uninfected followed by immunoblotting analyses were performed with the indicated antibodies. The data represent the mean ± SD of the three independent experiments (**p < 0.01). Con control