Porcine Teschovirus 2 3Cpro Evades Host Antiviral Innate Immunity by Inhibiting the IFN-β Signaling Pathway

Abstract

Porcine teschovirus (PTV) circulates in pig populations, causing clinical diseases such as poliomyelitis, reproductive disorders, and pneumonia. However, the molecular mechanisms underlying the pathogenesis of PTV infection have not been fully elucidated. Here, we found that PTV infection does not activate the promoters of NF-κB or IFN-β. The expression of PTV 3C^pro inhibits the promoter activity of NF-κB and IFN-β stimulated by SeV and inhibits the downstream transcription of NF-κB and IFN-β by blocking the phosphorylation and nuclear translocation of NF-κB. Coimmunoprecipiation (co-IP) experiments demonstrated that 3C^pro and NF-κB interact. The degradation of NF-κB was unaffected by inhibitors targeting lysosomes (NH4Cl), proteasomes (MG132), or caspases (Z-VAD-FMK). The protease activity of 3C^pro, which relies on its catalytic active site, is vital for NF-κB cleavage and degradation. Loss of proteolytic activity in mutants abolished NF-κB degradation, impairing the ability of 3C^pro to suppress SeV-induced innate immunity and restore VSV-GFP replication, thereby underscoring its critical role in immune evasion by targeting NF-κB. This study reveals novel mechanisms underlying PTV-mediated suppression of host innate immunity.

Keywords: 3Cpro; NF-κB; inhibitor; porcine teschovirus; protease activity.

Figure 1

PTV infection inhibits the activity of the NF-κB and IFN-β promoters. (A) ST cells were cotransfected with NF-κB-Luc or IFN-β-Luc (1000 ng) and PRL-TK (50 ng), followed by 24 h of infection with PTV (MOI = 1, 5) or SeV (MOI = 1), and the fluorescence intensity was measured. (B) ST cells were cotransfected with NF-κB-Luc or IFN-β-Luc (1000 ng) and PRL-TK (50 ng), followed by 24 h of induction with SeV (MOI = 1) and 24 h of infection with PTV (MOI = 1, 5), and the fluorescence intensity was measured. **** p < 0.0001, ns indicates no significant difference.

Figure 2

PTV 3C^pro inhibits NF-κB and IFN-β mRNA expression and promoter activity. (A,B) Cotransfection of NF-κB-Luc or IFN-β-Luc (1000 ng) with PRL-TK (50 ng) and 3C^pro (200 ng, 500 ng) into 293T cells, followed by 12 h of induction with SeV (MOI = 1) and measurement of firefly luciferase activity, normalized to Renilla luciferase activity. 3C^pro (200 ng, 500 ng) was transfected into ST cells, which were then induced with SeV (MOI = 1) for 12 h, and the NF-κB or IFN-β mRNA levels were measured using fluorescence quantitative PCR. (C) NF-κB-Luc (1000 ng) with PRL-TK (50 ng) and 3C^pro (500 ng) were cotransfected into 293T cells, followed by cotransfection of MDA5, RIG-I, TBK1, and MAVS (700 ng) to induce promoter activity. After 24 h, firefly luciferase activity, normalized to Renilla luciferase activity, was measured. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 3

PTV 3C^pro inhibits NF-κB phosphorylation and nuclear translocation. (A) NF-κB (500 ng) and PTV 3C^pro (500 ng) were introduced into ST cells that were infected with SeV after 24 h, and protein fluorescence was observed through an indirect immunofluorescence assay. (B) PTV 3C^pro (500 ng) was introduced into ST cells infected with SeV after 24 h, and cytoplasmic and nuclear proteins were extracted to measure the levels of NF-κB. GAPDH and Lamin B proteins were used as internal references. Quantitative display is achieved through standardization of the ratio of NF-κB/internal references. (C) PTV 3C^pro (500 ng) was transfected into ST cells, followed by SeV (MOI = 1) stimulation after 12 h, and the cell lysate was collected at 0 h, 6 h, and 12 h. Western blotting was performed to detect the protein levels of NF-κB and phosphorylated NF-κB. GAPDH protein was used as the internal reference. Quantitative display is achieved through standardization of the ratio of p-NF-κB (NF-κB) to GAPDH.

Figure 4

Mechanism of 3C^pro-mediated degradation of NF-κB. (A) PTV 3C^pro (1000 ng) was transfected into ST or 293T cells, which were subsequently incubated for 24 h, and NF-κB protein levels were detected using western blotting. The asterisk (*) denotes the band corresponding to H-NF-κB cleaved by PTV 3C^pro. (B) 293T cells were cotransfected with NF-κB (or H-NF-κB) (1000 ng) and the 3C^pro plasmid (300 ng), and co-IP and western blotting were subsequently performed to measure the protein levels after 24 h of incubation. (C) 293T cells were cotransfected with NF-κB (or H-NF-κB) (1000 ng) and the 3C^pro plasmid (1000 ng), followed by 12 h of inhibition treatment and western blotting detection of protein levels. (D) Comparative sequence analysis of PTV 3C^pro with other members of the Picornaviridae family revealed the conserved catalytic site. On the basis of the enzyme activity sites of other viruses, H49 and C158 were predicted to be the enzyme activity sites of PTV 3C^pro. The asterisks (*) were used to separate consecutive 10-sequence intervals. (E) NF-κB (or H-NF-κB) (1000 ng), PTV 3C^pro, and its mutant (1000 ng) were transfected into 293T cells, which were then cultured for 24 h, after which NF-κB expression was assessed using western blotting. The asterisk (*) denotes the band corresponding to H-NF-κB cleaved by PTV 3C^pro.

Figure 5

The PTV 3C^pro mutant cannot antagonize innate immunity. (A) In 293T cells, NF-κB-Luc or IFN-β-Luc (1000 ng) and PRL-TK (50 ng) were cotransfected with 3C_H49A, 3C_C158A, or 3C_DM (500 ng), followed by SeV induction for 12 h prior to dual-luciferase activity measurement. (B,C) PTV 3C^pro and the variant (1000 ng) were transfected into 293T cells, which were subsequently subjected to VSV infection and poly(I:C) stimulation for 12 h, fluorescence observation, and quantification using fluorescence microscopy and flow cytometry. * p < 0.05, ns indicates no significant difference.