The VP1 Protein of Porcine Teschovirus Inhibits the Innate Immune Response to Viral Infection by Blocking MDA5 Activation

Figure 1. The PTV VP1 protein curbs SeV-mediated IFN-β activation: (a and b) reproduction of PTV in ST cells was examined by infecting the cells with PTV at a MOI of 1 for specified durations: (a) the expression levels of VP1 mRNA were quantified using qPCR at different time points (0, 6, 12, 24, and 48 hr) postinfection, (b) the levels of IFN-β mRNA expression were also measured by qPCR at the same time points; (c) HEK293T cells were introduced to VP1 plasmids (500 ng, 1,000 ng) via transfection 24 hr, then infected with SeV for 12 hr. The levels of IFN-β mRNA expression were again measured by qPCR; (d) HEK293T cells were cotransfected with the IFN-β-Luc, pRL-TK, and VP1 plasmids (500 ng, 1,000 ng) and then subjected to SeV infection. The luminescent signal was assessed after a 12-hr period utilizing a dual-luciferase detection method; (e) HEK293T cells transfected with an empty vector and a vector encoding VP1 protein for 24 hr, then infected with VSV–GFP and poly(I:C) for 12 hr.

Figure 2. The PTV VP1 protein inhibits NF-κB activation: (a) HEK293T cells underwent co-transfection with 500 ng and 1,000 ng of the NF-κB reporter, pRL-TK, and VP1 plasmids, respectively. Following transfection, the cells were either infected with SeV. After 12 hr incubation, luciferase activity was quantified using a dual-luciferase assay; (b and c) ST cells were subjected to transfection with the VP1 expression plasmid and an empty vector 24 hr, followed by infection with SeV for 0, 6, and 12 hr. The collected cells were subjected to Western blot to assess the protein levels of p-NF-κB and NF-κB: (b) following a 12 hr infection with SeV, the abundance of NF-κB in the cytoplasmic and nuclear fractions was assessed using western blot analysis, (c) GAPDH and LaminB1 as markers for cytoplasmic and nuclear fractions, respectively. Whole-cell lysate—WCL; (d) ST cells transfected with the plasmids as indicated for 24 hr, then infected with SeV for 12 hr. The subcellular localizations of His-NF-κB (red), HA-VP1 (green), and the nuclear marker DAPI (blue) were determined by immunofluorescence staining.

Figure 3. The PTV VP1 protein targets MDA5 and suppresses IFN-β production: (a–d) effects of VP1 on components of the RLR pathway. ST cells were transfected with various plasmids, including RIG-1, MDA5, MAVS, or TBK1, along with a control vector or a VP1-expressing plasmid. After 24 hr, the IFN-β activity was quantified using qPCR.

Figure 4. The PTV VP1 inhibits MDA5 function by directly binding to MDA5: (a) the capacity of VP1 to hinder MDA5-triggered IFN-β production was evaluated by transfecting ST cells with MDA5 and a vector expressing VP1, subsequently exposed to poly(I:C) treatment duration of 12 hr; (b) The impact of VP1 on the activation of the IFN-β promoter mediated by MDA5 was studied in HEK293T cells transfected with the IFN-β reporter, pRL-TK, and MDA5 along with a control vector or a VP1-expressing vector, followed by measurement of IFN-β activities employing a dual-luciferase reporter-based assay after 36 hr; (c) The Co-IP assay was performed to validate the interaction between VP1 and MDA5 in HEK293T cells. This involved cotransfecting His-tagged MDA5 and HA-VP1 into the cells. After 36 hr, the cell lysates were utilized for immunoprecipitation employing either anti-His or anti-HA mouse monoclonal antibodies, followed by immunoblotting using anti-His, anti-HA, and anti-GAPDH mouse monoclonal antibodies; (d) colocalization of MDA5 with VP1 was observed in ST cells through confocal microscopy analysis. Representative images of His-MDA5 colocalization with HA-VP1 were captured, and the intensity of each…

Figure 5. The PTV VP1 inhibits MDA5 function by direct binding of the 2CARD and Hel domain to MDA5: (a) a diagrammatic illustration of the distinct domains of MDA5 is displayed; (b and c) the MDA5 domain that interacts with VP1 is identified through CO-IP assays. HEK293T cells were simultaneously transfected with HA-tagged VP1 and constructs encoding either Flag-tagged 2CARD, Hel, or CTD, together with an empty vector. After transfection, the lysates were analyzed by immunoblotting using antibodies specific to either Flag or HA tags; (d) the inhibitory effects of VP1 on the induction of IFN-β facilitated by 2CARD, Hel, or CTD were assessed. HEK293T cells were subjected to transfection with the IFN-β-Luc and pRL-TK, along with a control vector or a VP1, 2CARD, Hel, and CTD plasmids. Luciferase signals were quantified employing a dual-luciferase reporter assay.

Figure 6. Model depicting PTV structural protein VP1-mediated viral suppression by blocking MDA5 and NF-κB activation during PTV infection. VP1 exhibits the ability to suppress the expression of IFN-β by obstructing the activation of NF-κB. The binding domain localization demonstrates the interaction between VP1 and the CARD and Hel domains of MDA5, potentially reducing its RNA ligand-binding capability. Pointed arrows represent activation, while blunt-ended lines indicate inhibition (created using https://www.biorender.com).