Fig 1: BVDV infection promotes the formation of the HK2-MAVS-VDAC1 complex to inhibit RLR-MAVS pathway activation. (A) Validation of plasmid expression for RIG-I, MAVS, HK2, and VDAC1 by western blot using protein-specific antibodies. (B) Co-IP analysis showing HK2-mediated inhibition of RIG-I/MAVS interaction in BT cells co-transfected with Myc-HK2, HA-RIG-I, and Flag-MAVS. (C) Co-IP analysis of VDAC1-mediated inhibition of RIG-I/MAVS interaction in BT cells co-transfected with Myc-VDAC1, HA-RIG-I and Flag-MAVS. (D) Disruption of the HK2/MAVS interaction upon poly(I:C) treatment, as assessed by Co-IP. (E) Disruption of the VDAC1/MAVS interaction upon poly(I:C) treatment, as assessed by Co-IP. (F) Suppression of endogenous RIG-I/MAVS interaction by the BVDV-induced HK2-MAVS-VDAC1 complex, analyzed by Co-IP. (G) Disruption of poly(I:C)-mediated endogenous RIG-I/MAVS interaction by the BVDV-induced HK2-MAVS-VDAC1 complex, analyzed by Co-IP. (H) Protein levels of TBK1, p-TBK1, IRF3, and p-IRF3 in HK2-overexpressing BT cells following poly(I:C) transfection. (I) Protein levels of TBK1, p-TBK1, IRF3, and p-IRF3 in VDAC1-overexpressing MDBK cells following poly(I:C) transfection. Data are representative of three independent experiments and are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.
Fig 2: Inhibition of the HK2-MAVS-VDAC1 complex formation enhances RLR signaling pathway-mediated IFN-β production. (A) Knockdown efficiency of HK2-targeting siRNAs as determined by qRT-PCR and western blot. (B) Knockdown efficiency of VDAC1-targeting siRNAs as determined by qRT-PCR and western blot. (C) Enhanced endogenous RIG-I/MAVS interaction in HK2-knockdown MDBK cells analyzed by CO-IP. (D) Enhanced endogenous RIG-I/MAVS interaction in VDAC1-knockdown MDBK cells analyzed by CO-IP. (E) Upregulation of IFN-β mRNA expression in HK2-knockdown MDBK cells measured by qRT-PCR. (F) Upregulation of IFN-β mRNA expression and protein secretion in VDAC1-knockdown MDBK cells measured by qRT-PCR. (G) With siRNA-mediated knockdown of VDAC1, the expression levels of interferon-stimulated genes (ISG20, ISG15, IFITM1, IFITM3, OAS1, and MX1) in poly I:C-pretreated MDBK cells upon BVDV infection, as measured by qRT-PCR. Data are representative of three independent experiments and are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.
Fig 3: Lactate suppressed the RLR signaling pathway by directly binding to MAVS. (A) Interaction between lactate and MAVS assessed by biotin-labeled lactate pull-down assay. (B) Lactate-induced translocation of MAVS from mitochondria to cytoplasm, as detected by western blot. (C) Co-IP analysis of the endogenous RIG-I–MAVS interaction in SO-treated MDBK cells upon BVDV infection, with or without exogenous lactate supplementation. (D) Co-IP analysis of the endogenous RIG-I–MAVS interaction in LDHA-knockdown cells upon BVDV infection, with or without adding exogenous lactate supplementation. (E) Western blot analysis of IRF3 phosphorylation and nuclear translocation following lactate treatment. (F–G) IFN-β mRNA expression (F) and protein secretion (G) in poly I:C-pretreated MDBK cells upon BVDV infection, with exogenous lactate supplementation. (H) Expression levels of interferon-stimulated genes (ISG20, ISG15, IFITM1, IFITM3, OAS1, and MX1) under the same conditions as in panel F. Data are representative of three independent experiments and are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant.
Fig 4: Schematic illustration of the mechanism by which BVDV infection inhibits the RIG-I/MAVS-mediated IFN-I production via the ROS–HIF-1α–glycolysis axis.
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