Fig 1.
SVA infection induces glycolysis in PK-15 cells.
(A, B) PK-15 cells were infected with SVA at an MOI of 1. Cell lysates were collected at 48 h.p.i. and the lactate and intracellular ATP levels were measured. (C) PK-15 cells were mock infected or infected with Heat-SVA and SVA at MOIs of 0.1 or 1. Cells were harvested at 48 h.p.i. The mRNA expression of HK2, PFKM, PGK1, PKM, HIF-1α, and SOD2 was analyzed by qRT-PCR. (D) Cells were infected with SVA at an MOI of 1 and lysed in RIPA buffer at 48 h.p.i. The expression levels of PGK1, PKM, and VP2 were analyzed by western blot. (E) The grayscale analysis of PGK1, PKM, and VP2 protein. All data represent the means ± SD (Student’s t test) (*P < 0.05, **P < 0.01, ns, not significant).
Fig 2.
Enhanced glycolysis promotes replication of SVA.
PK-15 cells were transfected with pEGFP-PDK3, pEGFP-PGK1, pEGFP-PKM, pEGFP-HIF-1α or co-transfected with all four vectors. Cells were infected with SVA at an MOI of 1. (A-E) The RNA of the cells was harvested and extracted at 6, 12 or 24 h.p.i, and intracellular mRNA levels of the SVA VP2 gene were analyzed by qRT-PCR. β-actin expression was used as an internal control. (F) Virus titers in PK-15 cells were detected using the Reed–Muench method. All data represent the means ± SD (Student’s t test) (*P < 0.05, **P < 0.01, ns, not significant).
Fig 3.
Inhibition of glycolysis inhibits replication of SVA.
(A) Schematic overview of glucose metabolism and the functional targets of glycolytic inhibitors (2DG, oxamate, and DCA) used in this study. (B) PK-15 cells were infected with SVA at an MOI of 1, incubated in the presence or absence of glycolytic inhibitors, and intracellular mRNA levels of the SVA VP2 gene were analyzed by qRT-PCR at 48 h.p.i. (C) Cells were infected with SVA at an MOI of 1, incubated in the presence or absence of glycolytic inhibitors, and lysed with RIPA at 48 h.p.i. The VP2 protein levels were detected by western blot. (D) The grayscale analysis of the VP2 protein. (E) PK-15 cells were infected with SVA at an MOI of 1, incubated in high (25 mM) or low (5 mM) glucose, and intracellular mRNA levels of SVA VP2 gene were measured by qRT-PCR. (F) Cells were infected with SVA at an MOI of 1, incubated in high (25 mM) or low (5 mM) glucose and lysed with RIPA at 48 h.p.i. VP2 protein levels were determined by western blot. (G) The grayscale analysis of VP2 protein. All data represent the means ± SD (Student’s t test) (*P < 0.05, **P < 0.01, ns, not significant).
Fig 4.
Glycolysis induced by SVA infection inhibits RLR signaling.
(A-F) PK-15 cells were mock infected or infected with SVA at an MOI of 1 and incubated in the presence or absence of 2DG, DCA, or oxamate. Cells were harvested at 48 h.p.i. The mRNA expression of RIG-I, IFNβ, IFNα, IFIT1, IL-6, and ISG-15 were analyzed by qRT-PCR. (G-L) PK-15 cells were treated with high (25 mM) or low (5 mM) glucose and infected with SVA at an MOI of 1. Cells were harvested at 48 h.p.i. The mRNA expression of RIG-I, IFNβ, IFNα, IFIT1, IL-6, and ISG-15 were analyzed by qRT-PCR. β-actin expression was used as an internal control. All data represent the means ± SD (Student’s t test) (*P < 0.05, **P < 0.01, ns, not significant).
Fig 5.
Lactate produced by SVA-induced glycolysis inhibits RLR signaling.
(A-F) PK-15 cells were incubated in the presence or absence of sodium oxamate (10 or 25 mM), in the presence or absence of sodium lactate (5 or 10 mM), and with or without SVA infection at an MOI of 1. (A, B) The expression levels of the SVA VP2 gene were analyzed by qRT-PCR at 48 h.p.i. (C) The intracellular lactate levels were measured using lactate assay kits. (D) VP2 protein levels were measured by western blot. (E) The expression levels of the SVA VP2 gene were analyzed by qRT-PCR at 48 h.p.i. (F) Virus titers in PK-15 cells were detected using the Reed–Muench method. (G-L) The mRNA expression of RIG-I, IFNβ, IFNα, ISG-15, IL-6, and IFIT1 was analyzed by qRT-PCR. β-actin expression was used as an internal control. All data represent the means ± SD (Student’s t test) (*P < 0.05, **P < 0.01, ns, not significant).
Fig 6.
Lactate produced by SVA infection inhibits RLR signaling mainly through RIG-I.
(A, B) Lactate inhibits RLR signaling by destabilizing the interaction between MAVS and RIG-I. (C) Expression levels of RIG-I in PK-15 cells and RIG-I-KO PK-15 cells. (D-F) The mRNA expression of IFNβ, ISG15, IFNα, and IL-6 was analyzed by qRT-PCR. All data represent the means ± SD (Student’s t test) (*P < 0.05, **P < 0.01, ns, not significant).
Fig 7.
RLR signaling was inhibited by SVA through lactate in mice.
(A) Mice were orally challenged with SVA, treated with high (1.5 g/kg), low (0.2 g/kg), or no (control) glucose, and treated with or without sodium oxamate (750 mg/kg) or sodium lactate (1 g/kg). (B) At 7 d.p.i, the expression of the SVA VP2 gene in the heart, liver, spleen, duodenum, and kidney was analyzed by qRT-PCR. (C) At 7 d.p.i, the serum lactate was measured using lactate assay kits. (D) The expression of PGK1, PKM, and HIF-1α in mouse heart, liver, and spleen tissues was detected by western blot. (E) Grayscale analysis of detected proteins. (F-Q) The expression of the SVA VP2 gene, IFNβ, IFNα, ISG15, RIG-I, and IL-6 was analyzed by qRT-PCR. All data represent the means ± SD (Student’s t test) (*P < 0.05, **P < 0.01, ns, not significant).
Fig 8.
Histopathological changes of heart, liver, spleen, kidney and duodenum in mice.
Fig 9.
Schematic overview of SVA-induced glycolysis that facilitates virus replication by promoting lactate production, which attenuates the interaction between MAVS and RIG-I.