Foot-and-mouth disease virus activates glycolysis and hijacks HK2 to inhibit innate immunity and promote viral replication

Fig. 1. FMDV triggered glycolysis and HK2 expression. A PK-15 cells were infected with 0.5 MOI FMDV for 12 h, and the ECAR was monitored with a fluorescence microplate reader. B ELISA was used to measure the concentration of lactic acid. The error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (t tests). (C and D) qRT‒PCR analysis of the relative mRNA expression of HK2 and 3Dpol in FMDV-infected PK-15 cells at different times (0 h, 3 h, 6 h and 12 h). The error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (D) HK2 and FMDV VP1 protein expression in FMDV-infected PK-15 cells at different times, as determined by western blotting.

Fig. 2. HK2 enhanced glycolysis and FMDV replication in PK-15 cells. A PK-15 cells overexpressing the pCAGGS-HA or pCAGGS-HA-HK2 plasmid were infected with FMDV for 12 h, and the lactic acid content in the cells was measured by ELISA. The error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (t tests). B ELISA was used to measure the concentrations of ATP in FMDV-infected PK-15 cells overexpressing the pCAGGS-HA or pCAGGS-HA-HK2 plasmid. The error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (t tests). C PK-15 cells pretreated with the pCAGGS-HA plasmid, pCAGGS-HA-HK2 plasmid or 2-DG (50 mmol/L) were infected with FMDV for 12 h, and the ECAR levels were monitored with a fluorescence microplate reader. D–F PK-15 cells were treated with 50 mmol/L 2-DG for 4 h and then infected with FMDV for 6 h and 12 h. FMDV VP1 protein expression (D), FMDV 3Dpol mRNA expression (E) and virus titres (F) were measured by western blot, qRT‒PCR and TCID50 survey, respectively. The error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001…

Fig. 3. Interaction of HK2 with the FMDV VP3 protein. A PK-15 cells were co-transfected with pCAGGS-HA-HK2 and different FMDV protein expression plasmids (pEGFP-VP1, pEGFP-VP3, pEGFP-VP4 and pEGFP-2B) for 24 h, fixed, stained with DAPI (blue) and antibodies against GFP (green) and HA (red), and then examined by confocal microscopy. B pCAGGS-HA-HK2 and pEGFP-VP3 were transfected into 293T cells together. At 24 h after transfection, the cell lysates were immunoprecipitated with anti-GFP agarose beads. The bound proteins were eluted and detected by western blotting with anti-HA and anti-FLAG monoclonal antibodies. C PK-15 cells were transfected with 0 μg, 1 μg, 2 μg or 3 μg of pCAGGS-HA-HK2 together with pEGFP-VP3 for 24 h, and the expression of HK2 and VP3 was tested by western blotting.

Fig. 4. HK2 enhances FMDV replication through autophagy. A PK-15 cells were infected with FMDV for 12 h, and the number of endogenous LC3 puncta was determined by confocal microscopy. B PK-15 cells were transfected with dual fluorescence-tagged mRFP-eGFP-LC3 for 24 h, followed by FMDV infection for 12 h. Changes in the fluorescence of mRFP-eGFP-LC3 were observed by confocal microscopy. C PK-15 cells were transfected with pEGFP-C1 or pEGFP-HK2 for 24 h, followed by FMDV infection for 12 h. The number of endogenous LC3 puncta was determined by confocal microscopy. D PK-15 cells were co-transfected with dual fluorescence-tagged mRFP-eGFP-LC3 and pCAGGS-HA or with dual fluorescence-tagged mRFP-eGFP-LC3 and pCAGGS-HA-HK2 for 24 h, followed by FMDV infection for 12 h. Changes in the fluorescence of mRFP-eGFP-LC3 were observed by confocal microscopy. E and F PK-15 cells overexpressing pCAGGS-HA or pCAGGS-HA-HK2 were treated or not treated with bafilomycin A1 or CQ for 4 h and then infected with FMDV for 12 h. Western blotting was used to evaluate the protein expression of HA, VP1, P62, LC3B and β-actin (E), and FMDV 3Dpol mRNA levels were measured by qRT‒PCR (F).

Fig. 5. HK2 promoted FMDV replication by suppressing innate cellular immunity through autophagy. A–D PK-15 cells overexpressing pCAGGS-HA or pCAGGS-HA-HK2 were infected with or not infected with FMDV for 12 h. The mRNA levels of IFN-β, IL-6, ISG15 and ISG56 in the cells were measured by qRT-PCR. E–H PK-15 cells overexpressing pCAGGS-HA or pCAGGS-HA-HK2 were treated with bafilomycin A1 and CQ for 4 h, followed by FMDV infection for 12 h. The mRNA levels of IFN-β, IL-6, ISG15 and ISG56 in the cells were measured via qRT‒PCR. The error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA).

Fig. 6. HK2 interacted with IRF3 and IRF7 and inhibited their expression. A The targets closely related to HK2 were predicted by BioGRID database. B and C PK-15 cells were co-transfected with pEGFP-HK2 and pCAGGS-HA-IRF3/IRF7, pEGFP-HK2 and pCAGGS-HA, pEGFP-C1 and pCAGGS-HA-IRF3/IRF7 or pEGFP-C1 and pCAGGS-HA for 24 h, fixed, stained with DAPI (blue) and antibodies against GFP (green) and HA (red), and then examined by confocal microscopy. D and E HEK-293T cells were co-transfected with pEGFP-HK2 and pCAGGS-HA-IRF3/IRF7, pEGFP-HK2 and pCAGGS-HA, pEGFP-C1 and pCAGGS-HA-IRF3/IRF7 or pEGFP-C1 and pCAGGS-HA for 24 h. The bound proteins were eluted and detected by western blotting with anti-HA and anti-GFP monoclonal antibodies. (F and G) 0 μg, 1 μg, 2 μg and 3 μg of pEGFP-HK2 were transfected into PK-15 cells together with 1 μg of pCAGGS-HA-IRF3/IRF7. Western blotting was used to evaluate the protein expression of GFP, HA and β-actin. (H) 0 μg, 1 μg, 2 μg and 3 μg of pEGFP-HK2 were transfected into PK-15 cells, and the protein expression of GFP, IRF3, IRF7, and β-actin was detected by western blotting.

Fig. 7. HK2 utilizes autophagy to degrade IRF3/IRF7 during FMDV infection. A PK-15 cells were co-transfected with pEGFP-HK2 and pCAGGS-HA-IRF3/IRF7 and pRFP-LC3 or with pEGFP-C1 and pCAGGS-HA-IRF3/IRF7 and pRFP-LC3 for 24 h, infected with FMDV for 12 h, fixed, stained with antibodies against HA (blue), GFP (green) and RFP (red), and then examined by confocal microscopy. B and C PK-15 cells were pretreated with the autophagy activator rapamycin (100 nM) or the inhibitors CQ (20 μM) and bafilomycin A1 (0.4 μM) for 4 h and then co-transfected with pEGFP-HK2 and pCAGGS-HA-IRF3/IRF7 for 24 h, followed by FMDV infection for 12 h. Western blotting was used to evaluate the protein expression of GFP, HA, VP1, p62, LC3B and β-actin. D PK-15 cells were transfected with pEGFP-HK2 after 4 h of pretreatment with the autophagy activator rapamycin (100 nM) or the inhibitors CQ (20 μM) and bafilomycin A1 (0.4 μM). The protein expression of GFP, IRF3, IRF7, VP1, p62, LC3B and β-actin was detected by western blotting. E PK-15 cells were co-transfected with pEGFP-HK2 and Flag-p62, EGFP-C1 and Flag-p62, EGFP-HK2 and p3 × Flag-CMV, or EGFP-C1 and p3 × Flag-CMV for 24 h, fixed, stained with DAPI (blue) and…