Innate immune sensor LGP2 is cleaved by the Leader protease of foot-and-mouth disease virus
SK6 cells were co-transfected with a plasmid encoding DDK-poLGP2 or EV (2 μg) together with plasmids encoding LbWT, LbC51A or EV (1 μg). After 24 h, cells were infected with FMDV CS8 isolate at an MOI of 5. Supernatants were collected and cells lysed 8 h after infection. (A) Viral titers in supernatants were determined on IBRS2 cells. Data as mean ± SD of triplicates (n = 3). Cell lysates were analyzed by western blot for the indicated proteins. The N-terminal cleavage product of LGP2 is indicated with arrows. Bands corresponding to full-length eIF4G and the 110 KDa C-terminal cleavage fragment generated by Lpro are indicated. A minor band of slightly faster migration than p110 is observed in SK6 cells lysates and marked with an asterisk. (B) The fold induction of porcine IFN-β mRNA in cell lysates was determined by RT-qPCR normalized to GAPDH. Data as mean ± SD (n = 4). (C) IFN bioassay of supernatants. Antiviral activity is expressed as the reciprocal of the highest dilution needed to reduce the number of VSV plaques on IBRS-2 cells by 50%. When indicated, supernatants were previously treated with a monoclonal antibody anti-IFN-α. A representative IFN bioassay is shown. Student’s t test; *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.