FIG 4.

Lpro deISGylase activity during FMDV infection of porcine cells. (A) LFPKαVβ6 cells were mock transfected or transfected with either GFP or the ISG15 machinery (DDK-ISG15, E1 [UBE1L], E2 [UbcH8], and E3 [HERC5]). At 24 h posttransfection, proteins were resolved by SDS-PAGE and Western blot was applied to detect DDK-tagged free or conjugated ISG15 and UbcH8. (B) LFPKαVβ6 transfected as indicated in A were infected with FMDV A12WT, LLV, or LproW105A. At 6 hpi, cell lysates were prepared, and proteins were resolved in 3% to 8% SDS-PAGE, followed by Western blotting to detect DDK-tagged free or conjugated ISG15 and UbcH8, FMDV VP1, eIF4G, and GAPDH. (C) DUB activity of A12-WT, A12-LLV, and A12 Lpro W105A virus. LFPKαVβ6 cells were transfected with an HA-Ub expression plasmid, and 24 h posttransfection, cells were infected with the indicated viruses. Cells were lysed 4.5 h postinfection and analyzed by Western blotting to detect HA-tagged Ub conjugates, FMDV VP1, and tubulin. (D) LFPKαVβ6 cells were mock infected or infected with either A12-WT, A12-LLV, or A12-LproW105A virus, and lysates were collected at 2, 4, 6, and 24 hpi. Proteins were resolved by SDS-PAGE, and Western blot was applied to detect FMDV VP1, eIF4G, G3BP1, and tubulin. For each figure, one representative blot is shown out of three independent experiments.