Fig 1.
Effect of the FMDV leader protease on LGP2.
(A) HEK293 cells were mock-transfected (-) or co-transfected with a plasmid encoding hLGP2-Myc-DDK and empty vector (EV), LbWT or LbC51A. Cells were lysed 24 h later and analyzed by western blot for the indicated proteins with the specified antibodies. The N- and C-terminal cleavage products of LGP2 are indicated with arrows. (B) HEK293 cells were mock-transfected (-), transfected with EV, or with plasmids encoding LbWT or LbC51A and lysed 24 h later (left and middle panels); BHK-21 cells were mock-infected (-) or infected with FMDV O1BFS at an MOI of 5 and lysed 5 h later (right panel). Lysates were analyzed by western blot for detection of eIF4G cleavage products (arrowhead). (C) HEK293 cells were mock-transfected (-) or co-transfected with 500ng of a plasmid encoding DDK-METTL3 and 500ng of empty vector (EV), LbWT or LbC51A.
Fig 2.
Expression of catalytically active FMDV Lbpro induces LGP2 cleavage.
HEK293 cells were co-transfected with a plasmid encoding hLGP2-Myc-DDK and (A) LbWT or EV and lysed at the indicated times after transfection, (B) EV or increasing amounts of a plasmid encoding LbWT (0, 0.2, 2, 20, 200 or 2000 ng) and lysed 24 h later. (C) HEK293 cells were co-transfected with plasmids encoding hLGP2-Myc-DDK and LbWT in the presence of zVAD or MG132 caspase and proteasome inhibitors, respectively. In control cells, apoptosis was induced with puromycin. Cells were lysed 24 h later. (D) HEK293 cells were co-transfected with plasmids encoding DDK-poLGP2 and LbWT, LbC51A, EV or water (-). Control cells were co-transfected with plasmids encoding hLGP2-Myc-DDK and LbWT. (E) HEK293 cells were co-transfected with plasmids encoding DDK-poLGP2 and LbWT, LbC51A or EV and lysed at the indicated times after transfection. (F) HEK293 cells were co-transfected with plasmids encoding hLGP2-Myc-DDK or DDK-poLGP2 and LbWT and lysed 24 h later. (G) HEK293 cells were co-transfected with plasmids encoding DDK-poLGP2-Myc and LbWT or LbC51A and lysed 24 h later. (H) Porcine SK6 cells were mock-transfected (-) or co-transfected with plasmids encoding DDK-poLGP2 and LbWT, LbC51A or EV and lysed 24 h later. Cell lysates were analyzed by western blot for the indicated proteins using the specified antibodies. The N- and C-terminal cleavage products of LGP2 are indicated with arrows. A putative C-terminal cleavage product of poLGP2 harboring the Myc tag is indicated by an asterisk.
Fig 3.
Interaction and cellular co-localization of Lbpro and LGP2.
(A) SK6 cells were co-transfected with the indicated plasmids and lysed 24 h later. Lysates were subjected to IP and analyzed by western blot. The N-terminal cleavage product of LGP2 is indicated with an arrow. HC denotes the 50 KDa IgG heavy chain band. (B) Confocal microscopy images of BHK-21 cells at 20 h after co-transfection with plasmids encoding hLGP2-Myc-DDK and FMDV LbWT or LbC51A mutant. Control cells transfected with individual plasmids are shown (bottom). Primary antibodies used for LGP2 and Lb detection were a monoclonal anti-FLAG and a polyclonal anti-Lpro, respectively. Co-localization of LGP2 (green) and Lb (red) was assessed by histogram profiles of merged images. Nuclei were stained with DAPI. Scale bars, 10 μm.
Fig 4.
FMDV infection induces LGP2 cleavage.
SK6 cells were transfected with a plasmid encoding (A) hLGP2-Myc-DDK or (B) DDK-poLGP2 and 24 h later infected with type-O or type-C FMDV isolates at an MOI of 5. Cells were lysed at different times after infection and analyzed by western blot using the indicated antibodies. Viral titers in the supernatants of transfected/infected cells at each time point are depicted. The N- and C-terminal cleavage products of LGP2 are indicated with arrows.
Fig 5.
Effect of Lbpro and LGP2 co-expression on type-I IFN responses against FMDV infection.
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.
Fig 6.
Mutation of the putative target sequence in LGP2 abolishes cleavage by Lbpro.
(A) Schematic representation of LGP2 showing the three subdomains in the conserved DExD/H helicase domain (Hel1, Hel2i and Hel2) and the C-terminal domain (CTD). The position and sequence of the conserved helicase motif VI (in red) and surrounding residues in the human and porcine proteins are indicated. Amino acid substitutions in the mutant version of hLGP2 (hLGP2MT) compared with WT sequence are indicated in the corresponding positions. (B) HEK293 cells were transfected with a plasmid encoding hLGP2-Myc-DDK (hLGP2WT) or its mutant version as indicated above (hLGP2MT) either alone or together with a plasmid encoding LbWT. The amount of DNA in all transfections was balanced with EV (-). Cell lysates were analyzed 24 h later by western blot for the indicated proteins using the specified antibodies. The N- and C-terminal cleavage products of LGP2 are indicated with arrows.