Degradation of Host Proteins and Apoptosis Induced by Foot-and-Mouth Disease Virus 3C Protease

Figure 1. FMDV 3C degrades host proteins involved in NF-κB signaling. (A–C) HEK293T cells were transfected with empty vectors or Flag-3C-expressing plasmids (1 µg) together with indicated HA-tagged adaptors expressing plasmids (2 µg). After 24 h, total protein extracts were collected and analyzed by western blotting using antibodies specific to Flag, HA, or β-actin. WB, western blotting.

Figure 2. FMDV 3C degrades host proteins involved in NF-κB signaling in a dose-dependent manner. (A–C) Indicated HA-tagged adaptors expressing plasmids (2 μg) and increasing amounts of Flag-3C-expressing plasmids (0, 0.5, and 1.0 μg) were co-transfected into HEK293T cells. After 24 h, total protein extracts were collected and analyzed by western blotting using antibodies specific to Flag, HA, or β-actin. WB, western blotting.

Figure 3. The protease activity of FMDV 3C is involved in the downregulation of host proteins involved in NF-κB signaling. (A–C) HEK293T cells were transfected with empty vectors, wild type 3C, or 3C mutants (H46Y, D84N, C163G, H205R) (1 μg) together with indicated adaptors (2 μg). After 24 h, total protein extracts were collected and analyzed by western blotting using antibodies specific to Flag, HA, or β-actin. WB, western blotting.

Figure 4. Expression of FMDV 3C induces cell apoptosis. (A) HEK293T cells were transfected with the indicated plasmids for 24 h or treated with apoptosis inducers for 12 h. Cells were visualized using a Laser scanning confocal microscope Leica TCS SP8 (Leica Microsystems, Wetzlar, Germany), 400× magnification. (B) A cell population with AbFlourTM 488 annexin V and PI staining. HEK293T cells were transfected with the indicated plasmids for 48 h or treated with apoptosis inducers for 24 h. Cells were collected for analysis of the percentage of cells with apoptotic characteristics by flow cytometry. (C) Analysis of apoptosis occurrence in HEK293T cells from (B). (D) PK-15 cells were transfected with the indicated plasmids for 24 h. Expression levels of CASP3 and CASP8 were assessed by RT-qPCR. The results are presented as relative fold changes in mRNA levels compared to mock cells. The results represent the average of three replicates. Error bars represent SEM; *: p < 0.05; **: p < 0.01; ***: p < 0.001, compared to mock cells.

Figure 5. Apoptosis inducers cause similar degradation of host proteins involved in NF-κB signaling as that induced by FMDV 3C. (A) Cytotoxic effect in HEK293T cells transfected with the indicated plasmids were analyzed by CCK8 assay. The results represent the average of three replicates. Error bars represent SEM; ns, no significance (p > 0.05), compared to mock cells. (B–D) HEK293T cells were transfected with the indicated adaptors (2 μg) for 16 h and then were treated with apoptosis inducers for 6 h. Total protein extracts were collected and analyzed by western blotting with the indicated antibodies.

Figure 6. CrmA abolishes the ability of 3C to downregulate host proteins involved in NF-κB signaling and inhibits FMDV replication. (A–D) HEK293T cells were transfected with empty vectors or CrmA (1 μg) together with 3C (100 ng) and the indicated adaptors (2 μg). After 24 h, total protein extracts were collected and analyzed by western blotting using antibodies specific to Flag, HA, or β-actin. (E) PK-15 cells were transfected with empty vectors or CrmA in increasing dosage for 24 h and were infected with FMDV (6 or 12 h; MOI = 0.1). Genomic copy numbers of FMDV were quantified using the quantitative RT-qPCR assay. The results represent the average of three replicates. Error bars represent SEM; *: p < 0.05, compared to mock cells.

Figure 7. 3C induces the degradation of host proteins involved in NF-κB signaling, independent of the activity of caspase3, caspase8, and caspase9. (A–C) Caspase activity was assessed after treatment with specific caspase inhibitors. The results represent the average of three replicates. Error bars represent SEM; *: p < 0.05; **: p < 0.01; ***: p < 0.001, compared to mock cells. (D–L) HEK293T cells (1 × 106) were treated with DMSO, Z-DEVD-FMK, Z-IETD-FMK, or Z-LEHD-FMK (20 mM) for 2 h. Then, the indicated expression plasmids of host proteins (2 μg) and 3C or empty vectors (100 ng) were co-transfected for 24 h. Total protein extracts were collected and analyzed by western blotting using antibodies specific to Flag, HA, or β-actin.