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. 2018 Apr 26;9:817. doi: 10.3389/fmicb.2018.00817

FIGURE 1.

FIGURE 1

Non-structural 3C protease binds caspase-8 and caspase-9. (A) Cell morphologic analysis and nuclear morphologic analysis were performed in HepG2 cells because they are easy to transfect. Analyses were performed 36 h after transfection with 4 μg of pEGFP-3C (3C) or the corresponding control vector pEGFP (Vector). Bar = 20 μm. (B) The expression and activity of caspase-3, caspase-8, and caspase-9 proteins after transfection of HepG2 cells with VR1012-3C-HA or the corresponding control vector VR1012-HA (Vec) was assessed at 36 h by Western blot analysis (top) and by activity assay (lower). Histone is shown as a loading control. Luminescence values in each cell were calculated and normalized to 1.0 in vector-transfected cells. The results indicate the means ± SD of three independent experiments. ∗∗∗P < 0.001. (C) Immunoprecipitation of caspases with viral protein 3C. HepG2 cells were collected 36 h after transfection with 10 μg of VR1012-3C-HA (3C) or the corresponding control vector VR1012-HA (Vec). Anti-HA affinity matrix was used for immunoprecipitation of HA-tagged proteins, which were analyzed by Western blotting for caspase-3, caspase-8, caspase-9, and HA (for input). The results are representative of three independent experiments. (D) Caspase-8, caspase-9, and caspase-3 activity were analyzed by each activity assay after caspase-8 inhibitor and caspase-9 inhibitor treatment, respectively. Luminescence values in each group were calculated and normalized to 1.0 in VR1012-transfected and control-treated cells (Vec). The results indicate the means ± SD of three independent experiments. ∗∗∗P < 0.001. (E) Caspase-8, caspase-9, and caspase-3 activity were analyzed by each activity assay after 3C and 3C mutant transfection. Luminescence values in each group were calculated and normalized to 1.0 in VR1012-transfected cells (Vec). The results indicate the means ± SD of three independent experiments. ∗∗∗P < 0.001. (F) Immunoprecipitation of caspases with viral protein 3C or 3C mutant with loss of proteolytic activity. HepG2 cells were collected 36 h after transfection with 10 μg of VR1012-3C-HA (3C), VR1012-Cys147Glu-3C-HA (3C-M) or the corresponding control vector VR1012-HA (Vec). Anti-HA affinity matrix was used for immunoprecipitation of HA-tagged proteins, which were analyzed by Western blotting for caspase-3, caspase-8, caspase-9, and HA (for input). Cas-3, caspase-3; Cas-8, caspase-8; Cas-9, caspase-9; His, histone.