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. 2019 Oct 29;93(22):e01273-19. doi: 10.1128/JVI.01273-19

FIG 8.

FIG 8

WRN is a substrate of HCV NS3/4A protease. (A) An NS3/4A-dependent cleavage of WRN protein. A WRN cleavage assay was performed by cotransfecting 293T cells with plasmids encoding NS3/4A-V5His or NS3pd/4A-V5His and an EGFP-WRN fusion protein (approximately 210 kDa) with myc and His tags at the C terminus. Twenty-four hours posttransfection, the cells were treated with MG132 for 24 h and harvested for Western blot analysis. The cleavage assay in HCVR and control Huh7 cells was also analyzed 72 h after transfection of the plasmid encoding the EGFP-WRN fusion protein. (B) WRN cleavage in cells expressing NS3/4A protease and the WRN mutant protein EGFP-WRN(ARm)-mycHis. (C) NHEJ assay in TR-NS3/4A and TR-NS3pd/4A cells. The overall and precise NHEJ efficiencies in TR-NS3/4A and TR-NS3pd/4A cells were measured by luciferase assay. The luminescence value of firefly luciferase was normalized against the internal EGFP fluorescence control. Statistical analysis is shown (n = 3). P values define significant differences between the NHEJ efficiencies of TR-NS3/4A and TR-NS3pd/4A cells. **, P < 0.01; ***, P < 0.001.