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. 2015 Feb 16;13(1):94–102. doi: 10.1038/cmi.2014.131

Figure 5.

Figure 5

TRIM22 is an ubiquitin ligase of NS5A. (a) TRIM22 specifically degrades NS5A. 293T cells are transfected. HCV proteins are transfected together with TRIM22 and ubiquitin. Western blotting was performed 48 h after plasmid transfection. (b) Exogenous NS5A: Huh-7 cells transfected with HA-NS5A plasmid (100 ng/well in a 24-well plate) were transfected with FLAG-TRIM22 (upper left: 0, 200 and 1000 ng/well in a 24-well plate) and HIS-Ubiquitin (40 ng/well in a 24-well plate) plasmids or treated with IFNα (upper right: IFNα 0, 50 and 100 IU/ml triggered 24 h after HA-NS5A plasmid transfection). After 48 h, samples were collected for western blotting. TRIM22 degraded NS5A (100 ng/well in a 24-well plate) in a dose-dependent manner. Endogenous NS5A: Huh-7-Con-1 replicon cells were transfected with FLAG-TRIM22 (lower left: 0, 200 and 1000 ng/well in a 24-well plate) and HIS-Ubiquitin (40 ng/well in a 24-well plate) or treated with IFNα (lower right: 50 IU/ml). Samples were detected by western blotting 48 h after plasmid transfection or IFNα treatment. TRIM22 degraded endogenous NS5A (100 ng/well in a 24-well plate) in a dose-dependent manner. (c) Upper panel: FLAG-TRIM22 (1.5 µg/well, six-well plate), HA-NS5A (1.5 µg/well, six-well plate) and HIS-Ubiquitin (0.3 µg/well, six-well plate) were expressed in 293T cells. Immunoprecipitation of NS5A with anti-HA antibody and western blotting of ubiquitin with an anti-HIS antibody were performed 24 h after plasmid transfection. TRIM22 efficiently ligated ubiquitin to NS5A. Lower panel: endogenous ubiquitination of NS5A was detected. Huh-7-Con-1 replicon cells were treated with IFNα (50 IU/ml). Twenty-four hours after IFNα treatment, cells were harvested and immunoprecipitated with an anti-NS5A antibody. Then, western blotting was performed with an anti-ubiquitin antibody. (d) MG132 (20 nM) successfully blocked the endogenous ubiquitination of NS5A and NS5A degradation. HCV, hepatitis C virus; IFN, interferon; TRIM, tripartite motif.