The Ubiquitin-Specific Protease 18 Promotes Hepatitis C Virus Production by Increasing Viral Infectivity

Figure 4

USP18 overexpression does not affect HCV replicons nor microRNA122 expression. AB12-A2 or sbJFH1-B2 cells were seeded overnight (6-well plates) before transfection with Lipofectamine 2000 in OptiMem with 4 μg USP18 WT or USP18 C64S plasmid DNA. 24 hours posttransfection, protein was collected (RIPA buffer) or cells were treated with IFNα (Recombinant, Sigma) at 1, 100, 500, or 1000 IU/ml in DMEM without antibiotics and incubated for 72 h before RNA isolation. Total RNA was collected and subjected to real-time PCR assessment using primers specific for HCV Con1b (a) or HCV JFH1 5 UTR (b). (c) Huh7.5 cells were transfected with 4 μg blank vector pcDNA-DEST53, 4 μg USP18 WT, or 4 μg USP18 C64S for 48 hours before treatment with IFNα (10 IU/ml). MicroRNA 122 levels were determined by a kit and normalized to U6 at different time points indicated post-IFNα treatment. Results are presented as ().

(a)

(b)

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