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

Figure 5

USP18 promotes HCV infectivity and CD81 expression in Huh7.5 cells. (a) Huh7.5 cells were seeded at /ml, 2 ml per well in 6-well plates in antibiotic-free medium for 24 hours before 1 μg, 2 μg, 4 μg, and 6 μg pcDNA3.1-USP18 or 4 μg empty vector pcDNA3.1 was transfected into each well. 48 hours posttransfection, total RNA was extracted; USP18 and CD81 expression was quantified by qRT-PCR and normalized to GAPDH expression as previously described. (b) Huh7.5 cells were seeded at /ml, 2 ml per well in 6-well plates in antibiotic-free medium for 24 hours before 4 μg pcDNA3.1-USP18 was transfected into each well. (B-1) Lower-left and lower right quadrants indicate that the cells were transfected with empty vector pcDNA3.1 or pcDNA3.1-USP18 at 72 hours, with the percentage of cells in respective quadrants. All dot blots show CD81 expression (vertical axis) according to USP18 expression (horizontal axis). Upper-left and right quadrants show the range of isotype control for PE- and Alexa 488-conjugated antibodies. (B-2) Huh7.5 cells with transfected empty vector pcDNA3.1 (open histogram, dark green line) or pcDNA3.1-USP18 DNA (filled histogram) were stained with a mouse anti-flag primary/FITC-conjugated goat anti-mouse secondary antibody, rabbit anti-human USP18 primary/Alexa 488-conjugated goat anti-rabbit secondary antibody, and PE-conjugated anti-human CD81 monoclonal antibody. The cells transfected with pcDNA3.1-USP18 were also stained with mouse and rabbit IgG and isotype control antibodies (open histogram, brown line). (B-3) Comparison of USP18/CD81 expression in or on Huh7.5 cells with sham control, transfected pcDNA3.1 control, and pcDNA3.1-USP18 DNA. Bars indicate MFI of USP18 . Untreated: untreated control; pcDNA3.1: transfected with 4 μg empty vector pcDNA3.1; 1 μg, 2 μg, 4 μg, and 6 μg: transfected with 1 μg, 2 μg, 4 μg, or 6 μg pcDNA3.1-USP18; sham control: untreated Huh7.5 cells only. Results are presented as (). ; ; .

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