FIG 6.

SKP2 is an E3 ligase for NS5A. (A) IP analysis of SKP2 and ISG12a (left) or NS5A (right) from HEK293T cells coexpressing exogenous SKP2 and ISG12a or NS5A. (B) IP analysis of SKP2 and endogenous ISG12a from HLCZ01 cells transfected with pcDNA3.1a-myc/His-SKP2 plasmid followed by HCV infection for 48 h. (C) Ubiquitination assay and Western blotting of lysates from HEK293T cells expressing exogenous NS5A and HA-ubiquitin (HA-ub) with or without SKP2 silencing for 48 h. MG132 (25 μM) was added into cells for 6 h prior to harvesting cell lysates and IP experiments were conducted with anti-Flag antibody followed by the ubiquitination assay of NS5A protein. (D) Cells were treated as described for panel C followed by the ubiquitination assays of NS4B (left) and NS5B (right) proteins. (E) HLCZ01 cells were infected by HCV (MOI, 0.1) for 24 h followed by transfection with pSilencer-SKP2 or pSilencer vector plasmid for 48 h. The ubiquitination assay was conducted as described for panel C. (F and G) FL-neo cells were cotransfected with p3×FLAG-ISG12a and pcDNA3.1a-myc/His-SKP2 (F) or pSilencer-SKP2 (G) plasmid for 48 h followed by Western blotting of NS5A. (H) Huh7.5 and HLCZ01 cells were transfected with pcDNA3.1a-myc/His-SKP2 or pSilencer-SKP2 plasmid for 48 h. The MTS assay was performed, and error bars represent SD from triplicate experiments. V5, Flag, and HA were the tags used.