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. 2018 Jun 13;92(13):e00639-18. doi: 10.1128/JVI.00639-18

FIG 2.

FIG 2

HCV NS5A protein enhances the interaction between HSC70 and HNF-1α. (A) Huh-7.5 cells (1.5 × 106 cells/10-cm dish) were infected with HCV J6/JFH1 at a multiplicity of infection (MOI) of 1. At 4 h postinfection, cells were transfected with pCAG-FLAG-HSC70 and cultured for 48 h. Cells were harvested and assayed for immunoprecipitation with mouse anti-FLAG M2 MAb, followed by immunoblotting with rabbit anti-FLAG PAb (3rd panel) or rabbit anti-NS5A PAb (4th panel). Input samples were immunoblotted with either rabbit anti-FLAG PAb (1st panel) or rabbit anti-NS5A PAb (2nd panel). (B) Huh-7.5 cells (1.5 × 106 cells/10-cm dish) were transfected with either FLAG-HNF-1α or empty plasmid together with pCAG-myc-HSC70-HA as indicated, and cultured. At 48 h after transfection, cells were harvested. Cell lysates were assayed for immunoprecipitation with rabbit anti-c-myc PAb, followed by immunoblotting with mouse anti-FLAG MAb (3rd panel) or mouse anti-HSC70 MAb (4th panel). Input samples were immunoblotted with either anti-FLAG MAb (1st panel) or anti-HSC70 MAb (2nd panel). (C) Huh-7.5 cells were plated at 1.5 × 106 cells/10-cm dish and cultured for 12 h. Cells were transfected with increasing amounts of pEF1A-NS5A-myc-His6 together with either pCAG-FLAG-HNF-1α or pCAG-FLAG-HNF-1α Q130A. At 48 h after transfection, the cells were harvested. The cell lysates were immunoprecipitated with mouse anti-HSC70 MAb, followed by immunoblotting with rabbit anti-FLAG PAb (1st panel, right), mouse anti-HSC70 MAb (2nd panel, right), or mouse anti-c-myc MAb (3rd panel, right). Input samples were analyzed by immunoblotting with anti-FLAG PAb (1st panel, left), anti-HSC70 MAb (2nd panel, left), or anti-c-myc MAb (3rd panel, left). (D) Huh-7.5 cells (1.5 × 106 cells/10-cm dish) were infected with HCV J6/JFH1 at an MOI of 1. At 4 h postinfection, cells were transfected with either pCAG-FLAG-HNF-1α or pCAG-FLAG-HNF-1α Q130A and cultured for 48 h. Cells were harvested and assayed for immunoprecipitation with mouse anti-HSC70 MAb. Bound proteins were immunoblotted with rabbit anti-FLAG PAb (2nd panel) or mouse anti-HSC70 MAb (4th panel). Input samples were immunoblotted with anti-FLAG PAb (1st panel), anti-HSC70 MAb (3rd panel), or anti-NS5A rabbit PAb (5th panel). (E) Huh-7.5 cells (1.5 × 106 cells/10-cm dish) were transfected with pCAG-FLAG-HNF-1α. At 24 h posttransfection, cells were infected with HCV J6/JFH1 at an MOI of 0.5 for 12 h, 24 h, 36 h, or 48 h. Cells were harvested and assayed for immunoprecipitation with mouse anti-HSC70 MAb. Bound proteins were immunoblotted with rabbit anti-FLAG PAb (2nd panel) or mouse anti-HSC70 MAb (4th panel). Input samples were immunoblotted with anti-FLAG PAb (1st panel), anti-HSC70 MAb (3rd panel), or rabbit anti-NS5A PAb (5th panel).