FIG 5.

HCV disturbs the trimeric complex of RAD51, RAD51AP1, and UAF1 via NS5A and accumulates RAD51 in the cytoplasm. (A) HEK293T cells were cotransfected with V5-tagged UAF1 and either vector, Flag-tagged RAD51, Xpress-tagged RAD51AP1, or increasing amounts of Myc-tagged NS5A. At 36 h posttransfection, total cell lysates were immunoprecipitated with an anti-Flag antibody, and bound proteins were immunoblotted with the indicated antibodies. (B) HEK293T cells were cotransfected with V5-tagged UAF1 and either vector, Flag-tagged RAD51, Xpress-tagged RAD51AP1, or increasing amounts of Myc-tagged NS4B. At 36 h posttransfection, cell lysates were immunoprecipitated with an anti-Flag antibody, and bound proteins were immunoblotted with the indicated antibodies. (C) Huh7 cells were cotransfected with the indicated combinations of plasmids. At 24 h posttransfection, cells were treated with 25 μM cisplatin. At 16 h posttreatment, cell lysates were immunoprecipitated with an anti-Flag antibody, and bound proteins were immunoblotted with the indicated antibodies. (D) Huh7 cells were cotransfected with the indicated combinations of plasmids. At 24 h posttransfection, cells were either mock infected or infected with Jc1 for 4 h, followed by treatment with 25 μM cisplatin. At 16 h posttreatment, cell lysates were immunoprecipitated with an anti-Flag antibody, and bound proteins were immunoblotted with the indicated antibodies. (E) Huh7.5 cells were either mock infected or infected with Jc1. Cell lysates harvested at 6 days postinfection were immunoprecipitated with an anti-UAF1 antibody. Bound proteins were immunoblotted with anti-RAD51 and anti-RAD51AP1 antibodies. (F) HCV induces cytoplasmic distribution of RAD51 protein. Huh7.5 cells were either mock infected or infected with Jc1. At 6 days postinfection, both cytosolic and nuclear fractions were immunoblotted with the indicated antibodies. (G) Huh7.5 cells were either mock infected or infected with Jc1. If more than 90% of cells were infected with Jc1 (upper panel), immunofluorescence staining was performed. Arrows indicate the position of the heavy chain, and the arrowhead denotes the light chain of IgG.