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. 2011 Sep;85(17):8777–8788. doi: 10.1128/JVI.02533-10

Fig. 5.

Fig. 5.

Both the binding and isomerase activities of Pin1 are essential for HCV replication. (A) Huh7.5 cells were transfected with the indicated combinations of expression plasmids. (Top panel) At 48 h after transfection, cell lysates were immunoprecipitated (IP) with an anti-Flag monoclonal antibody, and bound proteins were then detected by an immunoblot (IB) assay. (Lower panels) Protein expression in the same cell lysates was verified with the indicated antibodies. Pin1 WT-SR, siRNA-resistant mutant Pin1; Pin1 S16A-SR, siRNA-resistant binding-defective mutant Pin1; Pin1 C113A-SR, siRNA-resistant isomerase-inactive mutant Pin1. The asterisk indicates the IgG heavy chain. (B) Huh7.5 cells were transfected with the indicated siRNA. At 2 days after siRNA transfection, cells were infected with Jc1 for 4 h and were then transfected with 2 μg of a plasmid expressing wild-type Pin1 (Pin1 WT), Pin1 S16A-SR, Pin1 C113A-SR, or Pin1 WT-SR. Intracellular RNAs isolated at 2 days after Pin1 transfection were analyzed by qPCR. (C) Protein expression levels of both HCV and host cells were determined by immunoblot analysis using the indicated antibodies. (D) The effects of Pin1 mutants on HCV infectivity were determined by focus-forming assays. Experiments were carried out in duplicate. Error bars indicate standard deviations.