(Top panel) Cell lysates were immunoprecipitated (IP) with an anti-Pin1 polyclonal antibody, and bound proteins were then detected by immunoblot (IB) analysis. HCV is an enveloped virus with a positive-sense, single-stranded Nog RNA genome of 9.6 kb. The HCV genome encodes a single precursor polyprotein, which is cleaved by both cellular and viral proteases to generate three structural (core, E1, and E2) and seven nonstructural (p7; NS2 to NS5B) proteins. Although HCV is a highly prevalent pathogen, no protective vaccine is available yet. Current standard therapy is pegylated alpha interferon (IFN-) combined with ribavirin. However, this therapy shows some side effects and results in a sustained virological response in only a small portion of patients. Thus, there is an urgent need to develop more-effective therapeutic strategies for HCV-associated chronic hepatitis. Peptidyl-prolyl isomerase NIMA-interacting 1 (Pin1) was first discovered in a screen for molecules regulating mitosis (34). Pin1 consists of 163 amino acids and contains two functional domains: the N-terminal WW binding domain and the C-terminal peptidyl-prolyl isomerase domain (12, 13, 32, 33). The N-terminal WW binding domain is responsible for binding to specific proteins that are phosphorylated at Ser/Thr-Pro motifs, whereas the C-terminal isomerase domain promotes the isomerization of the bound peptide. Such conformational changes have significant effects on the phosphorylation status, subcellular localization, protein stability, and functions of many Pin1 substrates (12, 13, 32, 33). Accordingly, Pin1 plays important roles in many cellular events, including cell cycle progression, cell proliferation, transcriptional regulation, and neoplastic transformation. This protein has also been linked to several diseases, such as cancer, Alzheimer’s disease, and asthma. Pin1 is overexpressed in many human cancers, including HCC (11); it has been found to be overexpressed in more than 50% of HCCs. All cases with Pin1 overexpression also showed -catenin accumulation, and 68% of cases showed concomitant -catenin and cyclin D1 accumulation (16). Furthermore, overexpression of Pin1 in a nontransformed human liver cell line leads to hepatocyte transformation, and inhibition of Pin1 expression suppresses HCC tumorigenesis (18). It has been reported recently that Pin1 interacts with a specific serine-proline motif of hepatitis B virus (HBV) X protein (HBx) to enhance hepatocarcinogenesis in HBV patients (17). In the present study, we demonstrate for the first time that Pin1 interacts directly with the HCV NS5A and NS5B (NS5A/5B) proteins and plays unique roles in HCV replication. In addition, juglone (5-hydroxy-1,4-naphthoquinone), a natural inhibitor of Pin1, impairs the interaction between Pin1 and the HCV NS5A/5B proteins and inhibits HCV propagation. Therefore, Pin1 may be a potential target for HCV treatment. MATERIALS AND METHODS Plasmids and DNA transfection. Plasmids expressing Myc-tagged NS4B, Myc-tagged NS5A, and Myc-tagged NS5B have been described previously (3, 19). Full-length human Pin1 cDNA was amplified from the Nepicastat (free base) (SYN-117) pCNS-D2-Pin1 plasmid (21C Frontier Human Gene Bank) and was subcloned into the pGEX-4T1 (Amersham Biosciences) and p3Flag-CMV10 (Sigma-Aldrich) vectors to generate the GST-Pin1 and Flag-Pin1 expression plasmids, respectively. Pin1 mutants were generated by site-directed mutagenesis (Stratagene) using the primers listed in Table 1 according to the manufacturer’s instructions. Small interfering RNA (siRNA)-resistant mutant Pin1 contains two silent mutations in the siRNA binding site. To generate siRNA-resistant binding-defective mutant Pin1 (13) and siRNA-resistant isomerase-inactive mutant Pin1 (31), the substitution mutations S16A and C113A, respectively, were introduced into siRNA-resistant mutant Nepicastat (free base) (SYN-117) Pin1. For the cloning of human cyclophilin A (CypA) and CypB (mature form), total RNAs were extracted from Huh7.5 cells and were used for reverse transcription-PCR (RT-PCR) with the primer sets CypA-F/CypA-R and CypB-F/CypB-R (Table 1). PCR products were inserted into the EcoRI and BamHI sites of plasmid p3Flag-CMV10. For DNA transfection, cells were transfected with the expression plasmid by using a polyethyleneimine reagent (Sigma-Aldrich) as we described previously (19). Table 1. List of primers used in this study binding and coimmunoprecipitation assays were performed as we described elsewhere (4). Confocal microscopy. Either Huh7 or Huh7.5 cells cultured on glass slides were fixed with 4% paraformaldehyde in PBS at room temperature for 30 min. After two washes Nepicastat (free base) (SYN-117) with PBS, fixed cells were permeabilized Nepicastat (free base) (SYN-117) with 0.1% Triton X-100 in PBS for 5 min and were incubated with a mouse anti-Pin1 antibody, a mouse anti-Flag antibody, a rabbit anti-NS5A antibody, or a rabbit anti-NS5B antibody for 2 h. After three washes with PBS, cells were further incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse or anti-rabbit IgG (American Qualex,.