elaprevir or boceprevir is also a serious problem, since it is expected that these resistant viruses will exhibit a resistant phenotype against other NS3-4A inhibitors developed in the future. 17429684 Therefore, a new type of anti-HCV reagent without severe side effects or emergence of resistant virus is still needed, although several anti-HCV candidates, such as NS5A and NS5B inhibitors, are currently in phase IIIII development. To date, human hepatoma cell line HuH-7-derived cells are used as the only the preferred culture system for robust HCV replication, and most studies on anti-HCV reagents are currently Anti-HCV Activities of N-89 and N-251 carried out using an HuH-7-derived cell culture system. We also developed an HuH-7-derived drug assay system, in which genome-length HCV-RNA encoding renilla luciferase efficiently replicates. Such reporter assay systems could save time and facilitate the mass screening of antiHCV reagents, since the values of luciferase correlated well with the level of HCV RNA after treatment with anti-HCV reagents. Furthermore, OR6 assay system became more useful as a drug assay system than the HCV subgenomic replicon-based reporter assay systems developed to date, because the older systems lack the core-NS2 regions containing structural proteins order UPF 1069 likely to be involved in the events that take place in the HCV-infected human liver. Indeed, by the screening of preexisting drugs using the OR6 assay system, we have identified mizoribine, statins, hydroxyurea, and teprenone as new anti-HCV drug candidates, indicating that the OR6 assay system is useful for the discovery of anti-HCV reagents. On the other hand, we recently found a new human hepatoma cell line, Li23, that enables efficient HCV-RNA replication and persistent HCV production, and we developed Li23-derived assay systems that are comparable to the OR6 assay system. Since we indicated that the gene expression profile of Li23 cells was distinct from that of HuH-7 cells, we expected that anti-HCV targets in Li23-derived cells might be distinct from those in HuH-7-derived cells. Indeed, we recently found that 10 mM of RBV efficiently inhibited HCV-RNA replication in the ORL8/ORL11 assays, but not in the OR6 assay. This finding led us to clarify the anti-HCV mechanism of RBV. Furthermore, we demonstrated that plural assay systems including OR6 and ORL8 were required for the objective evaluation of anti-HCV reagents. In that study, we observed that the antimalarial drug artemisinin possessed weak anti-HCV activity, as reported previously. From these results, we considered that antimalarial drugs might be good candidates for anti-HCV reagents, since the proliferation of both HCV and malaria generally occurs in hepatocytes. Materials and Methods Cell Culture RSc and D7 cells were derived from the cell lines HuH-7 and Li23, respectively, 22634634 were cultured as described previously. HuH-7-derived OR6, AH1R, and 1B-4R cells harboring genome-length HCV-RNA and HuH7-derived polyclonal sOR, and RSc-JRN/35B cells harboring an HCV subgenomic replicon were cultured with medium in the presence of G418 as described previously. Li23-derived ORL8, ORL11, 1B-4RL, and KAH5RL cells harboring genome-length HCV-RNA were maintained with medium in the presence of G418 as described previously. Li23derived polyclonal sORL8 and sORL11 cells harboring an HCV replicon, which were established by the transfection of ORN/35B/QR,KE,SR RNA into the cured OL8 and OL11 cells, respectively, were also cultur