title: Resistance to cyclosporin A derives from mutations in hepatitis C virus nonstructural proteins.
abstract: Cyclosporine A (CsA) is an immunosuppressive drug that targets cyclophilins, cellular cofactors that regulate the immune system. Replication of hepatitis C virus (HCV) is suppressed by CsA, but the molecular basis of this suppression is still not fully understood. To investigate this suppression, we cultured HCV replicon cells (Con1, HCV genotype 1b, FLR-N cell) in the presence of CsA and obtained nine CsA-resistant FLR-N cell lines. We determined full-length HCV sequences for all nine clones, and chose two (clones #6 and #7) of the nine clones that have high replication activity in the presence of CsA for further analysis. Both clones showed two consensus mutations, one in NS3 (T1280V) and the other in NS5A (D2292E). Characterization of various mutants indicated that the D2292E mutation conferred resistance to high concentrations of CsA (up to 2 μM). In addition, the missense mutation T1280V contributed to the recovery of colony formation activity. The effects of these mutations are also evident in two established HCV replicon cell lines-HCV-RMT ([1], genotype 1a) and JFH1 (genotype 2a). Moreover, three other missense mutations in NS5A-D2303H, S2362G, and E2414K-enhanced the resistance to CsA conferred by D2292E; these double or all quadruple mutants could resist approximately 8- to 25-fold higher concentrations of CsA than could wild-type Con1. These four mutations, either as single or combinations, also made Con1 strain resistant to two other cyclophilin inhibitors, N-methyl-4-isoleucine-cyclosporin (NIM811) or Debio-025. Interestingly, the changes in IC50 values that resulted from each of these mutations were the lowest in the Debio-025-treated cells, indicating its highest resistant activity against the adaptive mutation.
authors.affiliation: Advanced Medical Research Laboratory, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 227-0033, Japan; Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Arai
authors.affiliation: Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Tsukiyama-Kohara
authors.affiliation: Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
authors.forename: Asako
authors.initials: A
authors.lastname: Takagi
authors.affiliation: Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Tobita
authors.affiliation: Division of Gastroenterology, Showa University Fujigaoka Hospital, 1-30, Aoba-ku, Fujigaoka, Yokohama 227-8501, Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Inoue
authors.affiliation: Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. Electronic address: kohara-mc@igakuken.or.jp.
authors.forename: Michinori
authors.initials: M
authors.lastname: Kohara
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D021983:Cyclophilins / Q000037:antagonists & inhibitors; D016572:Cyclosporine / Q000494:pharmacology*; D024882:Drug Resistance, Viral; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology; D006801:Humans; D009154:Mutation; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D021983:Cyclophilins; D016572:Cyclosporine; D017361:Viral Nonstructural Proteins; C090438:(melle-4)cyclosporin; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C499715:alisporivir
keywords: Cyclophylin inhibitor; Cyclosporine A; Debio-025; Hepatitis C virus; Mutation