title: Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells.
abstract: Dicistronic, selectable subgenomic replicons derived from the Con1 strain of hepatitis C virus (HCV) are capable of autonomous replication in cultured Huh7 cells (Lohmann et al., Science 285:110-113, 1999). However, adaptive mutations in the NS3, NS5A, and/or NS5B proteins are required for efficient replication of these RNAs and increase by orders of magnitude the numbers of G418-resistant colonies selected following transfection of Huh7 cells. Here, we demonstrate that a subgenomic replicon (NNeo/3-5B) derived from an infectious molecular clone of a second genotype 1b virus, HCV-N (Beard et al., Hepatology 30:316-324, 1999) is also capable of efficient replication in Huh7 cells. G418-resistant cells selected following transfection with NNeo/3-5B RNA contained abundant NS5A antigen and HCV RNA detectable by Northern analysis. Replicon RNA in one of three clonally isolated cell lines contained no mutations in the NS3-NS5B polyprotein, confirming that adaptive mutations are not required for efficient replication in these cells. However, the deletion of a unique 4-amino-acid insertion that is present within the interferon sensitivity-determining region (ISDR) of the NS5A protein in wild-type HCV-N drastically decreased the number of G418-resistant colonies obtained following transfection of Huh7 cells. This effect could be reversed by inclusion of a previously described Con1 cell culture-adaptive mutation (S2005-->I), confirming that this natural insertion has a controlling role in determining the replication capacity of wild-type HCV-N RNA in Huh7 cells. Additional selectable, dicistronic RNAs encoding NS2-NS5B, E1-NS5B, or the full-length HCV polyprotein were also capable of replication and gave rise to G418-resistant cell clones following transfection of Huh7 cells. We conclude that RNA derived from this documented infectious molecular clone has a unique capacity for replication in Huh7 cells in the absence of additional cell culture-adaptive mutations.
authors.affiliation: Department of Microbiology & Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019, USA.
authors.forename: Masanori
authors.initials: M
authors.lastname: Ikeda
authors.forename: MinKyung
authors.initials: M
authors.lastname: Yi
authors.forename: Kui
authors.initials: K
authors.lastname: Li
authors.forename: Stanley M
authors.initials: SM
authors.lastname: Lemon
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D006526:Hepatitis C / Q000821:virology; D006801:Humans; D019868:Kanamycin Kinase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
chemical_list: D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D019868:Kanamycin Kinase