title: Site-specific mutation of the interferon sensitivity-determining region (ISDR) modulates hepatitis C virus replication.
abstract: The number of amino acid substitutions in the interferon sensitivity-determining region (ISDR) in the nonstructural 5A (NS5A) gene of hepatitis C virus (HCV) is closely associated with the interferon (IFN) response and viral load. Several HCV replicon-based studies have reported that ISDR sequences had an influence on viral replication in vitro. However, it is unclear as to how different ISDR sequences affect HCV replication. Various clinically observed ISDR sequences were introduced into HCV replicons and their contribution to viral replication was investigated using a colony formation assay and/or a transient replication assay. A mapping study of the ISDR was performed to identify the amino acid positions that critically affect replication. While no colonies were formed in the colony formation assay using HCV replicons with few mutations (0, 1 and 3) in the ISDR, numerous colonies (>200) appeared when using constructs with six mutations. Introduction of various distinct ISDR sequences with multiple mutations resulted in replication enhancement in transient assays. A mapping study identified several specific sites in the ISDR that critically affected replication, including codon 2209 which, in patients, was closely associated with a strong response to IFN. ISDR sequences associated with a clinical IFN response and viral load modulated the replication of HCV replicons, suggesting the importance of the ISDR sequence in HCV infection.
authors.affiliation: Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Yushima, Bunkyo, Tokyo, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Kohashi
authors.forename: S
authors.initials: S
authors.lastname: Maekawa
authors.forename: N
authors.initials: N
authors.lastname: Sakamoto
authors.forename: M
authors.initials: M
authors.lastname: Kurosaki
authors.forename: H
authors.initials: H
authors.lastname: Watanabe
authors.forename: Y
authors.initials: Y
authors.lastname: Tanabe
authors.forename: C-H
authors.initials: CH
authors.lastname: Chen
authors.forename: N
authors.initials: N
authors.lastname: Kanazawa
authors.forename: M
authors.initials: M
authors.lastname: Nakagawa
authors.forename: S
authors.initials: S
authors.lastname: Kakinuma
authors.forename: T
authors.initials: T
authors.lastname: Yamashiro
authors.forename: Y
authors.initials: Y
authors.lastname: Itsui
authors.forename: T
authors.initials: T
authors.lastname: Koyama
authors.forename: N
authors.initials: N
authors.lastname: Enomoto
authors.forename: M
authors.initials: M
authors.lastname: Watanabe
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D007372:Interferons / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D007372:Interferons; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase