title: Development of recombinant hepatitis C virus with NS5A from strains of genotypes 1 and 2.
abstract: Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays multiple and diverse roles in the viral lifecycle, and is currently recognized as a novel target for anti-viral therapy. To establish an HCV cell culture system with NS5A of various strains, recombinant viruses were generated by replacing NS5A of strain JFH-1 with those of strains of genotypes 1 (H77; 1a and Con1; 1b) and 2 (J6CF; 2a and MA; 2b). All these recombinant viruses were capable of replication and infectious virus production. The replacement of JFH-1 NS5A with those of genotype 1 strains resulted in similar or slightly reduced virus production, whereas replacement with those of genotype 2 strains enhanced virus production as compared with JFH-1 wild-type. A single cycle virus production assay with a CD81-negative cell line revealed that the efficient virus production elicited by replacement with genotype 2 strains depended on enhanced viral assembly, and that substitutions in the C-terminus of NS5A were responsible for this phenotype. Pulse-chase assays revealed that these substitutions in the C-terminus of NS5A were possibly associated with accelerated cleavage kinetics at the NS5A-NS5B site. Using this cell culture system with NS5A-substituted recombinant viruses, the anti-viral effects of an NS5A inhibitor were then examined. A 300- to 1000-fold difference in susceptibility to the inhibitor was found between strains of genotypes 1 and 2. This system will facilitate not only a better understanding of strain-specific roles of NS5A in the HCV lifecycle, but also enable the evaluation of genotype and strain dependency of NS5A inhibitors.
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Okamoto
authors.forename: Takahiro
authors.initials: T
authors.lastname: Masaki
authors.forename: Asako
authors.initials: A
authors.lastname: Murayama
authors.forename: Tsubasa
authors.initials: T
authors.lastname: Munakata
authors.forename: Akio
authors.initials: A
authors.lastname: Nomoto
authors.forename: Shingo
authors.initials: S
authors.lastname: Nakamoto
authors.forename: Osamu
authors.initials: O
authors.lastname: Yokosuka
authors.forename: Haruo
authors.initials: H
authors.lastname: Watanabe
authors.forename: Takaji
authors.initials: T
authors.lastname: Wakita
authors.forename: Takanobu
authors.initials: T
authors.lastname: Kato
mesh_terms: D019943:Amino Acid Substitution; D000998:Antiviral Agents / Q000494:pharmacology; D002219:Carbamates; D045744:Cell Line, Tumor; D024882:Drug Resistance, Viral / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D011759:Pyrrolidines; D014633:Valine / Q000031:analogs & derivatives; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000502:physiology*; D019065:Virus Assembly / Q000235:genetics / Q000502:physiology*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D002219:Carbamates; D007093:Imidazoles; D011759:Pyrrolidines; D014633:Valine; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C549273:daclatasvir