title: Biological properties of purified recombinant HCV particles with an epitope-tagged envelope.
abstract: To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Takahashi
authors.forename: Daisuke
authors.initials: D
authors.lastname: Akazawa
authors.forename: Takanobu
authors.initials: T
authors.lastname: Kato
authors.forename: Tomoko
authors.initials: T
authors.lastname: Date
authors.forename: Masayuki
authors.initials: M
authors.lastname: Shirakura
authors.forename: Noriko
authors.initials: N
authors.lastname: Nakamura
authors.forename: Hidenori
authors.initials: H
authors.lastname: Mochizuki
authors.forename: Keiko
authors.initials: K
authors.lastname: Tanaka-Kaneko
authors.forename: Tetsutaro
authors.initials: T
authors.lastname: Sata
authors.forename: Yasuhito
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Masashi
authors.initials: M
authors.lastname: Mizokami
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Suzuki
authors.forename: Takaji
authors.initials: T
authors.lastname: Wakita
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D000939:Epitopes / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006031:Glycosylation; D016174:Hepacivirus / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D014761:Viral Hepatitis Vaccines / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D014771:Virion / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D000939:Epitopes; D014761:Viral Hepatitis Vaccines