title: Production of infectious chimeric hepatitis C virus genotype 2b harboring minimal regions of JFH-1.
abstract: To establish a cell culture system for chimeric hepatitis C virus (HCV) genotype 2b, we prepared a chimeric construct harboring the 5' untranslated region (UTR) to the E2 region of the MA strain (genotype 2b) and the region of p7 to the 3' UTR of the JFH-1 strain (genotype 2a). This chimeric RNA (MA/JFH-1.1) replicated and produced infectious virus in Huh7.5.1 cells. Replacement of the 5' UTR of this chimera with that from JFH-1 (MA/JFH-1.2) enhanced virus production, but infectivity remained low. In a long-term follow-up study, we identified a cell culture-adaptive mutation in the core region (R167G) and found that it enhanced virus assembly. We previously reported that the NS3 helicase (N3H) and the region of NS5B to 3' X (N5BX) of JFH-1 enabled replication of the J6CF strain (genotype 2a), which could not replicate in cells. To reduce JFH-1 content in MA/JFH-1.2, we produced a chimeric viral genome for MA harboring the N3H and N5BX regions of JFH-1, combined with a JFH-1 5' UTR replacement and the R167G mutation (MA/N3H+N5BX-JFH1/R167G). This chimeric RNA replicated efficiently, but virus production was low. After the introduction of four additional cell culture-adaptive mutations, MA/N3H+N5BX-JFH1/5am produced infectious virus efficiently. Using this chimeric virus harboring minimal regions of JFH-1, we analyzed interferon sensitivity and found that this chimeric virus was more sensitive to interferon than JFH-1 and another chimeric virus containing more regions from JFH-1 (MA/JFH-1.2/R167G). In conclusion, we established an HCV genotype 2b cell culture system using a chimeric genome harboring minimal regions of JFH-1. This cell culture system may be useful for characterizing genotype 2b viruses and developing antiviral strategies.
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
authors.forename: Asako
authors.initials: A
authors.lastname: Murayama
authors.forename: Takanobu
authors.initials: T
authors.lastname: Kato
authors.forename: Daisuke
authors.initials: D
authors.lastname: Akazawa
authors.forename: Nao
authors.initials: N
authors.lastname: Sugiyama
authors.forename: Tomoko
authors.initials: T
authors.lastname: Date
authors.forename: Takahiro
authors.initials: T
authors.lastname: Masaki
authors.forename: Shingo
authors.initials: S
authors.lastname: Nakamoto
authors.forename: Yasuhito
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Masashi
authors.initials: M
authors.lastname: Mizokami
authors.forename: Osamu
authors.initials: O
authors.lastname: Yokosuka
authors.forename: Akio
authors.initials: A
authors.lastname: Nomoto
authors.forename: Takaji
authors.initials: T
authors.lastname: Wakita
mesh_terms: D002460:Cell Line; D002678:Chimera / Q000145:classification / Q000235:genetics* / Q000502:physiology; D005818:Genetic Engineering; D016679:Genome, Viral; D005838:Genotype; D016174:Hepacivirus / Q000145:classification / Q000235:genetics* / Q000502:physiology; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D014771:Virion / Q000145:classification / Q000235:genetics* / Q000502:physiology; D019065:Virus Assembly