title: RNA polymerase activity and specific RNA structure are required for efficient HCV replication in cultured cells.
abstract: We have previously reported that the NS3 helicase (N3H) and NS5B-to-3'X (N5BX) regions are important for the efficient replication of hepatitis C virus (HCV) strain JFH-1 and viral production in HuH-7 cells. In the current study, we investigated the relationships between HCV genome replication, virus production, and the structure of N5BX. We found that the Q377R, A450S, S455N, R517K, and Y561F mutations in the NS5B region resulted in up-regulation of J6CF NS5B polymerase activity in vitro. However, the activation effects of these mutations on viral RNA replication and virus production with JFH-1 N3H appeared to differ. In the presence of the N3H region and 3' untranslated region (UTR) of JFH-1, A450S, R517K, and Y561F together were sufficient to confer HCV genome replication activity and virus production ability to J6CF in cultured cells. Y561F was also involved in the kissing-loop interaction between SL3.2 in the NS5B region and SL2 in the 3'X region. We next analyzed the 3' structure of HCV genome RNA. The shorter polyU/UC tracts of JFH-1 resulted in more efficient RNA replication than J6CF. Furthermore, 9458G in the JFH-1 variable region (VR) was responsible for RNA replication activity because of its RNA structures. In conclusion, N3H, high polymerase activity, enhanced kissing-loop interactions, and optimal viral RNA structure in the 3'UTR were required for J6CF replication in cultured cells.
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Asako
authors.initials: A
authors.lastname: Murayama
authors.forename: Leiyun
authors.initials: L
authors.lastname: Weng
authors.forename: Tomoko
authors.initials: T
authors.lastname: Date
authors.forename: Daisuke
authors.initials: D
authors.lastname: Akazawa
authors.forename: Xiao
authors.initials: X
authors.lastname: Tian
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Suzuki
authors.forename: Takanobu
authors.initials: T
authors.lastname: Kato
authors.forename: Yasuhito
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Masashi
authors.initials: M
authors.lastname: Mizokami
authors.forename: Takaji
authors.initials: T
authors.lastname: Wakita
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Toyoda
mesh_terms: D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005814:Genes, Viral; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D020365:RNA Helicases / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D014779:Virus Replication / Q000502:physiology*
chemical_list: D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases; D020365:RNA Helicases