Efficient replication of a full-length hepatitis C virus genome, strain O, in cell culture, and development of a luciferase reporter system

Masanori Ikeda, Ken Ichi Abe, Hiromichi Dansako, Takashi Nakamura, Kazuhito Naka, Nobuyuki Kato

Research output: Contribution to journalArticle

135 Citations (Scopus)

Abstract

Recently we reported a subgenomic hepatitis C virus (HCV) replicon derived from HCV (HCV-O strain) infected in non-neoplastic human hepatocyte PH5CH8. In this study, we developed a genome-length dicistronic HCV RNA from HCV-O. A cured HuH-7 cell line (sOc) was obtained from a cloned subgenomic replicon cell line (sO) by interferon (IFN) treatment and used for transfection with genome-length HCV RNA. One cloned cell line, O, was successfully selected by G418 treatment following the introduction of genome-length HCV RNA into sOc cells, and the robust expression of HCV RNA and proteins was confirmed. Oc, a cured cell line, was also obtained from the cloned cell line (O) by IFN treatment. The number of colonies increased drastically when genome-length HCV RNA was introduced into Oc cells. However, the cloned cured cell lines, sOc and Oc, differed in their colony formation efficiency despite their common origin. This result suggests that even a cloned cell line can change its characteristics during cell culture. Sequence analysis of HCV RNA from the O cells revealed an amino acid substitution in the NS3 helicase region (K1609E). This substitution worked as an adaptive mutation in transient reporter and colony formation assays. Using the advantages of this adaptive mutation and of Oc cells in colony formation, we established the first cell line in which genome-length dicistronic HCV RNA encoding a luciferase gene replicated efficiently. This culture system is useful tool for the study of HCV replication and mass screening for anti-HCV reagents.

Original languageEnglish
Pages (from-to)1350-1359
Number of pages10
JournalBiochemical and Biophysical Research Communications
Volume329
Issue number4
DOIs
Publication statusPublished - Apr 22 2005

Fingerprint

Luciferases
Viruses
Cell culture
Hepacivirus
Cell Culture Techniques
Genes
Genome
Cells
Cell Line
RNA
Replicon
Interferons
Substitution reactions
Mutation
Mass Screening
Amino Acid Substitution
Virus Replication
Transfection
Sequence Analysis
Hepatocytes

Keywords

  • Cured cells
  • Genome-length HCV RNA
  • Hepatitis C virus
  • Replication
  • Reporter assay

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Efficient replication of a full-length hepatitis C virus genome, strain O, in cell culture, and development of a luciferase reporter system. / Ikeda, Masanori; Abe, Ken Ichi; Dansako, Hiromichi; Nakamura, Takashi; Naka, Kazuhito; Kato, Nobuyuki.

In: Biochemical and Biophysical Research Communications, Vol. 329, No. 4, 22.04.2005, p. 1350-1359.

Research output: Contribution to journalArticle

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