Establishment of Hepatitis C Virus RNA-Replicating cell lines possessing ribavirin-resistant phenotype

Shinya Satoh, Kyoko Mori, Youki Ueda, Hiroe Sejima, Hiromichi Dansako, Masanori Ikeda, Nobuyuki Kato

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Background Ribavirin (RBV) is a potential partner of interferon-based therapy and recently approved therapy using direct acting antivirals for patients with chronic hepatitis C. However, the precise mechanisms underlying RBV action against hepatitis C virus (HCV) replication are not yet understood. To clarify this point, we attempted to develop RBV-resistant cells from RBV-sensitive HCV RNA-replicating cells. Methodology/Principal Findings By repetitive RBV (100 μM) treatment (10 weeks) of 3.5-year-cultured OL8 cells, in which genome-length HCV RNA (O strain of genotype 1b) efficiently replicates, dozens of colonies that survived RBV treatment were obtained. These colonies were mixed together and further treated with high doses of RBV (up to 200 μM). By such RBV treatment, we successfully established 12 RBV-survived genome-length HCV RNA-replicating cell lines. Among them, three representative cell lines were characterized. HCV RNA replication in these cells resisted RBV significantly more than that in the parental OL8 cells. Genetic analysis of HCV found several common and conserved amino acid substitutions in HCV proteins among the three RBV-resistant cell species. Furthermore, using cDNA microarray and quantitative RT-PCR analyses, we identified 5 host genes whose expression levels were commonly altered by more than four-fold among these RBV-resistant cells compared with the parental cells. Moreover, to determine whether viral or host factor contributes to RBV resistance, we developed newly HCV RNA-replicating cells by introducing total RNAs isolated from RBVsensitive parental cells or RBV-resistant cells into the HCV RNA-cured-parental or -RBV-resistant cells using an electroporation method, and evaluated the degrees of RBV resistance of these developed cells. Consequently, we found that RBV-resistant phenotype was conferred mainly by host factor and partially by viral factor. Conclusions/Significance These newly established HCV RNA-replicating cell lines should become useful tools for further understanding the anti-HCV mechanisms of RBV.

Original languageEnglish
Article numbere0118313
JournalPLoS One
Volume10
Issue number2
DOIs
Publication statusPublished - Feb 20 2015

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Hepatitis C virus
Ribavirin
Viruses
Hepacivirus
Cells
cell lines
RNA
Phenotype
phenotype
Cell Line
cells
chronic hepatitis C
Virus Replication
therapeutics
electroporation
genome
amino acid substitution
interferons
virus replication
Genes

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Establishment of Hepatitis C Virus RNA-Replicating cell lines possessing ribavirin-resistant phenotype. / Satoh, Shinya; Mori, Kyoko; Ueda, Youki; Sejima, Hiroe; Dansako, Hiromichi; Ikeda, Masanori; Kato, Nobuyuki.

In: PLoS One, Vol. 10, No. 2, e0118313, 20.02.2015.

Research output: Contribution to journalArticle

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