Genetic characterization of hepatitis C virus in long-term RNA replication using Li23 cell culture systems

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

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Background: The most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells. Methodology/Principal Findings: Li23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 59-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a timedependent manner, and the mutation rates in the 5′-terminus-NS2 and NS3-NS5B regions were 4.0-9.0×10-3 and 2.7-4.0×10-3 base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3-105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents. Conclusions/Significance: Long-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis.

Original languageEnglish
Article numbere91156
JournalPLoS One
Volume9
Issue number3
DOIs
Publication statusPublished - Mar 13 2014

Fingerprint

Hepatitis C virus
Viruses
Cell culture
Hepacivirus
cell culture
Cell Culture Techniques
RNA
Cells
Cultured Cells
cells
hepatoma
codons
Codon
cultured cells
genetic techniques and protocols
RNA replication
Hepatocellular Carcinoma
mutation
Cell Line
human cell lines

ASJC Scopus subject areas

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

Cite this

Genetic characterization of hepatitis C virus in long-term RNA replication using Li23 cell culture systems. / Kato, Nobuyuki; Sejima, Hiroe; Ueda, Youki; Mori, Kyoko; Satoh, Shinya; Dansako, Hiromichi; Ikeda, Masanori.

In: PLoS One, Vol. 9, No. 3, e91156, 13.03.2014.

Research output: Contribution to journalArticle

@article{5030f09251dc4869a649138b158907ef,
title = "Genetic characterization of hepatitis C virus in long-term RNA replication using Li23 cell culture systems",
abstract = "Background: The most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells. Methodology/Principal Findings: Li23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 59-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a timedependent manner, and the mutation rates in the 5′-terminus-NS2 and NS3-NS5B regions were 4.0-9.0×10-3 and 2.7-4.0×10-3 base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3-105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents. Conclusions/Significance: Long-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis.",
author = "Nobuyuki Kato and Hiroe Sejima and Youki Ueda and Kyoko Mori and Shinya Satoh and Hiromichi Dansako and Masanori Ikeda",
year = "2014",
month = "3",
day = "13",
doi = "10.1371/journal.pone.0091156",
language = "English",
volume = "9",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "3",

}

TY - JOUR

T1 - Genetic characterization of hepatitis C virus in long-term RNA replication using Li23 cell culture systems

AU - Kato, Nobuyuki

AU - Sejima, Hiroe

AU - Ueda, Youki

AU - Mori, Kyoko

AU - Satoh, Shinya

AU - Dansako, Hiromichi

AU - Ikeda, Masanori

PY - 2014/3/13

Y1 - 2014/3/13

N2 - Background: The most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells. Methodology/Principal Findings: Li23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 59-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a timedependent manner, and the mutation rates in the 5′-terminus-NS2 and NS3-NS5B regions were 4.0-9.0×10-3 and 2.7-4.0×10-3 base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3-105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents. Conclusions/Significance: Long-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis.

AB - Background: The most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells. Methodology/Principal Findings: Li23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 59-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a timedependent manner, and the mutation rates in the 5′-terminus-NS2 and NS3-NS5B regions were 4.0-9.0×10-3 and 2.7-4.0×10-3 base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3-105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents. Conclusions/Significance: Long-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis.

UR - http://www.scopus.com/inward/record.url?scp=84898715982&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84898715982&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0091156

DO - 10.1371/journal.pone.0091156

M3 - Article

C2 - 24625789

AN - SCOPUS:84898715982

VL - 9

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 3

M1 - e91156

ER -