Development of plaque assays for hepatitis C virus-JFH1 strain and isolation of mutants with enhanced cytopathogenicity and replication capacity

Yuko Sekine-Osajima, Naoya Sakamoto, Kako Mishima, Mina Nakagawa, Yasuhiro Itsui, Megumi Tasaka, Yuki Nishimura-Sakurai, Cheng Hsin Chen, Takanori Kanai, Kiichiro Tsuchiya, Takaji Wakita, Nobuyuki Enomoto, Mamoru Watanabe

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Abstract

HCV culture in vitro results in massive cell death, which suggests the presence of HCV-induced cytopathic effects. Therefore, we investigated its mechanisms and viral nucleotide sequences involved in this effect using HCV-JFH1 cell culture and a newly developed HCV plaque assay technique. The plaque assay developed cytopathic plaques, depending on the titer of the inoculum. In the virus-infected cells, the ER stress markers, GRP78 and phosphorylated eIF2-alpha, were overexpressed. Cells in the plaques were strongly positive for an apoptosis marker, annexin V. Isolated virus subclones from individual plaque showed greater replication efficiency and cytopathogenicity than the parental virus. The plaque-purified virus had 9 amino acid substitutions, of which 5 were clustered in the C terminal of the NS5B region. Taken together, the cytopathic effect of HCV infection involves ER-stress-induced apoptotic cell death. Certain HCV genomic structures may determine the viral replication capacity and cytopathogenicity.

Original languageEnglish
Pages (from-to)71-85
Number of pages15
JournalVirology
Volume371
Issue number1
DOIs
Publication statusPublished - 2008 Feb 5
Externally publishedYes

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Keywords

  • Apoptosis
  • ER stress
  • HCV cell culture
  • HCV-JFH1
  • NS5B RNA-dependent RNA polymerase
  • Plaque assay
  • Unfolded protein responses

ASJC Scopus subject areas

  • Virology

Cite this

Sekine-Osajima, Y., Sakamoto, N., Mishima, K., Nakagawa, M., Itsui, Y., Tasaka, M., Nishimura-Sakurai, Y., Chen, C. H., Kanai, T., Tsuchiya, K., Wakita, T., Enomoto, N., & Watanabe, M. (2008). Development of plaque assays for hepatitis C virus-JFH1 strain and isolation of mutants with enhanced cytopathogenicity and replication capacity. Virology, 371(1), 71-85. https://doi.org/10.1016/j.virol.2007.09.019