A new living cell-based assay system for monitoring genome-length hepatitis C virus RNA replication.
We previously developed a cell-based luciferase reporter assay system for monitoring genome-length hepatitis C virus (HCV) RNA replication (OR6 assay system). Here, we aimed to develop a new living cell-based reporter assay system using enhanced green fluorescent protein (EGFP). Genome-length HCV RNAs encoding EGFP were introduced into a subline of HuH-7 cells and G418 selection was performed. One cloned cell line, OGF7, was successfully selected from among the several G418-resistant cell lines obtained, and the robust expression of HCV RNA and proteins in OGF7 cells was confirmed. The fluorescent intensity of OGF7 cells was decreased by interferon-alpha treatment in a dose-dependent manner, and it correlated well with the HCV RNA concentration. We demonstrated that the interferon-alpha sensitivity in the OGF7 assay system measuring the fluorescent intensity was equivalent to that of the OR6 assay system, and that the OGF7 assay system was useful for quantitative evaluation of anti-HCV reagents. The OGF7 assay system is expected to be the most time-saving and inexpensive assay system for high-throughput screening of anti-HCV reagents.
Dansako
Hiromichi
H
Department of Molecular Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan.
Ikeda
Masanori
M
Abe
Ken-ichi
K
Mori
Kyoko
K
Takemoto
Kazunori
K
Ariumi
Yasuo
Y
Kato
Nobuyuki
N
D000998:Antiviral Agents / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D002470:Cell Survival; D002999:Clone Cells; D016679:Genome, Viral / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D012367:RNA, Viral / Q000096:biosynthesis*; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics*
D000998:Antiviral Agents; D016898:Interferon-alpha; D012367:RNA, Viral; D049452:Green Fluorescent Proteins