TY - JOUR
T1 - A new living cell-based assay system for monitoring genome-length hepatitis C virus RNA replication
AU - Dansako, Hiromichi
AU - Ikeda, Masanori
AU - Abe, Ken ichi
AU - Mori, Kyoko
AU - Takemoto, Kazunori
AU - Ariumi, Yasuo
AU - Kato, Nobuyuki
N1 - Funding Information:
We would like to thank T. Maeta and T. Nakamura for their helpful assistance with the experiments. This work was supported by grants-in-aid for a third-term comprehensive 10-year strategy for cancer control, and for research on hepatitis from the Ministry of Health, Labor, and Welfare of Japan. K.A. was supported by a Research Fellowship from the Japan Society for the Promotion of Science (JSPS) for Young Scientists.
PY - 2008/10
Y1 - 2008/10
N2 - 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-α treatment in a dose-dependent manner, and it correlated well with the HCV RNA concentration. We demonstrated that the interferon-α 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.
AB - 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-α treatment in a dose-dependent manner, and it correlated well with the HCV RNA concentration. We demonstrated that the interferon-α 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.
KW - Anti-HCV reagents
KW - Genome-length HCV RNA
KW - Green fluorescent protein
KW - Hepatitis C virus
KW - Living cell-based assay
KW - OGF7 assay system
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U2 - 10.1016/j.virusres.2008.06.001
DO - 10.1016/j.virusres.2008.06.001
M3 - Article
C2 - 18602954
AN - SCOPUS:49849095101
SN - 0168-1702
VL - 137
SP - 72
EP - 79
JO - Virus research
JF - Virus research
IS - 1
ER -
