Virus Research 137 (2008) 72–79
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Virus Research
journal homepage: www.elsevier.com/locate/virusres
A new living cell-based assay system for monitoring genome-length hepatitis
C virus RNA replication
Hiromichi Dansako, Masanori Ikeda, Ken-ichi Abe, Kyoko Mori, Kazunori Takemoto,
Yasuo Ariumi, Nobuyuki Kato
∗
Department of Molecular Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
article info
Article history:
Received 22 February 2008
Received in revised form 6 June 2008
Accepted 6 June 2008
Available online 21 July 2008
Keywords:
Hepatitis C virus
Genome-length HCV RNA
Living cell-based assay
Green fluorescent protein
OGF7 assay system
Anti-HCV reagents
abstract
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-␣ treatment in a dose-dependent manner, and it correlated well
with the HCV RNA concentration. We demonstrated that the interferon-␣ 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.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
Persistent hepatitis C virus (HCV) infection frequently causes
active liver disease in the form of chronic hepatitis (Choo et al.,
1989; Kuo et al., 1989), liver cirrhosis, and hepatocellular carci-
noma (Ohkoshi et al., 1990; Saito et al., 1990). HCV infection has
now become a serious health problem, with at least 170 million
people currently infected worldwide (Thomas, 2000). HCV is an
enveloped positive single-stranded RNA (9.6 kb) virus belonging
to the Flaviviridae (Kato et al., 1990; Tanaka et al., 1995). The HCV
genome encodes a large polyprotein precursor of approximately
3000 amino acid (aa) residues, which is cleaved co- and post-
translationally into at least 10 proteins in the following order: core,
envelope 1 (E1), E2, p7, non-structural protein 2 (NS2), NS3, NS4A,
NS4B, NS5A, and NS5B. These cleavages are mediated by the host
and virally encoded proteases (Hijikata et al., 1991, 1993; Kato,
2001). NS5B possessing an RNA-dependent RNA polymerase (RdRp)
activity is the central enzyme in replication of the HCV genome
(Kato, 2001).
In the recent past, interferon (IFN) was used as the main
treatment for patients with chronic hepatitis C. Currently, the com-
∗
Corresponding author. Tel.: +81 86 235 7385; fax: +81 86 235 7392.
E-mail address: nkato@md.okayama-u.ac.jp (N. Kato).
bination of pegylated-IFN (PEG-IFN) and ribavirin is the standard
therapy worldwide, although only 50% of patients show a sus-
tained virological response to this therapy (Hayashi and Takehara,
2006). Several clinical drugs have been proposed as adjuvants to
IFN, including cyclosporine A (CsA) (Watashi et al., 2003), mizorib-
ine (Naka et al., 2005), and statins (Ikeda et al., 2006; Ye et al., 2003).
Currently, NS3 proteinase/helicase activity and NS5B RdRp activity
have been considered as targets for the development of anti-HCV
reagents (e.g., the NS3 protease inhibitor BILN 2061 (Lamarre et al.,
2003). To date, however, we have not obtained HCV-specific drugs
possessing more effective anti-HCV activity than PEG-IFN. There-
fore, a more convenient high-throughput screening system is still
required to explore more effective anti-HCV reagents.
We previously developed a cell-based genome-length HCV RNA
replication system using Renilla luciferase as a reporter in order to
monitor the HCV RNA replication level (OR6 assay system) (Ikeda et
al., 2005; Naka et al., 2005). Other groups have also developed cell-
based subgenomic HCV replicon systems using secreted alkaline
phosphatase (Yi et al., 2002) or beta-lactamase (Murray et al., 2003)
as a reporter. However, these assay systems are still quite time- and
cost-intensive methods for measuring enzyme activity.
In the present study, we report a new living cell-based reporter
assay system that is able to monitor the level of genome-length
HCV RNA replication and to reduce both the time required and the
expense.
0168-1702/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.virusres.2008.06.001