Biology Reference
In-Depth Information
associated with development of chronic hepatitis, hepatic steatosis, cir-
rhosis, and hepatocellular carcinoma. There are currently 170 million or
more HCV carriers worldwide. In past years, anti-hepatitis C therapy has
modestly improved; however, a currently available combination therapy,
consisting of interferon (IFN)-
and the nucleoside analogue ribavirin,
shows a sustained response in only less than half of the treated patients.
The development of innovative treatment alternatives for patients
infected with HCV is urgently required, and a better understanding of
the mechanism of HCV replication should allow the identification of
novel targets for antiviral intervention specific to HCV.
α
Cell Culture Systems for HCV Replication
Studies on HCV replication have long been hampered by the lack of
efficient cell culture systems. Although many attempts have been made
to achieve full replication of HCV in cell cultures, all systems including
cell lines derived from human hepatoma, B cells and T cells, primary
hepatocytes and peripheral blood mononuclear cells suffer from low
virus yield and are not robust enough to allow genetic analyses of the
HCV life cycle. The development of HCV subgenomic replicons (self-
replicating RNAs) has allowed examination of viral RNA replication in
cell culture.
1
Expression systems of heterologous virus genes based on
RNA replicons have been established in a variety of positive-strand
RNA viruses such as polio virus,
2-5
alphaviruses semliki forest virus,
6
sindbis virus,
7-10
kunjin virus,
11
human rhinovirus 14,
12
and bovine viral
diarrhea virus.
13
In general, advantages of replicon systems are based on
(1) a high level of gene expression and RNA replication, (2) easy con-
struction of recombinants, and (3) the permitting of a wide host range.
The HCV replicons are typically composed of the 5
-nontranslated
region (NTR), which directs translation of the gene encoding the
neomycin phosohotransferase (Neor); the internal ribosome entry site
(IRES) of the encephalomyocarditis virus (EMCV), which directs trans-
lation of HCV NS3 through NS5B region; and the 3
′
-NTR.
Following transfection of RNA transcribed from the above bicistronic
constructs into a human hepatoma cell line Huh-7, antibiotic G418-
resistent cells could be obtained in which the subgenomic RNA repli-
cated autonomously. RNA replication was at first detected at low
′
