REVIEWAdvances in Genomic Research on Hepatitis C Viruswith a Useful Tool, Replicon System
Mitsuyasu Nakamura, Hidetsugu Saito, and Toshifumi Hibi

Division of Gastroenterology and Hepatology, Department of Internal Medicine,
School of Medicine, Keio University, Tokyo, Japan(Received for publication on August 31, 2007)(Revised for publication on December 3, 2007)(Accepted for publication on January 17, 2008)

Abstract: The research for hepatitis C virus (HCV) has long delayed by missing of in vitro
culture system. Since the development of replicon system, a replication system of subgenomic HCV RNAs in a hepatoma cell line, has been reported, many virological and clinicalfindings have been discovered. Recently, in addition of subgenomic replication system, hepatitis C virus full-length RNA replication has been possible, and a few cell culture systems
producing viral particles have been produced. These developments enabled us to investigate
the life cycle or intracellular circumstance of HCV production. By screening of newly synthesized drugs with this replicon system, several possible medicines have been establishedand clinical researches are now running. Among them, VX950 and SCH503034 are nearest to clinical use. Other possible agents for reducing viral replication such as cyclophyllin
inhibitors, inhibitors of sphingomyelin synthesis, HMG-CoA reductase inhibitors, or RNAdependent RNA polymerase inhibitors have been also investigated. Furthermore the mechanism for development of hepatocellular carcinoma in the HCV infected liver has been vigorously studied using the HCV replicon system. (Keio J Med 57 (2) : 75－ 83, June 2008)
Key words: hepatitis C virus, replicon, VX950, lipid raft, Rb

Introduction

mild. Infected subjects develop chronic hepatitis from acarrier state, and eventually, liver cirrhosis and hepatocellular carcinoma (HCC) over period of up to 30 ormore years. In Japan, it is estimated that there are 1.5
million HCV-infected people, and infection is more common among the elderly than among younger individuals.
About 30,000 patients die of HCC associated with HCV
infection, and HCC is currently ranked as the fourth
leading cause of death. The ideal treatment is eradicationof HCV from the body, and a combination of interferon(IFN) and ribavirin (RBV) is the current mainstream regimen.3
Presence of the hypervariable region (HVR) at the
N-terminus of the E1/E2/NS1 regions which is responsible for surface structure, is thought to play a role in persistent HCV infection. Nevertheless, heterogeneity of the
HVR does not correlate with hepatitis activity.4 With regard to HCV eradication by IFN, analysis of the viral

Hepatitis C virus (HCV) belongs to the Hepacivirus
genus of the Falviviridae family. It is a spherical particle,
55 to 65 nm in diameter, with an envelope, and consistsof the genome of a 9.6-kb plus strand RNA. It has shortuntranslated regions at both the 5’- and 3’- termini, andthe middle region, which accounts for about 95% of thewhole genome, encodes a precursor protein consisting ofroughly 3,010 amino acid residues. The coding regionsof viral structural proteins such as core as well as E1 andE2 are located at the N-terminus, while the non-structuralproteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B are
situated at the middle region near the C-terminus. 1, 2
HCV is categorized according to 6 genotypes and is further classified into several subtypes.
HCV is transmitted through blood and it often causes
persistent infection. Post-infection symptoms are often

Corresponding Author: Hidetsugu Saito MD, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan TEL: 3-3353-1211 Ext 62894, FAX: 3-3351-8705,
E-mail: hsaito@sc.itc.keio.ac.jp75Nakamura M, et al: HCV Replicon System

76
Open reading frame(ORF) 9030nt

HCV genome
Structuralprotein region

5' NTR

Nonstructural protein region

NS4A

p7

(2427 nt)

(6603nt)

3' NTR

341nt

C E1 E2

NS2

NS3

NS4B NS5A NS5B

HCV full-length replicon
5' NTR

ΔC

Neo

IRES

NS4A

p7

EMCV

3' NTRC E1 E2

NS2

NS3

NS4B NS5A NS5B

HCV subgenomic replicon
5' NTR

ΔC

NS4A

EMCV
Neo

IRES

3'
NS3

NS4B NS5A NS5B

Fig. 1 The structure of HCV genome, that of HCV full-length replicon and subgenomic repliconsensitivity to IFN revealed the presence of the IFN sensitivity-determining region (ISDR), which influences sensitivity to IFN, at the center of NS5A, a non-structuralprotein of HCV.5, 6 Meanwhile, the number of aminoacid mutation in the ISDR has been reported to have asignificant negative correlation with the amount of HCV
RNA.7
Since the replicon system has been developed, virological aspects of HCV including its life cycle were uncovered, and development of this system and more recentlyof a cell culture system has enabled the development of
HCV-specific inhibitors and quantitative measurement ofantiviral potency. Many of the newly developed inhibitors are under investigation in pre-clinical and clinical
trials, and these specific inhibitors will improve treatment opportunities of patients with chronic hepatitis C,
especially in difficult-to-treat patients including patients
who do not respond to Peg-IFN and RBV combination
therapy. In this mini-review, we introduced the HCV replicon system and summarized newly developed virological and clinical discoveries, following to the development of replicon and cell culture systems.
Cells Replicating an HCV
Gene-replication Unit (replicon)
In 1989, a group led by Houghton et al. at Chiron (asubsidiary of Novartis AG, Basel, Switzerland) succeeded in cloning the HCV genome for the first time.8 In
1997, Rice et al. successfully established an infectious

clone from cloned cDNA, which induced acute hepatitis
in chimpanzees.9 Chimpanzees which are the only reliable animal model for HCV infection, are difficult to obtain and expensive to maintain and are therefore not suitable for drug screening studies. As a result, the development of efficient replication systems in cultured cells has
been eagerly anticipated.
In 1999, Bartenshalger et al. established the first culture system in which HCV subgenomic RNA replicatedand maintained itself and in which HCV gene replication
was efficiently reproduced.10 This system was referred toas an HCV replicon system. The structure of HCV replicon RNA is shown in Fig. 1. In HCV subgenomic RNA,a structural protein region encoding the viral particle isreplaced with the code sequence of aminoglycosidephosphotransferase (Neor) that detoxifies neomycin harboring cytotoxicity. The internal ribosome entry site
(IRES) derived from encephalomyocarditis virus(EMCV) is inserted at the down-stream region of the
Neor gene. Due to this sequence, translation starts withinRNA, and non-structural protein, which is located downstream and involved in HCV gene replication, is efficiently translated.
RNA synthesized in vitro can be introduced into HCCderived HuH7 cells, and with RNA introduced cells canbe selected with neomycin. The HCV genome sequencethat can self-replicate in cells, and cells that enable theself-replication, are selected and replicon cells are thus
obtained (Fig. 2). To date, similar replicon cells havebeen established with several different HCV genome se-Keio J Med 2008; 57 (2): 75－ 83
T7

77
neo

NS3 4A 4B

5A

5B

in vitro RNA synthesis
transfection

HuH7cells

G418 (neomycin) selection
ER

nucleusHCVRNA(+)(+)(+)
replication( -)minus strand RNA

Fig. 2 Outline of HCV replicon system. Subgenomic replicon RNAs are produced from plasmid including T7 promoter by in vitrotranscription using T7 RNA polymerase. These RNAs are transfected into the human hepatoma cell line Huh-7. Only cells in which
the replicon self-replicates will carry the gene encoding neomycin phosphotransferase, which inactivates G418, to become resistant toG418.

quences.11-1 3 Thus, the development of the HCV replicon system has enabled the analysis of virus genome
replication at the cellular level.14
Research on the HCV Genome Replication System
Research on HCV genome replication started withanalysis of the RNA genome structure in replicon cells.
To date, it has been determined that a phenomenon called
adaptive mutation occurs in the RNA genome in cultured
cells.15 This is presumed to be a result of selected aminoacid mutation in HCV protein so that RNA can efficiently replicate and proliferate in cultured cells. The RNA
structure required for genome replication has been identified by artificial introduction of mutation (e.g., various
deletion mutations) into HCV subgenome RNA. Furthermore, replication systems are currently indispensable forelucidation of the pathogenesis of HCV infection and development of antiviral drugs. We previously found differences in specific sites in the HCV NS5B region between patients that did or did not respond to IFN-RBV
combination therapy,16 and are now exploring alterations
using the replicon system in vitro.17
Full-length HCV RNA Replication System
While the subgenomic replicon in vitro system is used
to express non-structural proteins necessary for replication, not only non-structural proteins but also structuralproteins are actually expressed in vivo. The influence of
HCV on host cell factors, such as IFN, other cytokines,

and signal transduction, are complex events mediated byfull-length HCV proteins. Different full-length repliconRNAs have been produced by some research groups.18, 19
In the full-length HCV RNA replication system, the presence of double strand RNA (dsRNA), consisting of the
HCV RNA plus strand and the minus strand, an intermediate replication product, enables easier evaluation of theinfluence of HCV, including the effect of dsRNA, on
host factors, compared with a replicon system with nonstructural regions using a variety of promoters. Since
structural proteins are required for the production of viralparticles, full-length HCV RNA replication systems haveadvanced research on in vivo properties of the virus, including packaging, budding, and infection20-24 (Fig.3).
Structure of the Replication Complex
Intracellular environments for HCV RNA synthesis
and replication were examined in the subgenome replicon system, and it was found that replicon HCV RNA
was localized in the cell membrane structure, especiallyat endoplasmic reticulum membrane, and partly in theregion surrounded by non-structural protein-like lipid
membrane, while most of the non-structural protein waspresent on the cytoplasmic side of the lipid membrane.24
This suggests that replicon RNA replicates in a structure
surrounded by lipid membrane, i.e. lipid raft. Similar results have also been reported with regard to RNA replication of plant viruses such as bromomosaic virus. Intracellulcar environments surrounded by lipid membraneNakamura M, et al: HCV Replicon System

78
HCV life cycle

unidentified
cellular receptors

endocytosis

host cells: hepatocyte

budding
Vesicle fusion at
Plasma membrane

uncoatingmaturation

HCVRNA(+)strand

Goldi body

viral protein
replication
complextranslationassemblyER

minus strand RNA

replication

nucleuscytoplasm

Fig. 3 HCV life cyclea) Virus binds to host cell receptors (CD81 and other unidentified candidates, such as low density lipoprotein receptor (LDL-R) andscavenger receptor class-B type-I (SR-BI)) and enters by endocytosis.
b) After uncoating nucleocapsid, IRES-mediated translation and polyprotein processing occurs on the endoplasmic reticulum (ER) .
c) RNA replication: formation of a replication complex on the altered ER membrane, and transcription and synthesis of (-) strand
RNA, which serves as a template for progeny (+) strand RNA.
d) Packing and assembly: structural protein (E1, E2 and core) and progeny (+) strand RNA assemble.
e) Virion maturation and particle release.

are potentially those for the HCV genome replication
complex.26, 27
JFH-1 Strain Replicon and Production of Infection
Virus Particles
Wakita et al. cloned an HCV gene from serum of a patient with fulminant hepatitis, a rare complication of typeC chronic hepatitis, and designated it as the JFH-1 strain
genotype 2a. Subgenomic2 8 and full-length replicons2 9were constructed from the JFH-1 strain, and productionof viral particles in the culture supernatant of HCV RNA
replicating cells was confirmed. In addition, an HCV
culture system with even higher infection efficiency wasestablished by combination of the JFH-1 strain and cured
cells established from replicon cell experiments. 3 0 , 3 1
However, the onset of fulminant hepatitis is extremelyrare in even HCV genotype 2a cases, suggesting that
JFH-1 strain was established from a very rare clinicalcase. The viral particles released from the JFH-1 replicon
can infect chimpanzees but do not cause hepatitis. The
establishment of JFH-1 strain which forms infectious viral particles opened a black box of HCV virology and biology. The role of CD81 and heparin sulfate proteoglycan on HCV entry into the cell has been demonstratedusing this system recently.31 On the other hand, in Japan,
the most prevalent HCV subtype is genotype 1b, and thevirus is most resistant to eradication by IFN in patientswith this genotype (1b). It is anticipated that a repliconthat produces infectious viral particles derived from genotype 1b HCV will be established.
Research and Application of Replicons and the
Full-length HCV RNA Replication System:
Screening of New Drugs with Replicons
Pegylated-IFN (Peg-IFN) in combination with RBV is
the current mainstream treatment for chronic hepatitis C,
however, such therapy has several limitations which remain to be overcome. Although viral eradication rates
have been improved by the combination of Peg-IFN andRBV compared with IFN monotherapy, the treatment iseffective in only half of patients infected with genotype
1b HCV with a high viral load. HCV dynamics at theearly treatment phase is associated with treatment efficacy Peg-IFN and RBV combination therapy, and earlysuppression of viral replication is thought to be important.3 3 In addition, adverse effects and lower response
rates can be a hindrance in the treatment of elderly patients.3 4 To solve these problems, screening with replicons is being used for the development of new anti-HCV
agents to replace Peg-IFN and RBV in clinical practice.
<Development of new treatments targeted at host factors>
Shimotohno et al. reported that cyclosporine A (CyA),

Keio J Med 2008; 57 (2): 75－ 83

an immunosuppressant widely used in clinical practice,suppressed HCV replication in the replicon system.3 5This HCV replication suppression is not related with thecytotoxicity of CyA or the function as an immunosuppressant, but is a specific action of the replicon cells
against HCV protein. The underlying mechanisms, binding to cyclophyllin and inhibition of peptidylprolyl isomerase enzyme activity, are thought to inhibit HCV replication.3 6, 3 7 It has been reported that CyA is effective
against HCV in vivo,3 8, 3 9 which has generated clinicalinterest.40
As mentioned previously, the HCV replication complex is created in the endoplasmic reticulum membrane,
and an HCV life cycle is formed. It has been suggested
that HCV replication can be regulated by targeting several molecules in the lipid raft, consisting of high-fluidity
lipids such as saturated fatty acids, cholesterol and sphingomyelin, in the area of high-fluidity portion formed
with unsaturated fatty acid bi-layered membrane. One
study reported suppression of HCV replicon replication
by myriocin, which interferes with the sphingomyelin
synthesis pathway.4 1 In addition, it was reported that
3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors suppress HCV replicon replication.42<Development of new treatments targeted at viral factors>
The development of new drugs targeted at NS3 encoding serine protease involved in viral replication3 8 and
NS5B encoding RNA polymerase4 3 is another strategy
that has been pursued. These drugs, which are expected
to directly exert antiviral activity, referred to as specifically-targeted antiviral therapies (STATCs). It has beenreported that serine protease activity encoded by NS3 inhibits not only the processing of viral protein but also
IFN signal transduction44, and clinical trials are ongoingfor clinical application of drugs targeted at this site.
VX9504 5, 4 6 and SCH5030344 7, 4 8 are among the NS3protease inhibitors under development. Since HCV ishighly mutatable, monotherapy using one of these drugs
results in immediate appearance of a resistant clone.49, 50
Therefore, clinical trials on VX950 with or without IFN
and in combination with RBV are ongoing in the U.S.
and European countries. Similarly, clinical trials of
SCH503034 with and without IFN are underway in patients refractory to a combination of Peg-IFN-α2b andRBV.
NM283, an RNA-dependent RNA polymerase inhibitor, was found to be effective in chimpanzees and cultured cells. However, since monotherapy with this agent
was not fully effective, combination with IFN was required and as a result, adverse effects due to high-dose
administration were reported.51, 52
Since toll-like receptor 9 agonists likely exert antiviral
activity by enhancing intrinsic IFN production, their ad-

79

ministration in combination with Peg-IFN and RBV improved early virological response rates.5 3 Moreover, inhibition of HCV replicon with siRNA has been reported.54 Treatment with ribozyme and antisense oligonucleotides seems to be effective in replicons, however, because of issues with stability and drug delivery, they
have not yet been sufficiently developed for clinical use
as anti-HCV agents.
Table 1 summarized several treatment targets and ongoing preclinical and clinical trials.
Regulation of Host Genes by HCV Protein
To date, it has been reported that viral proteins, such as
E1A5 5 and E1B5 6 of adenovirus, human immunodeficiency virus type 1 Tat protein,57 HTLV-1 Tax protein,58
human papilloma virus E65 9 and E76 0 protein, regulatetranscriptional activity of host cells by influencing transcription regulating factors such as CBP/p300, PCAF,
histone deacetylase complex, and SRC-1. On the other
hand, it has been reported that HCV core protein upregulates transcription of retinoic acid receptor-α (RAR-α) byisolating Sp110b, a transcriptional inhibitor of RAR-α,from intranuclear space to the surface of endoplasmic reticulum.61 RAR-α is known as a receptor for retinoic acid-induced apoptosis. Similarly, it is possible that HCV
protein regulates various other host genes.
HCV and Carcinogenesis (Rb gene and HCV)The precise mechanism underlying the developmentfrom HCV infection to carcinogenesis has not been elucidated, but a relationship with several oncogenes has
been proposed. Unlike hepatitis B virus (HBV), integration of the HCV gene into host cell DNA has not beenrecognized. Therefore, there are possibilities that HCV
protein produced by HCV infection affects functions ofhost cells and heightens carcinogenicity.
Resent studies have revealed that HCV infection affects the cell cycle regulating protein Rb. Rb protein affects cells in the G1-S phase62 and inhibits transcription
factor E2F.63 In addition, the protein is well known as atumor suppressor gene, and mutation in the Rb gene has
been reported in lung and ovarian cancer cells.6 4, 6 5 We
reported that the Rb gene bound to a specific site in the
NS5B-encoding polymerase required for HCV replication, which suppressed the expression of Rb protein.6 6
The HCV sequence for binding was the same as the sequence previously reported in adenovirus67 and papilloma virus6 8 that bound the Rb gene.6 9 These oncogenes
are thought to inhibit Rb gene function and the LxCxE
motif by directly binding with them (Fig. 4 and 5), whichsuggests the involvement of HCV in carcinogenesis.

Nakamura M, et al: HCV Replicon System

80

Table 1 Targets of therapy and emerging therapies in HCV
Target at viral factors

Virus entry
Post translation processing
HCV replication

HCV RNA translation

Target at host factors

anti E2monoclonal antibody
HCV-AB(XTL)68
NS3/4A serine protease inhibitors
VX950 (Telaprevir; phase 2), SCH503034(Boceprevir; phase 2), ITMN-191 (phase 1)
NS5B RNA dependent RNA polymerase inhibitors(a) nucleoside analogues
NM283 (Valopicitabine; phase 2), R1626 (prodrug ofR1479;
phase 2), MK-608 (preclinical), R1656, R7128 (preclinical)(b) non-nucleoside analogues
HCV-796 (phase 2), BILB1941 (phase 1), A-837093(preclinical), GS-9190 (phase 1)
NS5A inhibitors
A-831 (phase 1), A-689 (preclinical)
ribozyme
antisense oligonucleotide
siRNA
hepatitis C immunoglobulin
Civacir
Cyclosporin inhibitors
DEBIO-25 (phase 1), NIM811 (preclinical)
Myoricin,
HMG-CoA reductase inhibitors
TLR9 agonist
ER glucosidase inhibitors
Celgosivir (preclinical)

Virus entry
Replication

Production of IFN
Virus particle release

In addition of these therapies, several interferons have been on going as clinical studies (Albinterferon; phase 3, Pegamax; phase 1,
Locteron; phase 1/2, Omega interferon; phase 2).

・・・LXCXE・・・
HPV

E7

AdV

E1A

SV-40

Chromatin remodeling complexes
SWI/SNF
HDACs
Methylases

T
RB

HCV

P107

P130

NS5B

E2F1

E2F2

E2F3

Strong transcription activators

E2F4

E2F5

Weak transcription
activator

E2F6Transcriptional
repressor

Fig. 4 Various viruses which regulate Rb gene and E2Fgene. Rb protein binds to a transcriptional factor E2F and inhibits its
transcriptional activity. There are 6 kinds of E2F proteins in mammal genome, and they are divided into 3 categories depending to theirtranscriptional activity. Rb protein inhibits high active E2F1, E2F2 and E2F3. This E2F-dependent transcriptional inhibition of Rb
protein is regulated by chromatin remodeling complex such as SWI/SNF complex, histone deacetylase polycomb group and methylase.
Papilloma virus, adenovirus and SV40 T antigen also attenuate Rb protein-induced E2F inhibition. HDACs: histone deacetylasesKeio J Med 2008; 57 (2): 75－ 83

81

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LxCxE
HPV16.E7

ETTD

LYCYE

QLNDSSEHCV -Con1.NS5B wt

DCTM LVCGDDLVVICEHCV -N.NS5B wt

DCTM LVNGDDLVVICEHCV -BK.NS5B wt

DCTM LVNGDDLVVICEHCV -JFH1.NS5B wt

APTM LVCGDDLVVISE

GDD
Fig. 5 Molecular mimicry of amino acids between papilloma virus
and HCV. LxCxE domain of human papilloma virus E7 protein
and 314-318 amino acids alignment of NS5B region of HCV(Con1 strain and N strain). This region of HCV contains GDD
sequence necessary for viral polymerase activity.

Conclulsion
The aim of HCV eradication is to eventually reduce theoccurrence of HCC. It is therefore important to elucidate
the mechanisms by which HCV infection precipitates theonset of HCC. Clinical research in the past years has focused on the improvement of IFN-based treatment regimens. HCV-specific antiviral compounds, some of whichhave been developed by investigation using the repliconsystem, are a new perspective in the treatment of chronic
hepatitis C. Since the viral RNA-polymerase possesses ahigh error rate, HCV variants are continuously produced
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the other hand, innate and adaptive immune responseplay an important role in the control of HCV infection,
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in HCV infection, resulting a weak immunologic response for complete elimination of HCV infected hepatocytes. The replicon system is not a sufficient tool forinvestigating such an immunological mechanism. A potentially safe and stronger treatment for chronic hepatitis
C by combination of several drugs with different mechanisms is expected especially in Japan, where the patientsis becoming older, and in vitro replicon system will be
still expected to produce a useful information for making
a new drug design.

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