From Bench to BedsideThe hepatitis C virus replicon system: From basic research to clinical application
Introduction
With the discovery of the hepatitis C virus (HCV) in 1988 [1] and the implementation of tests for diagnosis of infections with this virus [2], it was expected that the development of antiviral therapies would have soon followed. Initial progress with respect to the delineation of the genomic organization of the virus and the characterization of viral proteins raised the hope that this could be realized in the near future. However, a major obstacle to this was the lack of systems to propagate the virus in tissue culture. Although infection of primary cell cultures and several cell lines had been achieved, the efficiency of these systems was so low that they were of very limited use (reviewed in [3]). Moreover, since viruses are obligate intracellular parasites and heavily depend on host cell functions for their replication, it was difficult to unravel the HCV life cycle in the absence of adequate cell culture systems.
A major step forward was therefore, the development of the replicon system in 1999 [4]. This system allowed for the first time the efficient propagation of genetically modified HCV RNAs (replicons) in a human hepatoma cell line and since then, became a well established tool in most laboratories working with this virus. Although the replicon system is instrumental to characterize at least parts of the HCV life cycle, the question arose as to what extent replicons reflect the in vivo situation and what clinical impact they may have. I will try to address these questions and begin with a short description of the current state of HCV replicon technology.
Section snippets
Principle of the replicon system
The first generation HCV replicons were derived from a consensus genome that was cloned from the liver of a patient chronically infected with a genotype 1b virus [4]. This genome, designated Con1, was genetically modified by replacements of the core to NS2 or core to p7 region by a selectable marker (neo) and an internal ribosome entry site from a picornavirus (encephalomyocarditis virus; Fig. 1). As a result, subgenomic, selectable replicons were obtained composed of two expression cassettes:
Improvements of the replicon system: from subgenomic RNAs to infectious virus production
With the advent of cell culture adaptive mutations and highly permissive cell clones, novel replicons could be established (Fig. 1). These include monocistronic replicons in which RNA translation and replication are exclusively controlled by HCV sequences, replicons that carry a reporter gene for easy read-out of RNA levels, or transient replication systems that no longer depend on time consuming G418 selection (reviewed in [13]). Moreover, HCV replicons derived from genotypes 1a and 2a could
How close do HCV replicons mimic the in vivo situation?
In principle, most of the steps of the viral replication cycle such as polyprotein expression and processing, formation of the replication complex, or RNA amplification should be properly recapitulated in the replicon system. Caution may be required however, in cases where processes are studied that are dominated by the host cell like IFN-induced antiviral programs or cell cycle dependency of HCV replication, because Huh-7 cells are immortalized tumour cells that differ from regular hepatocytes
Potential impact of HCV replicon technology for the clinic
In spite of these complications, HCV replicons are instrumental in studying the viral life cycle and have therefore, become a major tool in basic research. In addition, replicon technology has several applications with potential impact for the clinic.
Application for drug development
Perhaps the most important clinical application of HCV replicons is their use for the development of antiviral drugs as well as for testing drug combinations with respect to synergistic/antagonistic activity and to reduce the potential of drug resistance development. Owing to new replicon designs that allow the easy measurement of RNA replication by reporter assays (Fig. 1), the replicon system can be used for high throughput screening assays to identify lead compounds in large collections of
Future applications in the clinic
Apart from being a critical component for drug discovery, HCV replicons may become a very important tool for the determination of antiviral drug resistance. Owing to the high error rate of the NS5B RdRp, it is assumed that drug resistance will develop in treated patients. In fact, resistance has already been generated in the replicon system against inhibitors of the NS3 proteinase, the NS5B RdRp or both arguing that the same may happen in vivo [41], [42], [43], [44]. Replicons may therefore, be
Concluding remarks
In summary, the HCV replicon system is a valuable tool to study various aspects of the viral life cycle. Although these RNAs are propagated in tumour cells and highly efficient RNA replication often depends on cell culture adaptive mutations, we can assume that HCV replicons mimic the basic principle of HCV replication as it occurs in vivo. The recent demonstration of antiviral activity of a proteinase inhibitor in the replicon system and in HCV infected patients clearly supports this
Acknowledgements
I am grateful to Dr V. Lohmann for the critical reading of the manuscript and helpful suggestions and to Dr T. Wakita for permission to cite unpublished results. Work in the author's laboratory was supported by grants from the Sonderforschungsbereich 638 (Teilprojekt A5), the Bundesministerium für Bildung und Forschung (Kompetenznetz Hepatitis, contract no. 01KI0102), the European Union (QLK2-CT-2002-01329) and activities of the VIRGIL European Network of Excellence on Antiviral Drug Resistance
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