The hepatitis C virus minigenome: A new cellular model for studying viral replication
Introduction
The hepatitis C virus (HCV) affects around 200 million people worldwide and 3–4 million persons are infected each year. This infection will lead to death in 5–7% of patients infected with HCV as a consequence of liver disease. The virus was first identified by Choo et al. (1989) but until recently development of new treatment for this infection has been hampered by the lack of an efficient cellular system.
HCV is a member of the Flaviviridae family and has been classified as the sole member of a distinct genus called hepacivirus (Bartenschlager and Lohmann, 2000). It is an enveloped virus with a single-stranded positive-sense RNA genome that contains a single long ORF translated as a polyprotein of about 3010 amino acids (Clarke, 1997). This polyprotein is cleaved further into at least nine individual proteins, including the viral RNA-dependent RNA polymerase (NS5B). The ORF is flanked by two untranslated regions (UTR). The 341-nucleotides of the 5′UTR with the first nucleotides encoding the capsid protein form a highly structured domain, the internal ribosome entry site (IRES), which is essential for the initiation of the ORF translation. The 3′UTR is composed of a short variable region, a polyuridine tract of variable length and a 98-nucleotide sequence (3′X) which is highly conserved in various isolates (Tanaka et al., 1995). This region is required for viral infectivity (Yanagi et al., 1999) but its precise role in viral replication is still poorly understood. The NS5B polymerase is a 65 kDa protein responsible for RNA replication (Behrens et al., 1996) which is active in two steps. In a first step, the viral polymerase synthesises a minus-strand RNA that serves as a template for the synthesis of new plus-strand RNA molecules. In HCV infected cells, the enzyme is associated with other non-structural viral proteins (NS) in a replication complex associated with cellular membranes derived from the endoplasmic reticulum (Aizaki et al., 2004, Ali et al., 2002, Gosert et al., 2003). The NS3, NS4A NS4B and NS5A proteins, in association with the NS5B, are required for efficient viral replication in the human hepatocellular carcinoma cell line Huh7 (Lohmann et al., 1999).
Understanding of HCV replication is still limited and would undoubtedly benefit from the development of new cellular models. The replicon system described by Lohmann et al. (1999) has contributed greatly to a better understanding of the functions of viral proteins and of sequences involved in viral RNA replication. In particular, the involvement of the 5′ and 3′UTR in viral replication has been studied. Sequences required for replication have been identified in the 5′UTR (Friebe et al., 2001, Luo et al., 2003) and in the 3′UTR (Cai et al., 2004, Friebe and Bartenschlager, 2002, Yi and Lemon, 2003). The role of the NS proteins has also been largely documented (Lohmann et al., 2003, Ma et al., 2004, Maekawa et al., 2004, Shimakami et al., 2004) using the replicon model.
Recently, an important development resulted from reports describing the production of infectious genotype 2a HCV in human hepatoma cells Huh7 (Cai et al., 2005, Wakita et al., 2005). The ability of this virus to infect cultured cells will benefit substantially antiviral and vaccine discovery programs, however, this system requires the constant use of high laboratory safety standards making difficult its routine use for high throughput screening and other applications.
It should be stressed that the translation and replication mechanisms are linked in the replicon system thus impairing any thorough analysis of their mechanisms. In fact, the arrest of translation following HCV IRES modifications leads to a lower expression level of the replication complex. Consequently, the synthesis of a new minus and plus-strand RNA is reduced. Moreover, decreasing replication efficiency leads to a decrease in the amount of viral RNA molecules and thus of the translation level. To overcome this difficulty, a new cellular system was developed, where the viral translation and replication steps operate independently thanks to the constitutive expression of HCV NS proteins and the use of a minigenome construction as reporter transgene in hepatoma cells.
Section snippets
Cell culture
The human hepatoma cell line Huh7 was cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS) and gentamycine (50 μg/mL) at 37 °C in a 5% CO2 atmosphere. Huh7/Rep 5.1 cell line was established after electroporation of Huh7 cells with the Rep5.1 RNA replicon obtained by transcription of pFK-I389neo/NS3-3/5.1 plasmid (kindly provided by R. Bartenschlager) and selection with 500 μg/mL G418. The cells were aliquoted and frozen after 1 month of
Constitutive expression of the replication complex
The use of the replicon system has provided considerable significant information on HCV replication mechanisms. However, in this system the translation of viral proteins is always linked to viral replication. One strategy for avoiding this problem is to produce constitutively the replication complex by inserting the gene coding for the HCV non-structural proteins (NS3-NS5B) into the genome of Huh7 cells. To enhance replication, Huh7 cells permissive to HCV replication were used. They were
Discussion
A new cellular system for the study of HCV replication is described. It was shown that RNA minigenomes carrying a reporter gene inserted between the HCV 5′ and 3′UTR sequences could be efficiently replicated by the HCV replication complex. It was demonstrated that minimal RNA minigenomes could be replicated not only in the Huh7/NS3-5B cells producing constitutively HCV NS proteins, but also in the Huh7/Rep5.1 cells. This shows that RNA minigenomes could be replicated efficiently by the
Conclusion
A new model was developed for studying HCV replication in cellular systems. The use of a minimal RNA minigenome in cells constitutively expressing the HCV replication complex means the replication and translation mechanisms can now be dissociated. This provides a useful tool for studies aimed at the investigation of the poorly known basic molecular mechanisms involved in the replication and expression of the HCV genome. For example, it could be helpful for identifying cis-elements involved in
Acknowledgments
We thank Simon Litvak, IFR66, Bordeaux for critical reading of the manuscript and helpful discussions, Ralph Bartenschlager and Universitätsklinikum Heidelberg for providing us the replicon Rep5.1. This work was supported by grants from the Agence Nationale de Recherche contre le SIDA, the Ligue Régionale contre le Cancer, the Réseau Hépatite C, the CNRS and the University Victor Segalen Bordeaux 2.
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