Hepatitis C Virus
3. The HCV Replicon System
Propagation of HCV in cell culture has for a long time not been possible and overcome only gradually by different technical developments. The first achievement was the establishment of the HCV replicon system ( Lohmann et al., 1999 ). By definition replicons are molecules that are capable of self-amplification. In case of HCV, the first generation replicons were derived from a cloned HCV genome by deleting the structural region and insertion of a selectable marker (neomycin phosphotransferase, conferring G418-resistance). A second IRES-element was introduced to allow translation of the HCV nonstructural region. Upon transfection of synthetic RNAs derived from such a construct into the human hepatoma cell line Huh-7 and G418 selection we were able to generate cell lines harboring high amounts of self-replicating HCV RNAs and proteins.
Subsequent studies showed that the original replicon we generated initially from the cloned HCV genome replicated only at a low level and had to acquire cell culture adaptive mutations ( Lohmann et al., 1999; Lohmann et al., 2001; Blight et al., 2000; Krieger et al., 2001; Lohmann et al., 2003 ). These mutations enhanced RNA replication to a level that was sufficient to confer G418 resistance to the transfected cell. Cell culture-adaptive mutations were identified throughout the polyprotein coding sequence with a distinct clustering in the center of NS5A. The most efficient replicon we developed carries 3 adaptive mutations, two in NS4B and one in NS5A that enhance RNA replication cooperatively (Lohmann et al., 2003). Moreover, our recent data also show that the host cell itself contributes significantly to efficient RNA replication. By using a functional screening assay we identified Huh-7 cells that are up to 100-times more permissive than the original cells we used to establish the replicon system (Lohmann et al., 2003). These findings allowed the development of transient replication assays as well as cell lines carrying selectable full length HCV genomes (Pietschmann et al., 2002).
Since the first description in 1999, the HCV replicon system became a very important tool that has been used with great success to study viral RNA replication, identify host cell factors required for HCV replication, to delineate how HCV counteracts the innate immunity and to develop and evaluate antiviral drugs. Moreover, in the past few years the replicon system was instrumental to identify mutations conferring antiviral drug resistance which is of concern when using selective drugs for treatment of persistently infected patients.
Schematic representation of the method used to establish HCV-replicon containing cell lines. The structure of the replicon DNA construct is given in the top. The subgenomic RNAs derived thereform by in vitro transcription are composed of the HCV 5' NTR plus a small fragment of the core-coding region (small box), the neo gene, the EMCV-IRES, HCV NS2-5B or NS3-5B and the 3' NTR. Upon transfection of Huh-7 cells, only those supporting replication of the HCV-RNAs amplify the neo gene and develop resistance against the drug G418. Therefore, only these cells will form colonies whereas untransfected cells and cells that do not support replication of these RNAs will be eliminated during the selection.