Studying Hepatitis C Virus: Making the Best of a Bad Virus

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Abstract
After the landmark report of the first HCV cDNA clones in 1989 (30), many expected HCV molecular virology to quickly advance to a state rivaling that of other positive-strand RNA viruses. More than 15 years later, this predication is only beginning to be realized. Significant advances in understanding the key steps of the HCV life cycle have been made in recent years, although many steps remain enigmatic (Fig. 1). Early attempts to coax replication in cell culture provided glimmers of hope, but none of this work provided tractable systems that became widely adopted (see reference 130 for a review). It was not until 1997, after the discovery that the original HCV cDNA clones lacked a highly conserved 3′-terminal genome fragment (109), that the first full-length functional cDNA clones were reported (108). In the absence of permissive cell culture systems, the infectivity of RNA transcripts from these clones was assessed by intrahepatic inoculation of chimpanzees (108). Attempts to demonstrate replication of these RNAs in cell culture failed. Through the use of bioinformatics, chimpanzee infections, surrogate expression systems, and biochemical analysis, the structure of the HCV genome, the polyprotein processing mechanisms, the protein topology, and some protein functions were defined, all without the ability to monitor RNA replication in a cell culture environment (Fig. 2). The advent of the subgenomic genotype 1b (isolate Con1) replicon system, first reported by Lohmann et al. in 1999 (134), established persistent HCV RNA replication in a human hepatoma cell line (Huh-7) (Fig. 3A). The inefficiency with which RNA replication was initiated in this system limited its utility, but this breakthrough provided a basis for further optimization. Blight et al. isolated subclones of replicon-transduced Huh-7 cells cured by alpha interferon treatment that showed enhanced permissiveness for HCV RNA replication (19). The most famous of these subclones, Huh-7.5, appears to harbor a defect in the retinoic-acid inducible gene I (RIG-I) intrinsic cellular antiviral response pathway (204). In addition, sequencing of HCV RNAs in replicon-containing cell clones identified a spectrum of adaptive mutations in nonstructural (NS) proteins that could dramatically enhance RNA replication (17; reviewed in reference 9). Adaptive mutations that rendered other genotype 1 RNAs (such as 1a, strain H77) replication competent in cell culture were soon identified (18). Unfortunately, full-length HCV replicons incorporating these changes, despite robust RNA replication, failed to yield infectious virus (18, 84, 178).