Progress with hepatitis C

Investigation of the cell biology and molecular biology of HCV has been hampered by the lack of a conventional cell culture system, but recent technical developments promise to remove this impediment. The development of subgenomic replicons was the first breakthrough [1] although, in this system, replication occurs in Huh-7 cells (derived from a human hepatoma) without assistance from HCV structural proteins and so may not be fully authentic. Adaptation to this cell line occurs through point mutations in the non-structural genes NS3, NS5A and/or NS5B [2]. Full length genomes containing these mutations amplify to high levels in Huh-7 cells and express the HCV envelope glycoproteins E1 and E2 [3]. However, nuclease-resistant buoyant-density fractions containing HCV RNA do not represent authentic virions, because they are produced also in cells infected with subgenomic replicons [3]. Retroviral pseudotypes (HCVpp) can be made containing the envelope proteins of HCV [4–7]. Infectivity requires co-expression of E1 and E2 [4,6] heterodimers as non-covalently bound glycoprotein complexes modified by passage through the Golgi compartment [5]. None of the several putative cellular receptors for HCV confers permissivity to the Huh-7 cell line [4,6]. However, soluble CD81 and/or a mAb reactive with CD81, inhibit infectivity of HCVpp, implying that CD81 is an essential component of the cellular receptor complex [4,6–9]. In addition, siRNA experiments show that the level of surface expression of scavenger receptor class-B type I (SR-BI) correlates with permissivity for several distinct genotypes of HCV [7]. Antibodies to E2 can neutralise infectivity of HCVpp [4,6], while polyclonal human sera may react broadly with multiple genotypes, yet fail to neutralise all of them [7]. HCVpp can fuse with liposomes; if these contain fluorescent probes, then fusion can be monitored by dequenching [10]. This HCVpp fusion requires low pH, as does influenza haemagglutinin which was used as a control [10]. One particular strain of HCV (Japan Fulminant Hepatitis 1; JFH-1) can replicate in the Huh-7 cell line producing 10 TCID 50 per ml from a cloned genome [11–13]. The characteristics of such HCVcc, reassuringly agree with those described for HCVpp, with mAb against CD81 or E2 neutralising infectivity [11,12]. Studies of virus replication in this system show E1-E2 heterodimers in the ER but not at the plasma membrane [14]. Productive infection has also been produced by transfecting synthetic RNA corresponding to the genotype 1a strain H77-S [15]. Compared to the genotype 2a work [11], this system produced virus more slowly, with a higher proportion secreted into the supernatant (most of which was non-infectious) so that the specific infectivity was lower [15]. However, polyclonal sera from individuals infected with genotype 1a failed to neutralise the genotype 2a virus, suggesting that at least two serotypes may have been identified [15]. An improved cell line Huh7.5 has been identified and shown to propagate both subgenomic and full-length HCV RNA with adaptive mutations in NS3 and NS5A [16]. This subline has a mutation in retinoic acid inducible gene I (RIG-I), an interferon-inducible cellular RNA helicase [17]. The helicase domain of RIG-I normally binds HCV RNA and activates interferon regulatory factor 3 (IRF3); the absence of such activity in Huh 7.5 cells presumably explains their increased permissivity for HCV [17]. Interestingly, the NS3/4A protease normally blocks the activation of IRF-3 [18] suggesting parallels between the in vitro system and that found in humans. It also suggests that therapeutic inhibition of HCV protease may have clinical benefits beyond direct suppression of viral replication. A Huh-7 cell line with a chromosomally integrated HCV cDNA has been described which constitutively produces infectious virus [19]. Both E D I T O R I A L