While the widely used Huh-7 cell line and, particularly, the derivative cell line cured of the genotype 1b replicon known as Huh7-Lunet support robust replication of genotype 1b reporter replicons, these cell lines do not appear to support the generation of stable genotype 1a reporter replicon cell lines. Indeed, we were unable to generate any genotype 1a stable replicon colonies when using replicon RNAs encoding a luciferase reporter gene. In contrast, but consistent with what others have reported, we were readily able to establish genotype 1a replicon cell lines using replicons without a luciferase reporter. To facilitate the generation of luciferase-encoding genotype 1a replicons, we cured cell lines stably replicating a genotype 1a replicon without a reporter gene, using a combination of HCV inhibitors. The resulting cured Huh7 cell lines were remarkably more permissive to genotype 1a replicons with or without reporter genes; in fact, two out of five cured cell lines had permissivity to nonreporter genotype 1a replicons that was enhanced more than 100-fold compared to the permissivity of Huh7-Lunet cells during colony formation assays.
We also tested the ability of the 57C cured cell line to support the replication of genotype 1b and genotype 2a replicons. We found that this cell line supported robust replication of all three genotypes (1a, 1b, and 2a). However, in contrast to genotype 1a, which replicated 30-fold better in 57C than in Huh7-Lunet, genotype 1b showed little enhancement (approximately 3-fold), while genotype 2a replicated better in Huh7-Lunet cells. These results suggest the presence of cellular factors that are able to influence HCV replication in a genotype-specific manner, since permissivity was not globally enhanced for all genotypes.
After establishing these 1a reporter replicons, we next validated their utility in antiviral assays. We found that under standard antiviral screening conditions (e.g., 3-day drug treatment of replicon cells in 96-well plates), replicon cell lines encoding either the Renilla
luciferase had signal-to-noise ratios of >100, with robust Z
factors (>0.7). Furthermore, multiple luciferase cell lines, including those based on the H77 and SF9 1a HCV strains, were demonstrated to have antiviral susceptibilities for multiple drug classes similar to those observed in nonluciferase-containing cell lines. As genotype 1b and 2a luciferase replicons have been previously described (3
), these new genotype 1a replicons will allow side-by-side direct comparison of antiviral potency among the three genotypes in a high-throughput reporter format. Furthermore, these 1a replicons encode either Renilla
luciferase, which utilize coelenterazine-based substrates, whereas typical genotype 1b or genotype 2b replicons utilize firefly luciferase (which utilizes luciferin-based substrates); this opens the possibility to conveniently multiplex these new 1a replicons with 1b or 2a replicons using commercially available luminescence assay kits designed for this purpose.
With the compounds that we tested, we did not note any significant difference in antiviral sensitivity between the H77 and SF9 strains. However, these two 1a strains have a high level of overall homology (99.1% identity at the protein level). The NS3 protease domain and NS5B polymerase have particularly high homology, at 99.4% and 98.9% identity, respectively. NS5A and NS4B, which are emerging targets for drug development, have 98.4% and 97.3% homology, respectively. Overall, having two independent genotype 1a strains to validate inhibitor activity will be valuable for drug discovery efforts, especially given the natural heterogeneity of HCV and the significant impact polymorphisms have already been described to have on some HCV drug classes. We did, in contrast, confirm that a monomeric NS5A inhibitor had dramatically less potency against genotype 1a than against genotype 1b (>1,200-fold less). Such potency information is valuable for assessing potential variability in the clinic but might also yield valuable insight into inhibitor mechanism of action or aid molecular target identification for new drug classes.
Sequencing the 1a replicons stably transfected into Huh7-Lunet or 1a cured cell lines to explore the potential evolution of additional genotype 1a adaptive mutations did not reveal any high-frequency mutations in conjunction with the preexisting adaptive mutations. We observed the previously reported NS4A adaptive mutation K1691R and the nearby I1694T mutation twice each out of 12 sequenced 1a replicon cell lines. I1694, whose role in replication has not yet been characterized, is highly conserved among genotype 1a patient isolates. Positions 1715 (NS4B), 1289 (NS3), and 1646 (NS3) are also highly conserved among genotype 1a patient isolates, and mutations at these residues have an uncertain impact on HCV replication. It should also be noted that some clonal and polyclonal cell lines selected during these studies had no amino acid changes from the parental replicon sequences. Together, these observations suggest that factors specific to the cured cell lines, and not additional adaptive mutations in replicon sequences, are the greatest contributor to the enhanced replication phenotype of genotype 1a replicons in these cells.
In summary, the new luciferase replicon cell lines described in this report will enable convenient high-throughput screening and characterization of antivirals against genotype 1a HCV. The approach of creating cell lines with enhanced permissivity was crucial to the generation of the new 1a reporter replicon cell lines and did not rely on identifying adaptive mutations or using preengineered adaptive mutations beyond those already commonly described. Given the high prevalence of chronic genotype 1a infection, we envision that both the cured cell lines and the luciferase-encoding 1a replicons described herein will facilitate efforts to develop new therapies for HCV. Indeed, we have recently taken advantage of the enhanced permissivity of 1a cured cell lines by using them to phenotype chimeric replicons encoding target genes from clinical isolates (data not shown). In this context, these cell lines are particularly valuable, since such chimeric replicons often have significantly reduced replication fitness compared to that of laboratory HCV strains. Finally, these cell lines could be used, along with other permissive cell lines (e.g., Huh7-Lunet and Huh-7.5), to study cellular factors and pathways that govern HCV replication in a pangenotype or genotype-/subtype-specific manner.