the presence of 26EC90 of each compound and supernatant from these cells (virus producer cells) was harvested at 48 h pi and ?transferred to naive target cells. Luciferase expression was measured in both producer and target cells at 48 h pi. As expected, viral replication was inhibited in producer cells by both the control entry (EI) and replication (BMS-339) inhibitors (Fig. 8A). On the contrary, LY411575 and Inhs 14?7 had no effect on genome replication in infected producer cells but rather ?inhibited the infection of naive target cells (Fig. 8A) suggesting that these inhibitors target a late stage of the virus life cycle that affects
the production of infectious virus. LY411575, as well as Inhs 14?17, exhibited similar potency against genotype 1a, 1b and 2a HCVcc chimeras (Fig. 8B).
Resistance selection
Antiviral discovery is aided significantly using cell culture methodology to select for and characterize resistant viral variants. These studies can yield information regarding; the molecular target of inhibition (is it viral or cellular?), if the resistance pattern/ profile is unique or overlaps with other antivirals, and if the
Figure 6. Early/Entry Inhibitors. A. The potencies (EC50) of early inhibitors (Inhs 1?) against gt 1a/2a-Rluc virus (HCVcc) and HCV pseudo-particles (HCVpp) with the corresponding genotype 1 envelope glycoproteins were compared to identify virus entry inhibitors. Selectivity for HCVpp was confirmed using Vesicular stomatitis virus glycoprotein pseudo-particles (VSVpp) and cytotoxicity (CC50) using Cell-Titer Glo. A. Screen hits (Inhs 1?) that demonstrated similar potency against HCVcc and HCVpp and exhibited selectivity relative to VSVpp and cytotoxicity (entry inhibitors). A control entry inhibitor (EI) was included to confirm the predicted outcome for a bonafide entry inhibitor. B. Screen hits (Inhs 4?) that demonstrated reduced potency against HCVpp (HCVcc-specific early inhibitors). Genotype selectivity was assessed by comparing potency against HCVcc chimeras with genotype 1a, 1b or 2a structural proteins. C. Potency of the entry inhibitors (Inhs 1?) and (D) HCVcc-specific early inhibitors (Inhs 4 & 5) against genotype 1a, 1b and 2a HCVcc and corresponding cytotoxicity. doi:10.1371/journal.pone.0042609.g006
Figure 7. HCV Genome Replication Inhibitors. A. The genome replication inhibitors (Inhs 6?3) were confirmed by comparing potency (EC50) against the 1a/2a-Rluc virus and a corresponding JFH1 replicon. B & C. Genotype 2a and HCV selectivity were assessed by comparing inhibitor potency against genotype 2a, genotype 1a and BVDV replicons and compounds were subsequently grouped into genotype 2a-selective (B) and nonselective (
changes that emerge in the resistant viruses are already present in the population of viruses that are circulating in patients prior to anticipated therapy. We tested the ability of the gt1a/2a chimeric HCVcc virus to be used for the selection of resistant virus variants using a select series of control inhibitors and screen hits and these results along with corresponding genotype coverage are presented in Table 4. EI, an entry inhibitor with genotype 1a and 1b coverage [8] was used as a control for a compound that hits a viral target. As reported previously [8], a V336G substitution in E2 was selected by EI which conferred 45 fold resistance to the drug. In contrast, LY411575, which perturbs HCV core processing by blocking SPP-mediated Core processing, did not select for resistance through ten weeks of culture (Table 4). Of the five screen hits tested, resistance was observed with Inhs 2, 6 and 7 while no resistance was observed for Inhs 4 and 17. Two independent populations were obtained for Inh-2, the genotype 1selective entry inhibitory (Fig. 6), and whole-genome sequencing revealed one population had an L67F substitution in E1 while the other had a W333L substitution in E2 (Table 4). These substitutions conferred complete resistance by themselves (Table 4) or in combination (data not shown). Two populations exhibiting 30 and 100 fold resistance to Inh-6 were selected and sequencing revealed mutations clustered exclusively within NS5A. A combination of F28L, L31M, and F169L conferred 30 fold resistance while a combination of L31M, S38T, and Q123R conferred 100 fold resistance (Table 4). The contribution of each individual mutation has yet to be assessed; however, these results confirmed that Inh-6 represents a novel genotype 2a-selective NS5A inhibitor.
A single population exhibiting .10 fold resistance to Inh-7 was isolated and sequenced. A single G60S substitution in NS4B was identified, suggesting that Inh-7 was a novel genotype 2a-selective NS4B inhibitor. Resistance to Inh-4 or Inh-17 was not observed (Table 4). This finding coupled with the similar potencies observed against genotype 1a, 1b and 2a HCVcc chimeras suggests that these inhibitors may target cellular proteins. Taken together, these findings demonstrated that the gt 1a/2a-Rluc virus can be used for both screening and resistance studies and showed a clear correlation between targets identified through resistance and life cycle stage classification defined by the hit deconvolution strategy.
Discussion
A number of clinical candidates and approved therapies for HCV include agents that target genome replication and have been advanced using the subgenomic HCV replicon (Reviewed in [42]). The recognized need for combination therapies for HCV creates a desire to identify inhibitors acting at other stages of virus infection, including those requiring the virus structural proteins. These stages include, at least, genome encapsidation, release of nascent virions from cells, and spread to and infection of new cells. While retroviral pseudo-particle assays can be used to identify inhibitors of virus entry [8], it is expected that the profile of some entry inhibitors could vary between pseudo-particles and authentic virus. In addition, other steps of entry including the eclipse or uncoating of the virion core, as well as later stages of infection, require a whole HCV virus assay for discovery. Several HCV in vitro growth assays have been reported using the full-length genotype 2a or intragenotypic (2a) chimeric viruses
