screening formats using cell protection as a readout [19]. In a envelope glycoproteins (E1�E2). They had no activity against VSVpp pseudo-particles. Since the only HCV proteins contained in HCVpp are the envelope glycoproteins, these results suggest that the inhibitors target the viral proteins E1 and/or E2, or their functions are distinct from those involved in entry of VSVpp. Indeed, this was confirmed for Inh-2 as substitutions in either E1 or E2 conferred resistance to this compound. There are many possible explanations for the resistance to a single compound arising in either E1 or E2. The simplest is that mutation of a residue in one protein of the heterodimer can affect the conformation of the compound binding site in the other. Another straightforward possibility is that the compound binding interface is made up of both proteins.
interest that these mutations were selected independent of each other, in separate experiments, leading to questions as to whether or not they can co-exist on the same virus. Further studies are required to determine how this combination of mutations would affect viral fitness and resistance to Inh-2. It is interesting to note that Inh-1 was active against only genotype 1a, while Inhs 2 and 3 were active against both genotype 1a and 1b but not 2a. Due to the genetic heterogeneity among HCV isolates belonging to different genotypes, broad spectrum antivirals with activity spanning across subtypes and genotypes are desired. Incomplete coverage by leads targeting both HCV structural [8,9] as well as nonstructural [43] proteins has been reported. It is anticipated that medicinal chemistry efforts relating to the hits identified in this report might result in broad genotype coverage. The other 2 early inhibitors had no activity against HCVpp suggesting that these could target an HCVcc-specific entry event or an aspect of the early phase of the life cycle not recapitulated in the HCVpp model. It is of interest that these inhibitors had no effect on transfected whole-genome vRNA or HCV replicons (data not shown) suggesting they could target a stage in the life cycle that occurs between envelope fusion and the initiation of vRNA replication such as nucleocapsid uncoating or the events directly preceding the initiation of vRNA translation/replication. Importantly, the identification of both entry and potentially post-entry/ pre-replication inhibitors validated one of the goals of the infectious virus screening which was to probe novel targets previously inaccessible using other surrogate model systems. Eight of the screen hits highlighted here exhibited a similar profile to a genome replication inhibitor (BMS-339; protease inhibitor) as they blocked HCVcc replication. Replication was blocked when initiated by infection or by transfection of vRNA. They also exhibited similar potency against a genotype 2a replicon demonstrating that these were bonafide genome replication inhibitors. Six of these hits are likely to be DAAs since they were inactive in HCV gt1 or BVDV replicon assays conducted in Huh7 cells. This was confirmed for Inhs 6 and 7 which selected for resistance substitutions exclusively within NS5A or NS4B, respectively. The remaining 2 inhibitors had similar potencies in HCVcc genotype 2a, 1a and BVDV replicon assays suggesting a common and likely cellular target shared within the replication machinery of these flaviruses. The final 4 inhibitors were confirmed to be late-stage inhibitors by their ability to block the production of infectious virus, but not replication. All of the inhibitors also blocked the replication of HCVcc chimeras with genotype 1a, 1b or 2a Core-NS2. As a result, it was not possible to discern if the activity was linked to the structural or non-structural proteins. The possibility exists that inhibitors targeting structural proteins could be active at both early and late stages of replication. Nonetheless, no inhibitors that were active at early or late stages demonstrated activity against both. The selection and characterization of resistant viruses is a powerful tool to further understand the target, mechanism and spectrum of inhibitors. At the time this work was completed, we performed the selection of resistant viruses to a sampling of the HTS leads to demonstrate the utility and various results of the method. While inhibitors that target entry proteins and replication proteins can be readily selected, viruses resistant to inhibitors of host proteins were unable to be selected in these initial studies. Presumably selection conditions may be altered to enable selection for viruses that escape inhibition of host proteins, although it may be dependent on the particular host target [44,45]. In summary, we leveraged a genotype 1a/2a intergenotypic HCVcc chimeric reporter virus, capable of relatively high titer
