SelectT automated cell culture system (Tap Biosystems, Greenville, DE). Large-scale virus stocks were prepared as above and maintained at 280uC until use. For compound library screening, infections were performed in 384-well plates by pre-mixing 400 ffu of gt 1a/2a-Rluc virus with 16104 Huh-7.5 cells and dispensing 50 ml on top of 150 nl of compound in DMSO (final 0.3% DMSO) followed by incubation at 37uC. Renilla luciferase activity, reflecting the degree of virus replication, was measured 96 h after infection using the EnduRen reagent (Promega). Luciferase activity was measured using an EnVision Multilabel Reader (Perkin Elmer), Test compounds were serially diluted 3-fold in DMSO to give a final concentration range in the assay of 60 mM to 3 nM. Maximum activity (100% of control) and background were derived from control wells containing DMSO alone or from uninfected wells, respectively. The individual signals in each of the compound test wells were then divided by the averaged control values (wells lacking inhibitor), after background subtraction, and multiplied by 100% to determine percent activity. The corresponding % inhibition values were then calculated by subtracting this value from 100. Dose-response assays were performed in triplicate and average EC50 values (reflecting the concentration at which 50% inhibition of virus replication was achieved) were calculated using XLfit for Excel (ID Business Solutions, Burlington, MA). Cytotoxicity studies were performed in parallel using CellTiter-Glo (Promega). For high-content screening, virus and cells were prepared as above and 50 ul was dispensed into black 384 well optics plates (Thermo Scientific). At 96 h post-infection, cells were fixed for 20 min by adding 17 ul of 16% paraformaldehyde (Electron Microscopy Sciences) to achieve 4% final fixative. Cells were washed and permeabilized twice for 10 min each with PBS containing 1% TX-100 and 0.05% Tween-20 (wash buffer) and blocked for 15 min with PBS with 2% BSA (blocking buffer). Cells were incubated for 1 hr with 3 mg/ml antiHCV Core monoclonal antibody (ABR-Affinity Bioreagents) in 15 ml blocking buffer, washed 3 times with 100 ml of wash buffer and incubated with a 1/400 dilution of Alexa Fluor 488-labeled donkey anti-mouse secondary antibody (Invitrogen) in blocking buffer for 1 hr. Samples were washed three times with wash buffer and 0.5 mg/ml of Hoechst 33258 (Sigma) was added to the final wash to visualize nuclei. The total number of cells per well (Hoechst staining) and infected cells per well (HCV Core) was measured using a Cellomics ArrayScan HCS Reader (Thermo Scientific) and EC50 values calculated as described above. Corresponding Luciferase assays were performed on replica plates and reporter expression was normalized by calculating the relative light units per 100 infected cells.
triplicate and average EC50 values were calculated using XLfit for Excel (ID Business Solutions, Burlington, MA).
Viral RNA transfection and HCV replicon assays
In vitro transcribed gt 1a/2a-Rluc virus genomic viral RNA ?(vRNA) was electroporated into naive Huh-7.5 cells and transfected cells were seeded into 384 well plates (10,000 cells/well) in the presence or absence of inhibitors. Luciferase activity, reflecting the degree of vRNA replication, was measured 24 h after transfection using the EnduRen Reagent (Promega). 10,000 cells were used in this format to increase the Luciferase signal per well as this assay was only 24 h in length compared to the viral infection assays which were either 48 or 96 h. Subgenomic replicon assays using genotypes 1a, 1b and 2a HCV and BVDV replicons have been described previously [47]. All assays were performed in triplicate and average EC50 values were calculated using XLfit for Excel (ID Business Solutions, Burlington, MA).
HCVcc infectious virus release assay
Huh-7.5 cells were plated in 96-well plates at 16104 cells/well and infected 24 h later with gt 1a/2a-Rluc virus at an MOI.1 in the presence of inhibitors at 26 their respective EC90 values. Cells were incubated at 37uC for 48 h and supernatants were collected. Supernatants were clarified by centrifugation, diluted 20 fold and ?used to infect naive Huh-7.5 cells in 96 well plates. Renilla luciferase expression was measured in both producer cells and target cells 48 h post-infection as described above.
HCVcc resistance selection
The gt 1a/2a-Rluc virus was used to infect Huh-7.5 cells and inhibitor was added 12 h post-infection at 56 EC50. Infection was ?allowed to progress for 96 h and cells were split 1:1 with naive Huh-7.5 cells in the presence of inhibitor and cultured for another ?96 h. Viral supernatants were passed onto naive Huh-7.5 cells in the presence of inhibitor and HCVcc replication in the presence of inhibitor was monitored by determining the spread of virus infection, using immunofluorescence (HCV Core), at each passage. Generally, virus stocks were prepared when HCVcc was $10-fold resistant relative to wild-type parental virus. The HCV genome was amplified by RT-PCR (Invitrogen), cloned and amino acid changes that arose during inhibitor selection were identified by analysis of the DNA sequence compared to the parent and control passages in the absence of inhibitor.
