Development of a cell-based hepatitis C virus infection fluorescent resonance energy transfer assay for high-throughput antiviral compound screening

Abstract

A major obstacle in the treatment of chronic hepatitis C virus (HCV) infection has been the lack of effective, well-tolerated therapeutics. Notably, the recent development of the HCV cell culture infection system now allows not only for the study of the entire viral life cycle, but also for the screening of inhibitors against all aspects of HCV infection. However, in order to screen libraries of potential antiviral compounds, it is necessary to develop a highly reproducible, accurate assay for HCV infection adaptable for high-throughput screening (HTS) automation. Using an internally quenched 5-FAM/QXL 520 fluorescence resonance energy transfer (FRET) substrate containing the HCV NS3 peptide cleavage sequence, we report the development of a simple, mix-and-measure, homogenous, cell-based HCV infection assay amendable for HTS. This assay makes use of synchronized, nondividing human hepatoma-derived Huh7 cells, which support more-reproducible long-term HCV infection and can be readily scaled down to a 96-well-plate format. We demonstrate that this stable cell culture method eliminates common problems associated with standard cell-based HTS, such as cell culture variability, poor reproducibility, and low signal intensity. Importantly, this HCV FRET assay not only can identify inhibitors that act throughout the viral life cycle as effectively as more-standard HCV assays, such as real-time quantitative PCR and Western blot analysis, but also exhibits a high degree of accuracy with limited signal variation (i.e., Z' > or = 0.6), providing the basis for a robust HTS campaign for screening compound libraries and identifying novel HCV antivirals.

FIG. 1.

HCV NS3 protein accumulation parallels infection kinetics. (A) Huh7 cells were infected with JFH-1 HCVcc at an MOI of 0.01 FFU/cell. Culture supernatant, intracellular RNA, and cellular protein were collected at the indicated times p.i. Intracellular HCV RNA was analyzed by RTqPCR and is displayed as HCV RNA copies/μg total cellular RNA (means ± standard errors of the means; n = 3). Infectivity titers, expressed as FFU/ml, were determined by immunohistochemical analysis of 10-fold serially diluted culture supernatants on naïve Huh7 cells. The lower limit of detection of the assay was 10 FFU/ml. (B) HCV NS3, core, and cellular actin protein levels were determined by WB analysis.

FIG. 2.

FRET signal increases linearly with NS3 protein levels. (A) Recombinant NS3 standard curve showing NS3-dependent cleavage of quenched peptide substrate produces FRET signal proportional to NS3 levels. One unit of NS3 equals 3.036 ng of recombinant NS3/4A. (B) Serial dilutions of sgJFH replicon cell lysate (equal to 8 × 10³ to 1.5 × 10⁵ cells) exhibit FRET signals linear to cell numbers. (C) Huh7 cells infected with increasing MOIs (0.05, 0.1, 0.5, and 1.0 FFU/cell) of HCVcc JFH-1 and lysed at day 6 p.i. exhibit FRET signals proportional to the infection MOI. Shown are the relative fluorescent units (RFU) minus the HCV-negative background detected at cycle 20. Insets represent linear representation of data and R² values.

FIG. 3.

Diagram of the nondividing Huh7 cell culture system. To establish nondividing cultures, Huh7 cells are seeded at 80% confluence on collagen-coated plasticware. Upon reaching 90% confluence, media are replaced with complete DMEM supplemented with 1% DMSO, and cultures are incubated for an additional 20 days. Six days after the initiation of DMSO treatment, cultures reach steady-state cell numbers.

FIG. 4.

Reproducibility of HCV infection in nondividing Huh7 cell cultures. Huh7 cells were plated in a 96-well format, DMSO-treated for 20 days, and then infected with HCVcc JFH-1 at an MOI of 0.05 FFU/cell. (A) HCV RNA and NS3 protease activity were measured 6 days p.i. in 42 replicates. Intracellular HCV RNA was analyzed by RTqPCR and displayed as HCV RNA copies/μg total cellular RNA. NS3 protease activity, expressed as RFU, was determined by FRET analysis. (B) HCVcc infectivity titers on days 3, 5, 7, and 25 p.i., expressed as FFU/ml, were determined by immunohistochemical analysis of 10-fold serially diluted culture supernatant replicates on naïve Huh7 cells.

FIG. 5.

NS3 FRET analysis of HCVcc infection in nondividing Huh7 cells. (A) Huh7 cells were plated in a 96-well format, DMSO treated for 20 days, and then infected with HCVcc at MOIs of 0.01 (diamonds, white bars), 0.05 (squares, black bars), and 0.1 (circles, gray bars) FFU/cell. On indicated days p.i., triplicate wells were harvested. Intracellular HCV RNA was determined by RTqPCR (lines), and NS3 protease activity was monitored by FRET analysis (bars). Results are graphed as mean ± standard error of the mean. (B) Linear representation of HCV copies/μg RNA versus RFU generated from data points corresponding to cultures infected with HCVcc at an MOI of 0.05 FFU/cell. R² value was determined from data points corresponding to days 1 to 8 p.i.

FIG. 6.

Quantitative identification of inhibitors that act throughout the HCV life cycle. (A) HTS experimental design. (B) DMSO-Huh7 cells were infected with HCVcc at 0.05 FFU/cell and treated with 2.5 μM CsA, 100 U/ml IFN-α, 100 U/ml IFN-β, 100 U/ml IFN-γ, 10 μM MA, or 18.5 μM NM107. Compounds were added at 2 days p.i. and were replenished in fresh medium at day 4 p.i. At day 6 p.i., triplicate cultures were assayed for HCV RNA levels by RTqPCR and for NS3 protein levels by FRET. Data are presented as a percentage of mock-treated cells. (C) HCV NS3 and cellular actin protein levels in parallel cultures were determined by WB analysis. (D) DMSO-Huh7 cells were infected with HCVcc at 0.05 FFU/cell and treated with HCV inhibitors that act at different stages of HCV infection: 50 μg/ml α-CD81, 100 μg/ml α-E2, 2.5 μM CsA, 250 U/ml IFN-β, 18.5 μM NM107, 200 μM naringenin, and 500 μM NB-DNJ. Compounds were added at the time of infection and were replenished every 2 days over the 6-day assay. At day 6 p.i., triplicate cultures were assayed for HCV RNA levels by RTqPCR and for NS3 protein levels by FRET. Data are presented as a percentage of mock-treated cells.

FIG. 7.

HCV FRET assay Z′ analysis. (A) The Z′ equation used to measure the distance between the standard deviations for the positive (signal) and negative (noise) controls of the assay (σ, standard deviation; μ, average; C⁺, positive controls; C⁻, negative controls). (B to D) Graphical representation of FRET signal (RFU) from three representative plates (n = 46 separate samples) used to calculate the Z′ per plate. HCV-infected 96-well plates containing untreated and treated samples were analyzed. IFN-β (250 U/ml) was used as a positive inhibitor of HCV replication, as it consistently and reproducibly reduced NS3 protease activity. The standard deviations and the number distribution between the end-point signals obtained for the controls (untreated) versus the background (IFN treated) were measured, and the Z′ statistic was calculated.