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Southern Research Institute - Dr. Michael Murray
Georgetown University - Dr. Brent Korba

Hepatitis C Virus Assays

M. Murray, PhD., Southern Research Institute
B. Korba, Ph.D., Georgetown University

Southern Research Institute Details

HCV RNA Replicon Protocol

Introduction

We use the cell line Huh7 ET (luc-ubi-neo/ET), which contains a new HCV RNA replicon with a stable luciferase (LUC) reporter. This particular construct has not been described in the scientific literature. It is similar to the cell line 5-2 (1), but contains additional modifications that make the cell line more robust and provide stable LUC expression for antiviral screening. This composition of the replicon is shown diagrammatically in Fig. 1.

Fig. 1. Structure of the HCV RNA replicon of cell line ET. The HCV RNA replicon ET contains the 5’ NTR (IRES) of HCV (5’) which drives the production of a firefly luciferase (Luc), ubiquitin (Ubiq), and neomycin phosphotransferase (Neo) fusion protein. Ubiquitin cleavage releases the LUC and Neo genes. The EMCV IRES element (E-I) controls the translation of the HCV structural proteins NS3-NS5. The NS3 protein cleaves the HCV polyprotein to release the mature NS3, NS4A, NS4B, NS5A and NS5B proteins that are required for HCV replication. At the 3’ end of the replicon is the authentic 3’ NTR of HCV. Not drawn to scale.

The LUC reporter is used as an indirect measure of HCV replication. The activity of the LUC reporter is directly proportional to HCV RNA levels and positive control antiviral compounds behave comparably using either LUC or RNA endpoints. The use of the LUC endpoint is more economical than HCV RNA and can be used for high-throughput applications to screen libraries of compounds.

Primary HCV RNA replicon assay

First we examine the effect of drugs added in triplicate at a single high-test concentration of 20 mM on HCV RNA-derived LUC activity and cytotoxicity. Human interferon alpha-2b is included in each run as a positive control compound. Subconfluent cultures of the ET line are plated out into 96-well plates that are dedicated for the analysis of cell numbers (cytotoxicity) or antiviral activity and the next day drugs are added to the appropriate wells. Cells are processed 72 hr later when the cells are still subconfluent. Compounds that reduced the LUC signal by 50% or more relative to the untreated cell controls move forward in the program. Compound cytotoxicity is assessed as the percent viable cells relative to the untreated cell controls. This data is also reported.

HCV RNA replicon confirmatory assay

The HCV RNA replicon comfirmatory assay is then used to examine the effects of compounds at five half-log concentrations each (Fig. 2). Human interferon alpha-2b is included in each run as a positive control compound. Subconfluent cultures of the ET line are plated out into 96-well plates that are dedicated for the analysis of cell numbers (cytotoxicity) or antiviral activity and the next day drugs are added to the appropriate wells. Cells are processed 72 hr later when the cells are still subconfluent. Compound EC50 and EC90 values (antiviral activity) are derived from HCV RNA levels assessed as either HCV RNA replicon-derived LUC activity or as HCV RNA using TaqMan RT-PCR. Compound IC50 and IC90 values (cytotoxicity) are calculated using CytoTox-1 (Promega), a colorimetric assay used as an indicator of cell numbers and cytotoxicity when the LUC assay system is employed, while ribosomal (rRNA) levels determined via TaqMan RT-PCR are used as an indication of cell numbers in the RNA-based assay. Compound selectivity indices SI50 and SI90 values are calculated from the spreadsheets (Fig. 3).

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Fig. 2A. Standardized 5-2 cell line antiviral evaluation 96-well plate format for the assessment of drug cytotoxicity.

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Fig. 2B. Standardized 5-2 cell line antiviral evaluation 96-well plate format for the determination of antiviral activity.

Figure 3A. See PDF. Activity report for the assessment of antiviral activity in the ET cell line using a LUC endpoint and a colorimetric measure of cell numbers as an indicator of cytotoxicity.

References

1. Krieger, N., V. Lohmann, and R. Bartenschlager. 2001. Enhancement of hepatitis C virus RNA replicon replication by cell culture-adaptive mutations. J. Virol. 75:4614-4624.

Georgetown University Details

Introduction

A variety of cell-culture based anti-HCV analyses are available. Candidate compounds are initially assed in a primary screening assay. Compounds demonstrating reasonable antiviral and cytotoxicity profiles are then candidates for several additional follow-up analyses. For the primary screening assay, routinely 2-3 mg are requested for compounds with molecular weights in the range of standard nucleosides (e.g. 300-500). Additional compound may be required for follow-up analyses. Molecular weights and solubility information should be provided if available. If no preferred solvent is specified, 100% tissue culture DMSO will be used. Compounds should soluble in aqueous solutions (normal pH range) at a minimum of a 10X final testing concentration or in DMSO at a minimum 50X test concentration. EtOH is generally not well tolerated by the cell lines used for these studies, but final concentrations of EtOH of less than 0.03% are acceptable. Compounds which need to be tested in other solvents should be accompanied by a small amount of solvent (under a separate accession number) to control for cytotoxicity. For compounds in solution, approximately 0.25 ml of a 100X stock is minimally required.

Primary assay

Antiviral activity against HCV is assessed in a 3-day assay (Okuse, et al., 2005, Antivir. Res. 65:23; Korba, et al., 2008, Antivir. Res. 77:56) using the stably-expressing HCV replicon cell line, AVA5 (sub-genomic (CON1), genotype 1b) (Blight, et al., 2000, Science 290:1972) maintained as sub-confluent cultures on 96-well plates. Antiviral activity is determined by blot hybridization analysis of intracellular HCV RNA (normalized to the level of cellular B-actin RNA in each culture sample). Cytotoxicity is assessed by neutral red dye uptake in cultures maintained in parallel plates.

EC₅₀, EC₉₀ and CC₅₀ values are calculated by linear regression analysis (MS EXCEL^®, QuattroPro^®) using data combined from all treated cultures (Korba & Gerin, 1992, Antivir. Res. 19:55; Okuse, et al., 2005, Antivir. Res. 65:23). Standard deviations for EC₅₀ and EC₉₀ values are calculated from the standard errors generated by the regression analyses. EC₅₀ and EC₉₀ are drug concentrations at which a 2-fold, or a 10-fold depression of intracellular HCV RNA (relative to the average levels in untreated cultures), respectively, is observed. CC₅₀ is the drug concentration at which a 2-fold lower level of neutral red dye uptake (relative to the average levels in untreated cultures) is observed. The Selectivity index (S.I.) is calculated as CC₅₀/EC₉₀. Recombinant human interferon 2b (PBL laboratories, Inc.) is used as an assay control. Limited supplies of compounds currently in clinical trials that are directed against NS3 and NS5B are also available.

Secondary assay

This assay assesses activity against additional genotypes using the format descrived for the primary assay. Activity against the genotype 1b HCV is included for comparison. Currently available is a replicon cell line containing H/FL-Neo (genotype 1a (H77), full length construct) (Blight, et al., 2003, J. Virol. 77:3181). A genotype 2a construct (J6/JFH-1, full length) is currently being assessed for future inclusion. EC50, EC90, CC50 and S.I. values are calculated for each replicon cell line.

Tertiary assays

In addition to standard antiviral assays, several other types of anti-HCV activities can also be assessed.

Drug combination

Compounds are mixed at approximately equipotent concentrations and this molar ratio is maintained during serial dilution (Korba, 1996, Antivir. Res. 29:49; Iyer et al., 2004). Usually, three different ratios are used in one experiment. Cultures are treated with 6-8 serial dilutions of the mixtures, as with the corresponding monotherapies, as described for the primary assay. Evaluation of drug interactions in the combination treatments is conducted against the corresponding monotherapies in the same experiments using the Combostat® (Biosoft, Inc.) analysis software. For combination treatments, EC50, EC90, CC50 and S.I. (CC50/EC90) are presented for the first compound listed. The molar ratio of the compounds in each combination is also indicated.

Drug-resistant HCV

Since there are currently no licensed anti-HCV drugs for which resistance mutations have been identified, a panel of mutants conferring resistance to compounds in mid to late phase clinical trials has been compiled. This panel will continue to evolve as trials and licensing progress. Currently available as stable replicon-containing cell lines (Korba, et al., 2008, Antivir. Res. 77:56) are genotype 1 B NS5B S282T (Perra, et al., 2005, Nucleosides Nucleotides Nucleic Acids 24:767), and NS3 A156S and NS3 A156V (Courcambeck, et al., 2006, Antivir. Ther. 11:847) drug-resistant mutants. The genetic background is the same as that in the BB7 replicon (AVA5 cells) used in the primary assay. Activity against these mutants is assessed as decribed in the primary assay, except that semi-quantitative real-time PCR is used for the analysis of HCV RNA due to reduced replication levels.

Currently available for use in transient transfection assays are the following mutants constructed in the genotype 1a background used in the secondary assays: NS5B S282T (Pierra, et al., 2005, Nucleosides Nucleotides Nucleic Acids 24:767), and NS3 R155K (Courcambeck, et al., 2006, Antivir. Ther. 11:847). The genotype 1b mutants can also be assessed in this manner. For this assay (Korba, et al., 2008, AAC, in press) Huh7.5 cells are transfected with HCV RNA using Liofectamine 2000™ (Gibco, Inc.) in 6-well culture plates. Three days post-transfection, cultures are exposed to 125µg/mL G418 and test compounds. After 10-14 days, surviving colonies are fixed, stained, and counted. EC50 and EC90 values are calculated for each transfected RNA.