Screening of Hepatitis C Virus Inhibitors Using Genotype 1a HCV Replicon Cell Lines

Margaret Robinson1, Yang Tian1, Nikos Pagratis1, William E. Delaney1

1 Gilead Sciences, Foster City, California
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 17.7
DOI:  10.1002/9780471729259.mc1707s22
Online Posting Date:  August, 2011
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Abstract

Hepatitis C virus (HCV) replicons are the primary tools for identifying and evaluating anti‐HCV compounds during drug discovery research. Genotype 1a and 1b are the most common genotypes in North America and Europe. These genotypes display significant genetic divergence (∼20% at the nucleotide level and ∼14% at the amino acid level), which can translate into marked differences in drug susceptibility. Thus, it is critical that potential therapeutics be assessed against both genotypes to ensure antiviral efficacy in the broad genotype 1 patient population. This unit describes assays for screening HCV inhibitors using replicons with a focus on genotype 1a. Specific protocols are provided for screening 1a replicons that encode the Renilla luciferase gene and for replicons that do not encode exogenous reporter genes, both in 96‐well (manual) and in 384‐well (high‐throughput) formats. Curr. Protoc. Microbiol. 22:17.1.1‐17.1.29. © 2011 by John Wiley & Sons, Inc.

Keywords: hepatitis C virus (HCV); genotype 1a; drug screening; replicon

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Determination of Antiviral Activity (EC50 Values) in Genotype 1a HCV Replicons Using a Kinetic NS3 Protease Assay
  • Alternate Protocol 1: Determination of Antiviral Activity (EC50 Values) in Genotype 1a HCV Replicons Using an End‐Point NS3 Protease Assay
  • Basic Protocol 2: Screening Renilla Luciferase Reporter Replicons Using a Luciferase Assay (96‐Well Format)
  • Basic Protocol 3: High‐Throughput (384‐Well) Screening of Non‐Reporter Replicon Cell Lines Using the NS3 Protease Assay
  • Basic Protocol 4: High‐Throughput (384‐Well) Screening of Renilla Reporter Replicon Cell Lines Using Luciferase Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Determination of Antiviral Activity (EC50 Values) in Genotype 1a HCV Replicons Using a Kinetic NS3 Protease Assay

  Materials
  • Genotype 1a HCV replicon cell line (e.g., H77 replicon cell line, Apath; or any cell line with similar or higher levels of HCV replicon expression)
  • Complete Dulbecco's modified Eagle medium (DMEM; see recipe)
  • 1× Dulbecco's phosphate‐buffered saline (DPBS) without calcium and magnesium (Cellgro, cat. no. 21‐031)
  • 0.5% (10×) trypsin with EDTA⋅4Na (Invitrogen, cat. no. 15400‐054)
  • Replicon cell assay medium (see recipe)
  • Antiviral test compounds, suggested control compounds are the nucleoside, 2′C‐methyl adenosine (2′CMA), or the NS3 protease inhibitor BILN‐2061 (both available for purchase from Acme Biosciences)
  • DMSO
  • 5× cell lysis buffer (Promega, cat. no. E1531)
  • 5 M NaCl solution (Sigma Aldrich, cat. no. S6546)
  • TR‐FRET‐Q NS3 protease substrate (see recipe)
  • 37°C, 5% CO 2 humidified cell culture incubator
  • Cell culture flasks (e.g., Corning 175‐cm2, cat. no. 431080)
  • Aspirator
  • 10‐ml pipets
  • 50‐ml disposable tubes (BD Falcon, cat. no. 352097)
  • Microscope and hemacytometer
  • 96‐well plates for drug dilution (V‐bottom plate; VWR International, cat. no. 249662), media dilution (deep‐well; VWR International, cat. no. 47734‐788), and fluorescence assays (white plate with flat bottom; Sigma‐Aldrich, cat. no. CLS3610)
  • Multi‐channel pipettors (10‐µl, 200‐µl, 1000‐µl; e.g., Rainin P10, P200, P1000)
  • Microplate shaker (Thermo Scientific Barnstead/Lab‐Line titer plate shaker or similar)
  • Fluorescence plate reader (e.g., PerkinElmer Victor3 fluorescence/luminescence reader or similar, e.g., Biotek Synergy, Molecular Devices Analyst)

Alternate Protocol 1: Determination of Antiviral Activity (EC50 Values) in Genotype 1a HCV Replicons Using an End‐Point NS3 Protease Assay

  • NS3 substrate stop solution (see recipe)

Basic Protocol 2: Screening Renilla Luciferase Reporter Replicons Using a Luciferase Assay (96‐Well Format)

  Materials
  • Genotype 1a HCV replicon cell line expressing the reporter gene Renilla luciferase (Robinson et al., )
  • Renilla luciferase assay system (Promega, cat. no. E2820) containing:
    • Renilla luciferase assay lysis buffer (see recipe)
    • Renilla luciferase assay reagent (see recipe)
  • 1× Dulbecco's phosphate‐buffered saline (DPBS) without calcium and magnesium (Cellgro, cat. no. 21‐031)
  • 37°C, 5% CO 2 humidified cell culture incubator
  • 37°C water bath
  • Microplate shaker
  • Multi‐channel pipettors (10 µl, 200 µl, 1000 µl, e.g., Rainin P10, P200, P1000)
  • Luminescence plate reader (PerkinElmer Victor Light is used here, but there are additional instrument choices, e.g., Packard Top Count, PerkinElmer Envision, Biotek Synergy)
  • Additional reagents and equipment for preparation of cells (see protocol 1)

Basic Protocol 3: High‐Throughput (384‐Well) Screening of Non‐Reporter Replicon Cell Lines Using the NS3 Protease Assay

  Materials
  • Genotype 1a HCV replicon cell line
  • Replicon cell assay medium (see recipe)
  • HCV NS3 protease inhibitor ITMN‐191 (Selleck Chemicals, cat. no. S1183)
  • Puromycin (Sigma Aldrich, cat. no. P7255)
  • Test compounds
  • 100% DMSO
  • 5 mM calcein‐AM stock solution (Anaspec, cat. no. 89203)
  • 1× Dulbecco's phosphate buffered saline with Ca2+ and Mg2+ (DPBS) (Mediatech, cat. no. 21‐030‐CM)
  • 5× cell lysis buffer (Promega, cat. no. PR‐E1483)
  • 5 M NaCl solution (Sigma Aldrich, cat. no. S6546)
  • 250 µM TR‐FRET‐Q NS3 protease substrate stock solution (see recipe)
  • NS3 substrate stop solution (see recipe)
  • 384‐well black polystyrene cell culture treated assay plates (Greiner Bio‐one, cat. no. 781086)
  • 384‐well polypropylene compound storage plates (Thermo Scientific, cat. no. 4341)
  • 96‐well polypropylene compound storage plates (Corning, cat. no. 3357)
  • Dual‐pod BioMek FX automation workstation equipped with a 384‐multichannel pipettor and a span‐8, 8‐channel pipettor (Beckman Coulter)
  • BioMek FX AP384 P30 tips (Beckman Coulter, cat. no. 719223)
  • 37°C, 5% CO 2, 85% humidity incubator
  • Biotek ELX405 plate washer
  • MicroFlo Select bulk dispenser (Bio Tek Instruments) and 5‐µl cassettes
  • Envision plate reader (Perkin Elmer)
  • Additional reagents and equipment for cell maintenance and preparation (see protocol 1)

Basic Protocol 4: High‐Throughput (384‐Well) Screening of Renilla Reporter Replicon Cell Lines Using Luciferase Assay

  Materials
  • HCV replicon cell line (e.g., human liver Huh7 cell line carrying a Renilla luciferase reporter carrying HCV genotype 1a replicon; Robinson et al., )
  • Dual‐Glo luciferase buffer (Promega, cat. no. E298B)
  • Dual‐Glo Stop & Glo assay solution (Promega, cat. no. E314B)
  • 384‐well polypropylene compound storage plates (Thermo Scientific, cat. no. 4341)
  • 384‐well black polystyrene cell culture–treated assay plates (Greiner Bio‐one, cat. no. 781086)
  • Additional reagents and equipment for cell preparation and maintenance (see protocol 4)
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Figures

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Literature Cited

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