Cell‐Based Hepatitis C Virus Infection Fluorescence Resonance Energy Transfer (FRET) Assay for Antiviral Compound Screening

Xuemei Yu1, Susan L. Uprichard2

1 Department of Medicine, University of Illinois at Chicago, Chicago, Illinois, 2 Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 17.5
DOI:  10.1002/9780471729259.mc1705s18
Online Posting Date:  August, 2010
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Abstract

Hepatitis C virus (HCV) affects an estimated 3% of the population and is a leading cause of chronic liver disease worldwide. Since HCV therapeutic and preventative options are limited, the development of new HCV antivirals has become a global health care concern. This has spurred the development of cell‐based infectious HCV high‐throughput screening assays to test the ability of compounds to inhibit HCV infection. This unit describes methods that may be used to assess the in vitro efficacy of HCV antivirals using a cell‐based high‐throughput fluorescence resonance energy transfer (FRET) HCV infection screening assay, which allows for the identification of inhibitors that target HCV at any step in the viral life cycle. Basic protocols are provided for compound screening during HCV infection and analysis of compound efficacy using an HCV FRET assay. Support protocols are provided for propagation of infectious HCV and measurement of viral infectivity. Curr. Protoc. Microbiol. 18:17.5.1‐17.5.27. © 2010 by John Wiley & Sons, Inc.

Keywords: hepatitis C virus; viral lifecycle; Huh7 cells; dimethylsulfoxide (DMSO); fluorescence resonance energy transfer (FRET); NS3 protease; antivirals; high‐throughput screening

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Screening of Anti‐HCV Compounds in Hepatoma Cells
  • Basic Protocol 2: Quantification of HCV Infection Using a Cell‐Based NS3 Protease Assay
  • Support Protocol 1: Generation of HCVcc from In Vitro–Transcribed RNA
  • Support Protocol 2: Generation of HCVcc from Infectious Virus
  • Support Protocol 3: HCVcc Infectivity Titer Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Screening of Anti‐HCV Compounds in Hepatoma Cells

  Materials
  • Huh7 cells (Japan Health Science Research Resources Bank, cat. no. JCRB0403; http://www.jhsf.or.jp)
  • Huh7 cell maintenance medium (see recipe)
  • Huh7 cell maintenance medium with 1% (v/v) dimethyl sulfoxide (DMSO; tissue culture grade)
  • Compound library
  • HCVcc (stock titer ≥3.25 × 104; see protocol 3 and 2)
  • FRET lysis buffer (see recipe), prechilled to 4°C
  • 75‐cm2 or larger tissue culture flasks
  • 96‐well clear flat‐bottom black microtiter BioCoat tissue culture plates (BD Biosciences)
  • 96‐well U‐bottom microtiter plates
  • Plate seals
  • Additional reagents and equipment for growing mammalian cells (Phelan, )

Basic Protocol 2: Quantification of HCV Infection Using a Cell‐Based NS3 Protease Assay

  Materials
  • Sample plate ( protocol 1)
  • 5‐FAM/QXL 520 NS3 FRET peptide substrate (AnaSpec; resuspend and distribute into single‐use aliquots upon receipt)
  • 2× FRET assay buffer (AnaSpec)
  • 1 M dithiothreitol (DTT; appendix 2A)
  • Fluorescent microplate reader with 490‐nm excitation and 520‐nm emission filters
  • Shaking platform
  • Centrifuge with microtiter plate carrier

Support Protocol 1: Generation of HCVcc from In Vitro–Transcribed RNA

  Materials
  • pJFH‐1 (HCV JFH‐1 encoding plasmid; Kato et al., )
  • XbaI restriction endonuclease and compatible 10× buffer
  • Buffered phenol, pH 7 to 8 ( appendix 2A)
  • Molecular‐biology‐grade nuclease‐free distilled H 2O
  • 25:24:1 phenol/chloroform/isoamyl alcohol (see appendix 2A)
  • Chloroform
  • 3 M sodium acetate, pH 7.0 (see appendix 2A)
  • Isopropanol
  • 75% (v/v) ethanol
  • 100% (v/v) ethanol
  • High‐yield in vitro RNA transcription reagents (e.g., Ambion MEGAscript) including:
    • 10× RNA transcription reaction buffer (also see recipe and Lindenbach et al., )
    • dATP, dGTP, dCTP, and dUTP stock solutions (also see appendix 2A)
    • T7 RNA polymerase
    • Nuclease‐free water
    • 2 U/µl DNase (also see appendix 2A)
  • Gel electrophoresis apparatus and reagents including:
    • Molecular‐biology‐grade agarose
    • TE buffer ( appendix 2A)
    • 1% (w/v) ethidium bromide ( appendix 2A)
    • 10× sample loading buffer containing bromphenol blue ( appendix 2A)
    • DNA size ladder
  • Huh7 cells (Japan Health Science Research Resources Bank, cat. no. JCRB0403; http://www.jhsf.or.jp)
  • Huh7 cell maintenance medium (see recipe)
  • Dulbecco's phosphate buffered saline (DPBS) without calcium and magnesium (see recipe in appendix 2A; prepare with tissue‐culture‐grade H 2O and omit Ca2+ and Mg2+)
  • 0.25% trypsin (tissue culture grade) with 2.21 mM EDTA in HBSS (see appendix 2A for HBSS)
  • Serum‐free DMEM and/or Opti‐MEM (both available from Invitrogen), prechilled to 4°C
  • 75‐cm2 (T‐75) or 150‐cm2 (T‐150) tissue culture flasks
  • 50‐ and 15‐ml conical centrifuge tubes
  • Inverted light microscope
  • Refrigerated centrifuge, 4°C
  • 0.4‐cm electroporation cuvettes
  • Electroporator
  • 0.2‐µm cellulose acetate filters for sterilization
  • Additional reagents and equipment for determining DNA and RNA concentration (Gallagher and Desjardins, ), RNA purification by purification kit (Phenol‐Free Total RNA Purification Kit or Ribozol Plus RNA Purification Kit; AMRESCO Inc., http://www.amresco‐inc.com/), lithium chloride precipitation (Diaz‐Ruiz and Kaper, ), spin‐column chromatography, or phenol:chloroform extraction and isopropanol precipitation (Chomczynski and Sacchi, ; Kingston et al., ), and determination of HCVcc infectivity titer ( protocol 5)

Support Protocol 2: Generation of HCVcc from Infectious Virus

  Materials
  • Huh7 cells (Japan Health Science Research Resources Bank, cat. no. JCRB0403; http://www.jhsf.or.jp)
  • Huh7 cell maintenance medium (see recipe)
  • Post‐transfection generated JFH‐1 HCVcc ( protocol 3)
  • Dulbecco's phosphate buffered saline (DPBS) without calcium and magnesium (see recipe in appendix 2A; prepare with tissue‐culture‐grade H 2O and omit Ca2+ and Mg2+)
  • 0.25% trypsin (tissue culture grade) with 2.21 mM EDTA in HBSS (see appendix 2A for HBSS)
  • 75‐cm2 and 150‐cm2 tissue culture flasks
  • 50‐ml conical centrifuge tubes (BD Falcon)
  • Refrigerated centrifuge
  • 0.2‐µm cellulose acetate filter for sterilization

Support Protocol 3: HCVcc Infectivity Titer Analysis

  Materials
  • Huh7 cells (Japan Health Science Research Resources Bank, cat. no. JCRB0403)
  • Huh7 cell maintenance medium (see recipe)
  • HCVcc test samples (see protocols above)
  • Huh7 cell maintenance medium with 0.25% (w/v) methylcellulose (see recipe)
  • 4% PFA fixation buffer (see recipe)
  • Dulbecco's phosphate buffered saline (DPBS) without calcium and magnesium (see recipe in appendix 2A; prepare with tissue‐culture‐grade H 2O and omit Ca2+ and Mg2+)
  • DPBS (without Ca2+ and Mg2+) containing 0.3% hydrogen peroxide (prepare immediately before use)
  • Blocking buffer (see recipe)
  • HCV‐specific primary antibody (Ab), e.g.:
    • AR3A, human anti‐HCV E2 antibody (Law et al., )
    • E910, mouse anti‐HCV NS5A antibody (Lindenbach et al., )
    • C750, mouse anti‐HCV Core (Affinity Bioreagents)
  • Binding buffer (see recipe)
  • Appropriate HRP‐conjugated secondary antibody (Ab) such as:
    • DAKOCytomation EnVision+ System‐HRP labeled ready‐to‐use anti‐mouse antibody (for primary Ab raised in mouse)
    • Goat anti‐human HRP‐conjugated antibody (Pierce; for primary Ab raised in human)
  • AEC (3‐amino‐9‐ethylcarbazole) peroxidase substrate solution (BD Biosciences)
  • 50% (v/v) glycerol in H 2O
  • 96‐well flat‐bottom tissue culture plates
  • 96‐well U‐bottom microtiter plates
  • Inverted light microscope
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Figures

Videos

Literature Cited

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