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

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Abstract
Table of Contents
Materials
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Literature Cited

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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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., )
Xba I restriction endonuclease and compatible 10× buffer
Buffered phenol, pH 7 to 8 ( appendix 2A )
Molecular‐biology‐grade nuclease‐free distilled H₂ O
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 H₂ O 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 H₂ O 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 H₂ O 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 H₂ O

96‐well flat‐bottom tissue culture plates
96‐well U‐bottom microtiter plates
Inverted light microscope

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Figures

Figure 17.5.1 Sample plate layout for HTS anti‐HCV compound screening. This illustrates a standard plate layout in which two columns have been reserved for controls. However, compound libraries are often provided with only one empty column for the addition of controls. The layout chosen for individual screening campaigns will need to be adapted to accommodate these restrictions, and, if necessary, to avoid areas prone to “edge effect.”

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Figure 17.5.2 NS3 FRET peptide substrate. (A ) The 5‐FAM/QXL 520 NS3 FRET substrate is an internally quenched peptide with a fluorescent donor (5‐FAM) and acceptor (QXL) on opposing sides of the NS3 protease cleavage site. (B ) FRET protease assay. The donor absorbs energy at 490 nm and emits energy (i.e., fluorescence) at 520 nm. However, when in close contact on an intact peptide, the acceptor absorbs the 520‐nM energy emitted by the donor, preventing fluorescence. Cleavage of the peptide increases the distance between the fluorophores, resulting in proportional 5‐FAM fluorescence. Diagram and figure adapted from AnaSpec product information (http://www.anaspec.com/products/product.asp?id=30173&productid=13982).

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Figure 17.5.3 Sample plate layout for virus titration. 24 viral serial dilutions can be conveniently performed in each U‐bottom 96‐well microtiter plate. In this example, the wells in rows 1 and 5 are filled with the individual virus samples to be analyzed while the wells in rows 2 to 4 and 6 to 8 are each filled with 180 µl of Huh7 cell maintenance medium. Using a multichannel pipettor, three 1:10 dilutions can be made by transferring 20 µl between wells down each column, beginning with the undiluted virus row and moving on to the 1:10 row, the 1:100 row, and the 1:1000 row. It is important to thoroughly mix the virus dilution by pipetting up and down after each transfer, and to change pipet tips between each transfer.

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Videos

Literature Cited

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

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited