High‐Throughput Screening of Viral Entry Inhibitors Using Pseudotyped Virus

Arnab Basu1, Debra M. Mills1, Terry L. Bowlin1

1 Microbiotix, Worcester, Massachusetts
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 13B.3
DOI:  10.1002/0471141755.ph13b03s51
Online Posting Date:  December, 2010
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Abstract

Virus entry into a host cell is an attractive target for therapy because propagation of virus can be blocked at an early stage, minimizing chances for the virus to acquire drug resistance. Anti‐infective drug discovery for BSL‐4 viruses like Ebola or Lassa hemorrhagic fever virus presents challenges due to the requirement for a BSL‐4 laboratory containment facility. Pseudotyped viruses provide a surrogate model in which the native envelope glycoprotein of a BSL‐2 level virus (e.g., vesicular stomatitis virus) is replaced with envelope glycoprotein of a foreign BSL‐4 virus (e.g., Ebola virus). Because the envelope glycoprotein determines interaction of virus with its cellular receptors, pseudotyped viruses can mimic the viral entry process of the original virus. Moreover, they are competent for only a single cycle of infection, and therefore can be used in BSL‐2 facilities. Pseudotyped viruses have been used in high‐throughput screening of entry inhibitors for a number of BSL‐4 level viruses. This unit includes protocols for preparing pseudotyped viruses using lentiviral vectors and use of pseudotyped viruses for high‐throughput screening of viral entry inhibitors.Curr. Protoc. Pharmacol. 51:13B.3.1‐13B.3.17. © 2010 by John Wiley & Sons, Inc.

Keywords: pseudotyped virus; lentiviral vectors; high‐throughput screen

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of Ebola Pseudotyped Virus (GP/HIV)
  • Basic Protocol 2: High‐Throughput Screening (HTS) Assay to Screen Inhibitors of Pseudotyped Virus Entry
  • Alternate Protocol 1: High‐Throughput Screen for Inhibitors of Pseudotyped Virus Entry Using Nonadherent (Suspension) Cells
  • Basic Protocol 3: Determination of the Specificity of Inhibitors of Pseudotyped Virus Entry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generation of Ebola Pseudotyped Virus (GP/HIV)

  Materials
  • 293T cells: e.g., 293FT cells (Invitrogen, cat. no. R700‐07) or HEK293T cells (ATCC, cat. no. CRL‐11268) growing at low passage number in 75‐cm2 flasks
  • Complete DMEM medium (see recipe) with and without antibiotics
  • 0.25% (w/v) trypsin‐EDTA solution (e.g., Invitrogen)
  • Phosphate buffered saline (PBS; Invitrogen, cat. no. 10010‐03) without Ca2+ or Mg2+
  • 0.4% trypan blue stain in PBS
  • Serum‐free DMEM (Invitrogen)
  • Plasmid mixture: pNL4.3.Luc+.RE (NIH AIDS Research and References Reagent Program) and envelope expression plasmids like the Ebola Virus GP cloned in pCDNA3.1 plasmid (the procedure for cloning of Ebola GP or any other heterologous envelope glycoprotein into pCDNA3.1 plasmid is not described here; it can be performed following standard procedures: e.g., Lagging et al., ; Matsuura et al., ; Manicassamy et al., , )
  • Opti‐MEM I Reduced Serum Medium (Invitrogen, cat. no. 31985‐062)
  • Lipofectamine 2000 (Invitrogen, cat. no. 11668‐027)
  • DMEM (Invitrogen) with 2% FBS and without antibiotics
  • p24 ELISA kit (e.g., PerkinElmer)
  • 10‐cm cell culture plates
  • Inverted phase‐contrast microscope
  • Hemacytometer and coverslip
  • 5‐ml culture tubes (Falcon 2054 tubes, sterile; Becton‐Dickinson)
  • 0.45‐µm syringe filter
CAUTION: The generation and handling of pseudotyped virus using lentiviral vectors should be carried out in a proper biosafety containment facility. See Biosafety Considerations for Research with Lentiviral Vectors at (http://oba.od.nih.gov/oba/rac/Guidance/LentiVirus_Containment/pdf/Lenti_Containment_Guidance.pdf).NOTE: All solutions and equipment coming into contact with cells must be sterile. Proper sterile technique should be used.NOTE: All incubations must be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: High‐Throughput Screening (HTS) Assay to Screen Inhibitors of Pseudotyped Virus Entry

  Materials
  • 293FT cells (Invitrogen, cat. no. R700‐07), Vero cells (ATCC, cat. no. CCL‐81), BHK‐21 cells (ATCC, cat. no. CCL‐10), HeLa cells (ATCC, cat. no. CCL‐2), or other cell lines permeable to Ebola virus
  • Complete DMEM medium (see recipe)
  • DMEM with high glucose (4.5 g/liter; Invitrogen)
  • GP/HIV pseudotyped virus ( protocol 1): this assay is described using GP/HIV as an example; therefore, cells that are permeable to the Ebola virus infection are used; for other pseudotyped viruses, the choice of cells must be modified accordingly
  • Polybrene (hexadimethrine bromide; Sigma, cat. no. H9268)
  • Combinatorial small‐molecule compound libraries for screening (see annotations to step 10, below)
  • Control Ebola entry inhibitors: e.g., E‐64 (Calbiochem) or CA‐074 (Calbiochem)
  • DMSO vehicle
  • PBS without Ca2+ or Mg2+ (Invitrogen, cat. no. 10010‐031)
  • Complete DMEM (see recipe) without antibiotics or phenol red
  • britelite plus Ultra‐High Sensitivity Luminescence Reporter Gene Assay System (PerkinElmer, cat. no. 6016761)
  • 0.4% trypan blue stain in PBS
  • CulturPlate‐96, white (PerkinElmer, cat. no. 6005680) or ViewPlate‐96, white, (PerkinElmer, cat. no. 6005181)
  • Inverted phase‐contrast light microscope
  • Hemacytometer
  • TopSeal‐A (PerkinElmer)
  • Orbital shaker
  • Luminometer: e.g., Wallac EnVision 2102 Multilabel Reader or Wallac Microbeta Trilux Luminometer (both available from PerkinElmer)
  • Additional reagents and equipment for trypsinizing cells (see protocol 1, steps 1 to 10)
CAUTION: The handling of pseudotyped virus must be carried out using proper biosafety containment. See Biosafety Considerations for Research with Lentiviral Vectors at (http://oba.od.nih.gov/oba/rac/Guidance/LentiVirus_Containment/pdf/Lenti_Containment_Guidance.pdf).NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used.NOTE: Incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: Make sure that the target cells have not been passaged more than 20 times.

Alternate Protocol 1: High‐Throughput Screen for Inhibitors of Pseudotyped Virus Entry Using Nonadherent (Suspension) Cells

  • Suspension cells

Basic Protocol 3: Determination of the Specificity of Inhibitors of Pseudotyped Virus Entry

  Materials
  • 293FT cells (Invitrogen, cat. no. R700‐07) growing at low passage number in 75‐cm2 flasks
  • VSV‐G/HIV pseudotyped virus (see protocol 1)
  • Polybrene (hexadimethrine bromide; Sigma, cat. no. H9268)
  • Hit compounds from combinatorial small‐molecule compound libraries screened (see annotations to step 10 of protocol 2, above)
  • DMSO vehicle
  • Control compound, e.g., bafilomycin or other endosomal pH modulator
  • britelite plus Ultra‐High Sensitivity Luminescence Reporter Gene Assay System (PerkinElmer, cat. no. 6016761)
  • Confirmation plates containing “hits” (see protocol 2)
  • CulturPlate‐96, white (PerkinElmer, cat. no. 6005680) or ViewPlate‐96, white, (PerkinElmer, cat. no. 6005181)
  • Inverted phase‐contrast microscope
  • Additional reagents and equipment for trypsinizing cells (see protocol 1, steps 1 to 10), infecting cells with virus (see protocol 2, steps 4 to 10), and developing assay plates using the britelite plus Ultra‐High Sensitivity Luminescence Reporter Gene Assay System (see protocol 2, steps 11 to 18)
CAUTION: The handling of pseudotyped virus must be carried out using proper biosafety containment. See Biosafety Considerations for Research with Lentiviral Vectors at (http://oba.od.nih.gov/oba/rac/Guidance/LentiVirus_Containment/pdf/Lenti_Containment_Guidance.pdf).NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used.NOTE: Incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Figures

Videos

Literature Cited

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