Production and Use of HIV‐1 Luciferase Reporter Viruses

Carsten Münk1, Nathaniel R. Landau1

1 The Salk Institute for Biological Studies, La Jolla, California
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.5
DOI:  10.1002/0471141755.ph1205s22
Online Posting Date:  November, 2003
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Abstract

This unit describes the production and use of HIV‐1 luciferase reporter viruses. These viruses are used to rapidly and accurately quantify HIV‐1 in cell culture. Protocols are also presented for screening and characterization of small molecule inhibitors of HIV‐1 entry into cells.

Keywords: reporter virus; entry; infection; envelope glycoprotein; HIV‐1; luciferase

     
 
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Table of Contents

  • Basic Protocol 1: Production of HIV‐1 Luciferase Reporter Virus
  • Basic Protocol 2: Infect Cells with HIV‐1 Luciferase Reporter Virus
  • Basic Protocol 3: Evaluation of Cell Entry Inhibitors with HIV‐1 Luciferase Reporter Virus
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Production of HIV‐1 Luciferase Reporter Virus

  Materials
  • Confluent plate of HEK293 (ATCC# CRL‐1573) or HEK293T (ATCC# CRL‐11268) cells
  • PBS (Life Technologies)
  • Trypsin‐Versene (Biowhittaker)
  • DMEM‐10 (Biowhittaker, Life Technologies; also see unit 7.2)
  • Plasmid mixture: equal mixture of pNL‐Luc reporter (available from authors) and envelope expression plasmids (NIH AIDS Research and References Reagent Program; see Internet Resources)
  • 2 M CaCl 2 (see recipe)
  • 2× HBS (see recipe)
  • Gag p24 ELISA kit (e.g., Dupont)
  • 10‐cm plates
  • 2054 tubes, sterile (Becton‐Dickinson)
  • 0.45‐µm syringe filter

Basic Protocol 2: Infect Cells with HIV‐1 Luciferase Reporter Virus

  Materials
  • Cells (NIH AIDS Research and Reference Reagent Program; see Internet Resources)
  • Reporter virus, frozen (see protocol 1)
  • DMEM‐10 (unit 7.2)
  • Luc‐Lite luciferase assay reagent (Packard)
  • 96‐well culture dishes
  • Microtiter plate luminometer
  • Hemacytometer
  • 96‐well black microtiter plates
  • Transparent microplate adhesion sealing film (Packard)

Basic Protocol 3: Evaluation of Cell Entry Inhibitors with HIV‐1 Luciferase Reporter Virus

  Materials
  • Cells (NIH AIDS Research and Reference Reagent Program; see Internet Resources)
  • Medium (e.g., RPMI‐10; InVitrogen, Biowhittaker, Life Technologies; also see unit 7.2)
  • Viral entry inhibitor in DMSO
  • DMSO
  • Reporter virus, frozen (see protocol 1)
  • Luc‐Lite luciferase assay reagent (Packard)
  • Hemacytometer
  • 96‐well culture dishes
  • Multichannel micropipettor (optional)
  • 96‐well black microtiter plates
  • Transparent microplate adhesion sealing film
  • Microtiter plate luminometer
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Figures

Videos

Literature Cited

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   Connor, R.I., Chen, B.K., Choe, S., and Landau, N.R. 1995. Vpr is required for efficient replication of human immunodeficiency virus type‐1 in mononuclear phagocytes. Virology 206:935‐944.
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Internet Resources
  http://www.atcc.org
  Online catalog from the American Type Culture Collection.
  http://www.aidsreagent.org
  Online catalog from the National Institutes of Health AIDS Research and Reference Reagent Program.
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