Regulation of the HIV Promoter/Enhancer

Camillo Palmieri1, Francesca Trimboli1, Giuseppe Scala1, Ileana Quinto1, Peter B. Bressler2

1 Department of Clinical and Experimental Medicine, University of Catanzaro, Magna Grecia, Cantazaro, Italy, 2 National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 12.7
DOI:  10.1002/0471142735.im1207s54
Online Posting Date:  May, 2003
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Abstract

This unit describes adaptations of two molecular techniques that can be used to study the regulation of HIV expression. The first two protocols describe the chloramphenicol acetyltransferase (CAT) assay, in which the CAT reporter gene is attached to an HIV‐1 promoter and CAT activity is measured as an indication of the promoter's activity. The basic protocol is rapid, simple, and suited to analyzing multiple samples. An alternate protocol describes an assay for CAT function that involves separating the reaction products by thin‐layer chromatography (TLC). The second basic protocol describes an electrophoretic mobility shift assay for detecting proteins present in cell extracts that can bind to the HIV‐1 LTR (long terminal repeat). Such studies are central to current HIV research because it is important to know what agents induce and inhibit (or “down‐regulate”) HIV transcription.

     
 
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Table of Contents

  • Basic Protocol 1: Analysis of Promoter Activity by the Chloramphenicol Acetyltransferase (CAT) Assay
  • Alternate Protocol 1: Analysis of Promoter Activity using a Thin‐Layer Chromatography (TLC) CAT Assay
  • Alternate Protocol 2: Analysis of LTR Promoter Activity by Luciferase Assay
  • Basic Protocol 2: Electrophoretic Mobility Shift Assay
  • Basic Protocol 3: DNA‐Affinity Chromatography
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Promoter Activity by the Chloramphenicol Acetyltransferase (CAT) Assay

  Materials
  • Cells
  • Plasmid containing a bacterial CAT gene driven by the HIV‐1 LTR promoter (HIV LTR–CAT construct; e.g., pUC‐BENN‐CAT, AIDS Research and Reference Reagent Program; appendix 55)
  • Phosphate‐buffered saline (PBS; appendix 2A; culture medium used to grow cells without fetal bovine serum may also be used)
  • 100 mM Tris⋅Cl, pH 7.5 to 7.8 ( appendix 2A)
  • recipeTEN buffer (see recipe)
  • Dry ice
  • Chloramphenicol
  • 0.5 mCi/ml [3H]acetyl CoA (200 mCi/mmol) or 0.05 mCi/ml [14C]acetyl CoA (4.0 mCi/mmol; e.g., Du Pont NEN)
  • Econofluor liquid scintillation fluid (e.g., Du Pont NEN)
  • Water‐miscible scintillation fluid (e.g., Beckman Ready‐Safe or ICN Biomedicals Cytoscint)
  • 60‐mm tissue culture plate
  • 3‐ or 10‐ml scintillation vials
  • Additional reagents and equipment for transfection of eukaryotic cells (e.g., units 10.13 10.16)
CAUTION: Radioactive materials require special handling. See appendix 1Q and the institutional Radiation Safety Office for guidelines concerning proper handling and disposal.NOTE: All incubations of cells are performed in a humidified 37°C, 5% CO 2 incubator.

Alternate Protocol 1: Analysis of Promoter Activity using a Thin‐Layer Chromatography (TLC) CAT Assay

  • 1 M Tris⋅Cl, pH 7.5 to 7.8 ( appendix 2A)
  • 3.5 mg/ml acetyl CoA (prepare monthly and store at −20°C)
  • 0.05 mCi/ml [14C]chloramphenicol (60 mCi/mmol; e.g., Du Pont NEN)
  • Ethyl acetate
  • Chloroform
  • Methanol
  • Glass or polypropylene pipets
  • Vacuum concentrator or Speedvac evaporator with organic trap (Savant)
  • Thin‐layer chromatography (TLC) tank with Whatman 3MM filter paper or equivalent
  • 20 × 20–cm silica gel 60 TLC plates, rigid or flexible
  • Additional reagents and equipment for autoradiography ( appendix 3J)
CAUTION: Radioactive materials require special handling. See appendix 1Q and the institutional Radiation Safety Office for guidelines concerning proper handling and disposal.NOTE: All incubations of cells are performed in a humidified 37°C, 5% CO 2 incubator.

Alternate Protocol 2: Analysis of LTR Promoter Activity by Luciferase Assay

  • Plasmid containing a luc gene driven by the HIV‐1 LTR promoter (HIV LTR‐luc; e.g., pBlue 3′LTR‐luc, AIDS Research and Reference Reagent Program)
  • Phosphate‐buffered saline (PBS; appendix 2A), 4°C
  • recipeLuciferase lysis buffer (see recipe)
  • recipeLuciferase assay buffer (see recipe), fresh
  • recipe1 mM D‐luciferin (see recipe)
  • Luminometer
  • Borosilicate‐glass test tubes
  • Additional reagents and equipment for transfection of eukaryotic cells (units 10.13 10.16)

Basic Protocol 2: Electrophoretic Mobility Shift Assay

  Materials
  • Cells
  • Phosphate‐buffered saline (PBS; appendix 2A), 4°C
  • recipeCell lysis buffer with and without NP‐40 (see recipe)
  • recipeNuclear extraction buffer (see recipe)
  • 0.25 nmol/µl complementary oligonucleotides
  • recipe10× annealing buffer (see recipe)
  • 10 U/µl T4 polynucleotide kinase (PNK) and recipe10× buffer (see recipe)
  • 10 mCi/ml [γ‐32 P]ATP (3000 Ci/mmol)
  • Water‐miscible scintillation fluid (e.g., Neckman Ready‐Safe)
  • 40% (w/v) 29:1 acrylamide/bisacrylamide
  • 10× and 0.5× TBE (unit 10.4)
  • 10% ammonium persulfate (APS; make fresh weekly and store at 4°C)
  • TEMED
  • recipePoly(dI‐dC)⋅poly(dI‐dC) (see recipe)
  • recipe4× nuclear‐factor binding buffer (see recipe)
  • recipeDNA loading buffer (see recipe)
  • G‐25 spin column (Amersham Pharmacia Biotech)
  • 16‐cm‐long siliconized glass plates
  • 1.5‐mm spacers and comb
  • Needle and syringe
  • 65°C water bath
  • Whatman 3MM or equivalent filter paper
  • Additional reagents and equipment for trypan blue–exclusion test of cell viability ( appendix 3B), determination of protein concentration (e.g., unit 2.11), and autoradiography ( appendix 3J)
CAUTION: Radioactive materials require special handling. See appendix 1Q and the institutional Radiation Safety Office for guidelines concerning proper handling and disposal.

Basic Protocol 3: DNA‐Affinity Chromatography

  Materials
  • Streptavidin paramagnetic particles (e.g., Promega Z5481)
  • Washing buffer—e.g., PBS ( appendix 2A)/1% (v/v) Triton‐X 100
  • Phosphate‐buffered saline (PBS; appendix 2A), 4°C
  • 25 to 50 pmol/µl double‐stranded biotinylated oligonucleotides (unit 10.10)
  • recipe1× nuclear‐factor binding buffer (see recipe) containing 0.5% BSA and 0.1% IGEPAL (NP‐40)
  • Nuclear proteins
  • Elution buffer: 50 mM glycine⋅HCl, pH 2.2
  • Neutralization buffer: 2 M Tris⋅HCl, pH 8.8 ( appendix 2A)
  • recipePoly(dI‐dC)⋅poly(dI‐dC) (see recipe)
  • Magnetic separation stand (e.g., Promega)
  • Additional reagents and equipment for determining protein concentration (unit 2.11) and SDS‐PAGE (unit 8.1)
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Literature Cited

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