Human Immunodeficiency Viruses: Propagation, Quantification, and Storage

Paul J. Peters1, Kathryn Richards1, Paul Clapham1

1 Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15J.1
DOI:  10.1002/9780471729259.mc15j01s28
Online Posting Date:  February, 2013
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Abstract

Described in this unit are basic protocols frequently used in the research of human immunodeficiency viruses (HIVs). Provided are methods for propagating and quantifying HIV, as well as recommendations for long‐term storage. Background information about these methods is also provided and includes their advantages, disadvantages, and troubleshooting. Curr. Protoc. Microbiol. 28:15J.1.1‐15J.1.24. © 2013 by John Wiley & Sons, Inc.

Keywords: HIV‐1; HIV‐2; retroviruses; primary isolates; T cell line–adapted (TCLA) viruses; molecular clones; reporter viruses; focus‐forming unit; radioactive reverse transcriptase assay; p24 ELISA

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Propagation of Human Immunodeficiency Virus
  • Basic Protocol 1: Isolation of HIV from Blood
  • Basic Protocol 2: Propagation of Primary HIV Isolates
  • Basic Protocol 3: Propagation of T Cell Line–Adapted (TCLA) HIV
  • Basic Protocol 4: Production of Replication‐Competent HIV from Molecular Clones
  • Basic Protocol 5: Propagation of ‘Single‐Round’ Reporter Viruses
  • Quantification of HIV
  • Basic Protocol 6: Tissue Culture Infectious Dose 50 Quantification of HIV
  • Basic Protocol 7: Focus‐Forming Unit (FFU) Quantification of HIV
  • Basic Protocol 8: Quantification of HIV Using Reporter Genes
  • Basic Protocol 9: Radioactive Reverse Transcriptase Assay
  • Basic Protocol 10: Quantification of HIV by p24 ELISA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of HIV from Blood

  Materials
  • Blood from infected individual
  • Complete RPMI medium/10% FBS (see recipe) containing 5.0 µg/ml phytohemagglutinin (PHA; Sigma; store in aliquots at –20°C and add to medium just before use)
  • Complete RPMI medium/10% FBS (see recipe) containing 10 U/ml interleukin 2 (IL‐2; Roche Applied Science; store in aliquots at –20°C and add to medium just before use)
  • Blood from uninfected individual
  • Tabletop centrifuge (e.g., Thermo Scientific Sorvall Legend RT)
  • Plastic hemacytometer (preferred for safety reasons)
  • 25‐ or 75‐cm2 tissue culture flasks
  • 0.45‐µm low‐protein‐binding filters (Millipore; optional)
  • Cryovials
  • Dry ice/ethanol bath
  • –80° or –152°C freezer
  • Additional reagents and equipment for preparing PBMC ( appendix 4C), counting cells with a hemacytometer ( appendix 4A), and RT activity assay ( protocol 9), or p24 ELISA ( protocol 10)

Basic Protocol 2: Propagation of Primary HIV Isolates

  Materials
  • Blood from uninfected individual
  • Complete RPMI medium/10% FBS (see recipe) containing 5.0 µg/ml phytohemagglutinin (PHA; Sigma; store in aliquots at –20°C and add to medium just before use)
  • Complete RPMI medium/10% FBS (see recipe) containing 10 U/ml interleukin 2 (IL‐2; Roche Applied Science; store in aliquots at –20°C and add to medium just before use)
  • Cell‐free virus isolate seed stock (viral supernatant; protocol 1)
  • RPMI 1640 medium containing 10% FBS
  • Tabletop centrifuge (e.g., Thermo Scientific Sorvall Legend RT)
  • Plastic hemacytometer (preferred for safety reasons)
  • 25‐ or 75‐cm2 tissue culture flasks
  • 0.45‐µm low‐protein‐binding filters (Millipore; optional)
  • Cryovials
  • Dry ice/ethanol bath
  • –80° or –152°C freezer
  • Additional reagents and equipment for preparing PBMC ( appendix 4C), counting cells with a hemacytometer ( appendix 4A), and p24 ELISA ( protocol 10) or RT activity assay ( protocol 9)

Basic Protocol 3: Propagation of T Cell Line–Adapted (TCLA) HIV

  Materials
  • CD4+ T cell line, e.g., H9 (Popovic et al., ) or MOLT‐4 cl.8 (Kikukawa et al., ) or C8166 (Salahuddin et al., )
  • Seed stocks of T cell line–adapted viruses (available from the NIH AIDS Research and Reference Reagent Program; http://www.aidsreagent.org)
  • RPMI medium/10% FBS (see recipe)
  • 25‐ or 75‐cm2 tissue culture flasks
  • Cryovials
  • Tabletop centrifuge (e.g., Thermo Scientific Sorvall Legend RT)
  • Plastic hemacytometer (preferred for safety reasons)
  • Dry ice/ethanol bath
  • –80° or –152°C freezer
  • Additional reagents and equipment for counting cells with a hemacytometer ( appendix 4A), quantification by TCID 50 ( protocol 6), and p24 ELISA ( protocol 10), or RT activity assay ( protocol 9)

Basic Protocol 4: Production of Replication‐Competent HIV from Molecular Clones

  Materials
  • 293T cells [human embryonic kidney cell line transformed by sheared adenovirus 5 DNA and expressing SV40 T antigen; (Pear et al., )]
  • DMEM medium (e.g., Invitrogen) containing 4% FBS (prepare with nuclease‐free H 2O)
  • HIV cDNA: plasmid DNA containing HIV proviral cDNA (pNL4.3, pYU‐2, or pJRCSF or other clones are available from the NIH AIDS Research and Reference Reagent Program; http://www.aidsreagent.org/)
  • 2 M CaCl 2 (prepare with nuclease‐free H 2O)
  • Nuclease‐free (DEPC‐treated) H 2O ( appendix 2A)
  • 2× HEPES‐buffered saline (HBS), pH 7.05 (see recipe)
  • 6‐well tissue culture dishes or 60‐ or 100‐mm tissue culture plates
  • Clear plastic tubes
  • 15‐ or 50‐ml centrifuge tubes
  • Cryovials
  • Dry ice/ethanol bath
  • –80° or –152°C freezer

Basic Protocol 5: Propagation of ‘Single‐Round’ Reporter Viruses

  Materials
  • 293T cells [human embryonic kidney cell line transformed by sheared Adenovirus 5 DNA and expressing SV40 T antigen; (Pear et al., )]
  • DMEM medium (e.g., Life Technologies–Invitrogen) containing 4% FBS (prepare with nuclease‐free H 2O)
  • Reporter construct DNA (i.e., pNL4.3env, pNL4.3env nef GFP+, pNL4.3env nef luc+(available from the NIH Reagent Program; http://www.aidsreagent.org/)
  • pSVIIIenv (available with various primary isolate envelopes from the NIH Reagent Program; http://www.aidsreagent.org/), or pVSV‐G
  • Calcium phosphate transfection kit (optional; Promega)
  • 6‐well tissue culture plates
  • 15‐ml centrifuge tubes
  • Tabletop centrifuge (e.g., Thermo Scientific Sorvall Legend RT)
  • 0.45‐µm low‐protein‐binding filters (Millipore; optional)
  • Cryovials
  • Dry ice/ethanol bath
  • –80° or –152°C freezer
  • Additional reagents and equipment for transfection of cells (see protocol 4) and quantification of virus ( protocol 7)

Basic Protocol 6: Tissue Culture Infectious Dose 50 Quantification of HIV

  Materials
  • Viral stock to be assayed
  • Growth medium for cells
  • 5 × 105 cells/ml suspension of PHA and IL‐2 stimulated PBMC ( protocol 1)
  • 96‐well flat‐bottom plate

Basic Protocol 7: Focus‐Forming Unit (FFU) Quantification of HIV

  Materials
  • CD4+, coreceptor+ cells (e.g., GHOST/CCR5; available from NIH AIDS Research and Reference Reagent Program; http://www.aidsreagent.org)
  • Complete DMEM medium/4% FBS (see recipe)
  • Virus stock to be assayed (see Basic Protocols protocol 11 to protocol 44)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1:1 methanol:acetone, –20°C
  • PBS ( appendix 2A) containing 1% (v/v) FBS and 0.05% (w/v) sodium azide
  • p24 antibody: Monoclonal antibody specific for HIV‐1 p24 antigen (available from UK Centralized Facility for AIDS Reagents (UK CFAR, http://www.nibsc.ac.uk/spotlight/centre_for_aids_reagents.aspx: ARP#365 and UK CFAR ARP#366 for HIV‐1 or HIV‐2+ human sera (WHO Panel C for HIV‐2; also available from UK CFAR)
  • Secondary antibody: goat anti‐mouse β‐galactosidase conjugate for HIV‐1 or goat anti‐human β‐galactosidase conjugate for HIV‐2 (SouthernBiotech)
  • 0.5 mg/ml Xgal in N, N‐dimethylformamide
  • Yellow PBS (see recipe)
  • PBS ( appendix 2A) containing 0.05% (w/v) sodium azide
  • 48‐well tissue culture plate

Basic Protocol 8: Quantification of HIV Using Reporter Genes

  Materials
  • Target cells for titrating virus with a luciferase reporter gene: PBMC, macrophages, or other target cells that carry a luciferase reporter gene, e.g., HeLa TZM‐bl (CD4+, CXCR4+, CCR5+; Wei et al., ); available from the NIH AIDS Reagent Program at http://www.aidsreagent.org
  • Complete DMEM medium/4% FBS (see recipe)
  • Reporter virus (see protocol 5) carrying a luciferase reporter cloned into the nef gene of pNL4.3 env construct (available from the NIH AIDS Reagent Program at http://www.aidsreagent.org) or HIV virus stock (Basic Protocols protocol 11 to protocol 44)
  • Clear DMEM: (no phenol red) (Life Technologies–Invitrogen)
  • Bright‐Glo substrate (Promega)
  • 96‐well luminometer culture plate: white walls, clear bottom (Corning, cat. no. 3610)
  • Luminometer (e.g., Clarity from Bio‐Tek)

Basic Protocol 9: Radioactive Reverse Transcriptase Assay

  Materials
  • RRT assay solution A (see recipe)
  • Cell culture supernatants (infected and uninfected) for testing RT activity (Basic Protocols protocol 11 to protocol 44)
  • 10 µCi/ml [3H]TTP (sp. act. 10 to 25 Ci/mmol)
  • RRT assay solution B (see recipe)
  • RRT stop solution (see recipe)
  • 5% (w/v) Na 2HPO 4
  • 70% and 100% ethanol
  • Scintillation fluid (Emulsifier‐Safe from Perkin‐Elmer)
  • Plastic flexible 96‐well plate (Falcon)
  • Cell harvester (e.g., Skatron)
  • DE‐81 chromatography paper
  • Fan for air drying filters
  • Polystyrene board
  • Scintillation counter (e.g., Beckman)

Basic Protocol 10: Quantification of HIV by p24 ELISA

  Materials
  • Coating antibody: sheep anti–HIV p24‐I/II/III mixture (Aalto Bio Reagents, cat. no. D7320)
  • Coating buffer: 0.1 M NaHCO 3 (pH 8.5)/0.15 M NaCl
  • Supernatants to be assayed for HIV (Basic Protocols protocol 11 to protocol 44)
  • 5% (w/v) benzalkonium chloride
  • Tris‐buffered saline (TBS), pH 7.4 ( appendix 2A)
  • TBS, pH 7.4 ( appendix 2A), containing 0.05% (w/v) benzalkonium chloride
  • TBS, pH 7.4 ( appendix 2A), containing 2% (w/v) nonfat dry milk
  • p24 standard: 10 µg/ml (Aalto Bio Reagents, cat. no. AG 6054)
  • Nonfat dry milk
  • Sheep serum (Sigma)
  • 10% (v/v) Tween 20
  • Detection antibody: mouse anti‐p24 alkaline phosphatase (AP)–conjugated antibody (Aalto Bio Reagents, cat. no. BC 1071‐AP)
  • TBS ( appendix 2A) containing 0.05% Tween 20
  • ELISA Amplification System (Life Technologies–Invitrogen, cat. no. 19589‐019)
    • ELISA Amplification System wash buffer: Tris‐buffered saline (TBS); 0.05 M Tris‐HCl (pH 7.5)/ 0.15 M NaCl
    • Substrate
    • Amplifier
    • Stop solution: 0.3 M H 2SO 4
  • Maxi‐Sorp 96‐well plate (Nunc, cat. no. 435494) with lid
  • Automated ELISA plate washer (optional)
  • Spectrophotometer with microtiter plate reader
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Figures

Videos

Literature Cited

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