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Human Immunodeficiency Viruses: Propagation, Quantification, and Storage
Publication Name:
Current Protocols in Microbiology
Unit Number:
UNIT 15J.1
DOI:
10.1002/9780471729259.mc15j01s02
Print Publication Date:
August, 2006
Online Posting Date:
September, 2006 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.
Keywords: HIV-1; HIV-2; retroviruses; primary isolates; T-cell line adapted; molecular clones; reporter viruses; focus-forming unit; radioactive reverse transcriptase assay; p24 ELISA
Materials
Basic Protocol 1: Isolation of HIV from Blood
Materials
- Blood from infected individual
- Complete RPMI medium/20% FBS (see recipe) containing 0.5 µg/ml phytohemagglutinin (PHA; Sigma; store in aliquots at –20°C and add to medium just before use)
- Complete RPMI medium/20% FBS (see recipe) containing 20 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., Sorvall Legend RT)
- Plastic hemacytometer (preferred for safety reasons)
- 25- or 75-cm
2 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 (Kanof et al., 1996), counting cells with a hemacytometer (appendix 4A), and RT activity assay (Basic Protocol 9) or p24 ELISA (Basic Protocol 10)
Basic Protocol 2: Propagation of Primary HIV Isolates
Materials
- Blood from uninfected individual
- Complete RPMI medium/20% FBS (see recipe) containing 0.5 µg/ml phytohemagglutinin (PHA; Sigma; store in aliquots at –20°C and add to medium just before use)
- Complete RPMI medium/20% FBS (see recipe) containing 20 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; Basic Protocol 1)
- RPMI 1640 medium containing 20% FBS
- Tabletop centrifuge (e.g., Sorvall Legend RT)
- Plastic hemacytometer (preferred for safety reasons)
- 25- or 75-cm
2 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 (Kanof et al., 1996), counting cells with a hemacytometer (appendix 4A) and p24 ELISA (Basic Protocol 10) or RT activity assay (Basic Protocol 9)
Basic Protocol 3: Propagation of T Cell Line–Adapted (TCLA) HIV
Materials
- CD4
+ T cell line, e.g., H9 (Popovic et al., 1984) or MOLT-4 cl.8 (Kikukawa et al., 1986) or C8166 (Salahuddin et al., 1983) - 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-cm
2 tissue culture flasks - Cryovials
- Tabletop centrifuge (e.g., 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 (Basic Protocol 6), and p24 ELISA (Basic Protocol 10) or RT activity assay (Basic 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., 1993)
- DMEM medium (e.g., Invitrogen) containing 10% FBS (prepare with nuclease-free H
2 O) - HIV cDNA: plasmid DNA containing HIV proviral cDNA (pNL4.3, pYU-2, or pJRCSF (available from the NIH AIDS Research and Reference Reagent Program; http://www.aidsreagent.org/)
- 2 M CaCl
2 (prepare with nuclease-free H2 O) - Nuclease-free (DEPC-treated) H
2 O (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 Reporter Viruses
Materials
- 293T cells (human embryonic kidney cell line transformed by sheared Adenovirus 5 DNA and expressing SV40 T antigen; Pear et al., 1993)
- DMEM medium (e.g., Invitrogen) containing 10% FBS (prepare with nuclease-free H
2 O) - 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., 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 Basic Protocol 4) and quantification of virus (Basic Protocol 7)
Basic Protocol 6: Tissue
Culture
Infectious
Dose 50 Quantification of
HIV
Materials
- Viral stock to be assayed
- Growth medium for cells
- 5 × 10
5 cells/ml suspension of PHA and IL-2 stimulated PBMC (Basic 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/10% FBS (see recipe)
- Virus stock to be assayed (see Basic Protocols 1 to 4)
- 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
- Monoclonal antibody specific for HIV-1 p24 antigen (available from UK Centralized Facility for AIDS Reagents (CFAR, http://www.nibsc.ac.uk/spotlight/aidsreagent/index.html): ARP#365 and CFAR ARP#366 for HIV-1 or HIV-2
+ human sera (WHO Panel C for HIV-2; also available from CFAR) - Secondary antibody: goat anti-mouse -galactosidase conjugate for HIV-1 or goat anti-human -galactosidase conjugate for HIV-2 (Southern Biotechnology)
- 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., 2002); available from the NIH AIDS Reagent Program at http://www.aidsreagent.org - Complete DMEM medium/10% FBS (see recipe)
- Reporter virus (see Basic 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 1 to 4) - Phosphate-buffered saline (PBS; appendix 2A)
- Bright-Glo substrate (Promega)
- 96-well luminometer culture plate (Corning)
- 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 1 to 4)
- 10µCi/ml [
3 H]TTP (sp. act. 10 to 25 Ci/mmol) - RRT assay solution B (see recipe)
- RRT stop solution (see recipe)
- 5% (w/v) Na
2 HPO4 - 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
Basic Protocol 10: Quantification of HIV by P24 ELISA
Materials
- Coating antibody: sheep anti–HIV p24-I/II/III mixture (Cliniqa)
- Coating buffer: 0.1 M NaHCO
3 (pH 8.5)/0.15 M NaCl - Supernatants to be assayed for HIV (Basic Protocols 1 to 4)
- 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
- 10 µg/ml p24 standard (National Institute of Biological Standards and Controls; http://www.nibsc.ac.uk/)
- Nonfat dry milk
- Sheep serum (Sigma)
- 10% (v/v) Tween-20
- Mouse anti-p24 alkaline phosphatase (AP)–conjugated antibody (Cliniqa)
- TBS (appendix 2A) containing 0.05% Tween-20
- AMPAK enzyme amplification kit (Dako)
- AMPAK wash buffer
- Substrate
- Amplifier
- Stop solution
- Maxi-Sorp 96-well plate (Nunc) with lid
- Automated ELISA plate washer (optional)
- Spectrophotometer with microtiter plate reader
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Figures
-
Figure 15J.1.1Typical syncytia and cytopathic effect in C8166 T-cells infected with a TCLA HIV-1 strain. (A) Infected C8166 cells. (B) uninfected C8166 cells. -
Figure 15J.1.2Vectors used to produce envelope+ reporter viruses. (A) pSVIIIenv expression vector encodes envelope and rev proteins. (B) env– pNL4.3 is a full-length clone of HIV-1 which contains a premature stop codon in the envelope gene. Reporter genes are often incorporated into the nef gene. Thus, env– pNL4.3 encodes all the viral genes required for particle production except for envelope. Cotransfection of 293T cells with env– pNL4.3 and pSVIIIenv carrying the envelope of choice results in the production of high-titer envelope+ pseudotype virions. -
Figure 15J.1.3TCID50 calculation. The table shows the virus dilutions that resulted in viral replication. Viral replication may be detected by RT or p24 estimation of virus particles produced in the cell supernatant (see Basic Protocols 9 and 10). -
Figure 15J.1.4Infectivity of HIV-1 R5 viruses detected as foci. HIV-1 reporter viruses were used to infect NP2 cells with and without CD4 and the coreceptor CCR5. Reporter viruses were prepared as described in Basic Protocol 5. In this assay, infection was detected (in this case without using the reporter gene) by immunostaining for intracellular p24 antigen as described in Basic Protocol 7.
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