A2和A72 J-Lat细胞系中药物诱导的HIV-1转录活性的流式细胞术分析

Daniela  Boehm; Melanie Ott

doi:10.21769/BioProtoc.2290

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Peer-reviewed

Flow Cytometric Analysis of Drug-induced HIV-1 Transcriptional Activity in A2 and A72 J-Lat Cell Lines

A2和A72 J-Lat细胞系中药物诱导的HIV-1转录活性的流式细胞术分析

DB Daniela Boehm

MO Melanie Ott email

发布: 2017年05月20日第7卷第10期 DOI: 10.21769/BioProtoc.2290 浏览次数: 12047

评审: Emilie BesnardEmilie BattivelliAnonymous reviewer(s)

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Abstract

The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). A combination of antiretroviral treatments with latency-purging strategies may accelerate the depletion of latent reservoirs and lead to a cure (Geeraert et al., 2008). Current strategies to reactivate HIV-1 from latency include use of prostratin, a non-tumor-promoting phorbol ester (Williams et al., 2004), BET inhibitors (Filippakopoulos et al., 2010; Delmore et al., 2011), and histone deacetylase (HDAC) inhibitors, such as suberoylanilidehydroxamic acid (i.e., SAHA or Vorinostat) (Kelly et al., 2003; Archin et al., 2009; Contreras et al., 2009; Edelstein et al., 2009). As the mechanisms of HIV-1 latency are diverse, effective reactivation may require combinatorial strategies (Quivy et al., 2002). The following protocol describes a flow cytometry-based method to quantify transcriptional activation of the HIV-1 long terminal repeat (LTR) upon drug treatment. This protocol is optimized for studying latently HIV-1-infected Jurkat (J-Lat) cell lines that contain a GFP cassette. J-Lats that contain a different reporter, for example Luciferase, can be treated with drugs as described but have to be analyzed differently.

Keywords: Human immunodeficiency virus-1 (人体免疫缺陷病毒-1)

Latency (潜伏期)

Drug treatment (药物处理)

Transcriptional activation (转录激活)

HIV-1 LTR (HIV-1 LTR)

Flow cytometry (流式细胞术)

J-Lat cell lines (J-Lat细胞系)

Background

Studies that assess transcriptional activation or repression of the HIV-1 LTR generally use CD4⁺ T cells containing latent full-length HIV-1, such as NL4-3/E-/GFP-IRES–nef (Kutsch et al., 2002) or R7/E-/GFP (Jordan et al., 2003), which contains a frameshift mutation in the viral Env gene to prevent viral spread and expresses GFP in the Nef open reading frame allowing separation of actively infected GFP⁺ from GFP⁻ cells (uninfected or latently infected) by cell sorting (Jordan et al., 2003). To specifically investigate transcriptional activation of the HIV-1 LTR, we utilize the J-Lat cell line A72 containing only a latent LTR-GFP construct (Jordan et al., 2003). To determine if drug treatment specifically activates Tat, we utilize a J-Lat cell line harboring a latent lentiviral construct expressing Tat with GFP from the HIV-1 LTR (clone A2; LTR-Tat-IRES-GFP) (Jordan et al., 2003).

Materials and Reagents

A2 and A72 J-Lat cell culture
1. 75 cm² tissue culture flask (Corning, Falcon^®, catalog number: 353110 )
2. Tips
  0.1-10 µl (Fisher Scientific, Fisherbrand^TM, catalog number: 02-681-440 )
  1-200 µl (Fisher Scientific, Fisherbrand^TM, catalog number: 02-707-502 )
  101-1,000 µl (Fisher Scientific, Fisherbrand^TM, catalog number: 02-707-509 )
3. A2 and A72 J-Lat cells (Jordan et al., 2003)
4. RPMI (Mediatech, catalog number: 10-040-CV )
5. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-106 )
6. L-glutamine (Mediatech, catalog number: 25-005-Cl )
7. 100x penicillin/streptomycin (Mediatech, catalog number: 30-002-Cl )
Analysis of HIV-1 LTR transcriptional activation by flow cytometry
1. 96-well V-bottom tissue culture plates and lids (Thermo Fisher Scientific, Thermo Scientific ^TM, catalog numbers: N249570 and N163320 )
2. Tips
  0.1-10 µl (Fisher Scientific, Fisherbrand^TM, catalog number: 02-681-440 )
  1-200 µl (Fisher Scientific, Fisherbrand^TM, catalog number: 02-707-502 )
  101-1,000 µl (Fisher Scientific, Fisherbrand^TM, catalog number: 02-707-509 )
3. RPMI (Mediatech, catalog number: 10-040-CV )
4. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-106 )
5. L-glutamine (Mediatech, catalog number: 25-005-Cl )
6. 100x penicillin/streptomycin (Mediatech, catalog number: 30-002-Cl )
7. TNFα (PeproTech, catalog number: 300-01A )
8. JQ1 (Cayman Chemical, catalog number: 11187 )
9. Prostratin (Sigma-Aldrich, catalog number: P0077 )
10. Suberoylanilide hydroxamic acid (SAHA) (Sigma-Aldrich, catalog number: SML0061 )
11. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
12. 1x PBS (Mediatech, catalog number: 21-031-CV )
13. MACSQuant Running buffer (Miltenyi Biotech, catalog number: 130-092-747 )
14. ApoTox-Glo^TM Triplex Assay (Promega, catalog number: G6320 )
15. RPMI medium (see Recipes)
16. TNFα stock solution (see Recipes)
17. JQ1 stock solution (see Recipes)
18. Prostratin stock solution (see Recipes)
19. Suberoylanilide hydroxamic acid (SAHA) stock solution (see Recipes)

Equipment

Pipette
Biosafety cabinet level 2
CO₂ tissue culture incubator (Thermo Electron, model: Forma^TM Steri-Cult^TM CO₂ Incubators , catalog number: 3307)
Tabletop centrifuge (Beckman Coulter, model: Allegra X-14R ) for 96-well plates
MACSQuant VYB FACS analyzer (Miltenyi Biotech, model: MACSOuant^®VYB, catalog number: 130-096-116 )
SpectraMax MiniMax^TM 300 Imaging Cytometer (Molecular Devices, model: SpectraMax MiniMax 300 )

Software

FlowJo 9.9 or never (Tree Star)

Procedure

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如何引用

Boehm, D. and Ott, M. (2017). Flow Cytometric Analysis of Drug-induced HIV-1 Transcriptional Activity in A2 and A72 J-Lat Cell Lines. Bio-protocol 7(10): e2290. DOI: 10.21769/BioProtoc.2290.