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Flow Cytometric Analysis of HIV-1 Transcriptional Activity in Response to shRNA Knockdown in A2 and A72 J-Lat Cell Lines

A2和A72 J-Lat细胞系中HIV-1经shRNA沉默后转录活性的流式细胞分析

DB Daniela Boehm
MO Melanie Ott

发布: 2017年06月05日第7卷第11期 DOI: 10.21769/BioProtoc.2314 浏览次数: 10131

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). To eliminate viral reservoirs, one strategy focuses on reversing HIV-1 latency via ‘shock and kill’ (Deeks, 2012). The basis of this strategy is to overcome the molecular mechanisms of HIV-1 latency by therapeutically inducing viral gene and protein expression under antiretroviral therapy and to cause selective cell death via the lytic properties of the virus, or the immune system now recognizing the infected cells. Recently, a number of studies have described the therapeutic potential of pharmacologically inhibiting members of the bromodomain and extraterminal (BET) family of human bromodomain proteins (Filippakopoulos et al., 2010; Dawson et al., 2011; Delmore et al., 2011) that include BRD2, BRB3, BRD4 and BRDT. Small-molecule BET inhibitors, such as JQ1 (Filippakopoulos et al., 2010; Delmore et al., 2011), I-BET (Nicodeme et al., 2010), I-Bet151 (Dawson et al., 2011), and MS417 (Zhang et al., 2012) successfully activate HIV transcription and reverse viral latency in clonal cell lines and certain primary T-cell models of latency. To identify the mechanism by which BET proteins regulate HIV-1 latency, we utilized small hairpin RNAs (shRNAs) that target BRD2, BRD4 and Cyclin T1, which is a component of the critical HIV-1 cofactor positive transcription elongation factor b (P-TEFb) and interacts with BRD2, and tested them in the CD4+ J-Lat A2 and A72 cell lines. The following protocol describes a flow cytometry-based method to determine the amount of transcriptional activation of the HIV-1 LTR upon shRNA knockdown. This protocol is optimized for studying latently HIV-1-infected Jurkat (J-Lat) cell lines.

Keywords: Human immunodeficiency virus-1 (人类免疫缺陷病毒-1) search Latency (潜伏期) search shRNA knockdown (shRNA沉默) search Transcriptional activation (转录激活) search Flow cytometry (流式细胞术) search HIV-1 LTR (HIV-1 LTR) search J-Lat cells lines (J-Lat细胞系) search BRD2 (BRD2) search BRD4 (BRD4) search Cyclin T1 (细胞周期蛋白T1) search

Background

A72 J-Lat cells contain a latent HIV minigenome composed of just the HIV promoter in the 5’LTR that drives the expression of the fluorescent marker GFP (LTR-GFP; A72) while in A2 cells transcriptional activity is driven by the viral transactivator Tat (LTR-Tat-IRES-GFP; A2) (Jordan et al., 2001 and 2003). HIV transcription can be induced in both cell lines with TNFα mimicking T cell-receptor engagement. Cells were transduced with lentiviral vectors expressing two different shRNAs targeting each cellular protein or a scrambled control, followed by puromycin treatment to select successfully transduced cells. Cells were then stimulated with a suboptimal or saturating dose of TNFα or were left unstimulated for 24 h, followed by flow cytometry of GFP to assess transcriptional activation of the HIV-1 LTR.

Materials and Reagents

  1. Production of shRNA containing virus particles
    1. 175 cm2 tissue culture flask (Corning, Falcon®, catalog number: 353112 )
    2. Tips
      0.1-10 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-681-440 )
      1-200 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-502 )
      101-1,000 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-509 )
    3. 15 ml conical tube (Fisher Scientific, FisherbrandTM, catalog number: 05-539-5 )
    4. 10 cm dish (Corning, catalog number: 353803 )
    5. 10 ml syringes (BD, catalog number: 309604 )
    6. 0.45 µm syringe filter (EMD Millipore, catalog number: SLHV033RS )
    7. HEK293T cells (ATCC, catalog number: CRL-3216 )
    8. shRNA-expressing lentiviral vectors (Sigma-Aldrich) to knockdown:
      a.BRD2: TRCN0000006308 and TRCN0000006310
      b.BRD4: TRCN0000021424 and TRCN0000021428
      c.Cyclin T1: TRCN0000013673 and TRCN0000013675
      d.Control: pLKO.1 vector containing scramble shRNA
      e.Lentiviral packaging construct pCMVdelta R8.91 (Naldini et al., 1996)
      f.VSV-G glycoprotein-expressing vector (Naldini et al., 1996)
    9. DMEM (Mediatech, catalog number: 10-013-CV )
    10. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-106 )
    11. L-glutamine (Mediatech, catalog number: 25-005-Cl )
    12. 100x penicillin/streptomycin (Mediatech, catalog number: 30-002-Cl )
    13. 1x PBS (Mediatech, catalog number: 21-031-CV )
    14. Trypsin-EDTA (Mediatech, catalog number: 25-052-Cl )
    15. Chloroquine diphosphate salt (Sigma-Aldrich, catalog number: C6628 )
    16. HEPES (Sigma-Aldrich, catalog number: H3375 )
    17. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
    18. Dextrose (Fisher Scientific, catalog number: BP350-1 )
    19. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
    20. Sodium phosphate dibasic (Na2HPO4) (Fisher Scientific, catalog number: BP332-500 )
    21. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016 )
    22. Glycerol (Sigma-Aldrich, catalog number: G5516 )
    23. Nuclease-free H2O (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9937 )
    24. Lenti-XTM p24 Rapid Titer Kit (Takara Bio, Clontech, catalog number: 632200 )
    25. 25 mM chloroquine (see Recipes)
    26. HBSS buffer (see Recipes)
    27. 2 M CaCl2 (see Recipes)
    28. Glycerol shock solution (see Recipes)

  2. Infection of A2 and A72 J-Lat cells
    1. 75 cm2 tissue culture flask (Corning, Falcon®, catalog number: 353110 )
    2. 6 well tissue culture plates (Corning, Falcon®, catalog number: 353224 )
    3. Posi-ClickTM 1.7 ml microcentrifuge tubes (Denville Scientific, catalog number: C2170 )
    4. Tips
      0.1-10 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-681-440 )
      1-200 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-502 )
      101-1,000 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-509 )
    5. A2 and A72 J-Lat cells (Jordan et al., 2003)
    6. RPMI (Mediatech, catalog number: 10-040-CV )
    7. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-106 )
    8. L-glutamine (Mediatech, catalog number: 25-005-Cl )
    9. 100x penicillin/streptomycin (Mediatech, catalog number: 30-002-Cl )
    10. Polybrene® (Santa Cruz Biotechnology, catalog number: sc-134220 )
    11. Puromycin (Sigma-Aldrich, catalog number: P7255 )
    12. Polybrene solution (see Recipes)

  3. Analysis of HIV-1 LTR transcriptional activation by flow cytometry
    1. 96-well tissue culture plates and lids (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 249570 and 163320 )
    2. Tips
      0.1-10 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-681-440 )
      1-200 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-502 )
      101-1,000 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-509 )
    3. A2 and A72 J-Lat cells (Jordan et al., 2003)
    4. 1x PBS (Mediatech, catalog number: 21-031-CV )
    5. RPMI (Mediatech, catalog number: 10-040-CV )
    6. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-106 )
    7. L-glutamine (Mediatech, catalog number: 25-005-Cl )
    8. 100x penicillin/streptomycin (Mediatech, catalog number: 30-002-Cl )
    9. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
    10. TNFα (PeproTech, catalog number: 300-01A ), make stock solution of 100 ng/μl in sterile H2O
    11. 7AAD, Propidium iodide or one of the Zombie viability dyes
    12. JQ1 (Cayman Chemical, catalog number: 11187 ), make stock solution of 10 mM in DMSO
    13. MACSQuant Running buffer (Miltenyi Biotech, catalog number: 130-092-747 )
    14. ApoTox-GloTM Triplex Assay (Promega, catalog number: G6320 )

  4. Confirmation of shRNA knockdown efficiency by SDS-PAGE and Western blot
    1. Posi-ClickTM 1.7 ml microcentrifuge tubes (Denville Scientific, catalog number: C2170 )
    2. Nitrocellulose membrane 0.2 µm (Bio-Rad Laboratories, catalog number: 1620112 )
    3. Whatman paper (GE Healthcare, catalog number: 3030-917 )
    4. High-performance chemiluminescence film (GE Healthcare, catalog number: 28906839 )
    5. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
    6. NP-40 (Igepal CA-630) (Sigma-Aldrich, catalog number: I3021 )
    7. Na-deoxycholate (Deoxycholic acid, sodium salt) (Fisher Scientific, catalog number: BP349-100 )
    8. Sodium dodecyl sulfate (SDS) (Fisher Scientific, catalog number: BP166-500 )
    9. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
    10. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
    11. Ethylenediaminetetraacetic acid (EDTA) (Fisher Scientific, catalog number: S311-500 )
    12. Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: S6521 )
      Note: This product has been discontinued.
    13. DCTM Protein assay (Reagents A, B and S, Bio-Rad Laboratories, catalog numbers: 5000113 , 5000114 , 5000115 )
    14. Mini-Protean® TGXTM Gels (Bio-Rad Laboratories, catalog number: 4569034 )
    15. PageRulerTM Prestained protein ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 26617 )
    16. Antibodies for Western blot
      1. Rabbit polyclonal anti-BRD2 (Cell Signaling, catalog number: 5848 )
      2. Rabbit polyclonal anti-BRD4 (Abcam, catalog number: ab75898 )
      3. Rabbit polyclonal anti-Cyclin T1 (Santa Cruz Biotechnology, catalog number: sc-10750 )
      4. Rabbit polyclonal anti-α-Tubulin (Abcam, catalog number: ab15246 )
      5. Goat-anti-Rabbit IgG, HRP conjugated (Bethyl Laboratories, catalog number: A120-201P )
    17. Tween® 20 (Fisher Scientific, catalog number: BP337-500 )
    18. Glycine (Fisher Scientific, catalog number: BP381-5 )
    19. Methanol (Fisher Scientific, catalog number: A433P-4 )
    20. Blotting-grade blocker (dry milk) (Bio-Rad Laboratories, catalog number: 1706404 )
    21. Lumi-light Western blotting substrate (Roche Diagnostics, catalog number: 12015200001 )
    22. RIPA buffer (see Recipes)
    23. Western blot running buffer (see Recipes)
    24. Western blot transfer buffer (pH 8.3) (see Recipes)
    25. TBS-T buffer (see Recipes)

Equipment

  1. Pipette
  2. Biosafety cabinet ‘Level 2’
  3. Tabletop centrifuge for Eppendorf tubes (Eppendorf, model: 5415 D )
  4. Tabletop centrifuge for 96-well plates, Eppendorf, 15 ml and 50 ml tubes; used for spininfection (Beckman Coulter, model: Allegra X-14R )
  5. CO2 tissue culture incubator, 37 °C (Thermo Electron, model: FormaTM Steri-CultTM CO2 Incubators, catalog number: 3307)
  6. MACSQuant VYB FACS analyzer (Miltenyi Biotech, model: MACSOuant® VYB , catalog number: 130-096-116)
  7. Mini-PROTEAN® Electrophoresis System (Bio-Rad Laboratories, catalog-numbers: 1658006FC and 1703935 )
  8. PowerPacTM HC (Bio-Rad Laboratories, model: Power Pac HC High-Current Power Supply , catalog number: 1645052)
  9. Rocker II (Boekel Scientific, catalog number: 260350 )
  10. SpectraMax MiniMaxTM 300 Imaging Cytometer (Molecular Devices, model: SpectraMax MiniMax 300 )

Software

  1. FlowJo 9.9 or never (Tree Star)

Procedure

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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Boehm, D. and Ott, M. (2017). Flow Cytometric Analysis of HIV-1 Transcriptional Activity in Response to shRNA Knockdown in A2 and A72 J-Lat Cell Lines. Bio-protocol 7(11): e2314. DOI: 10.21769/BioProtoc.2314.

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分类

细胞生物学 > 单细胞分析 > 流式细胞术

分子生物学 > DNA > 转染

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