Cells
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Pancoll (PAN biotech, Aidenbach, Germany, lymphocytes separation medium) density gradient centrifugation. B lymphocytes were obtained by negative cell selection using the B-cell isolation kit II (MiltenyBiotec, Bergisch-Gladbach, Germany) according to the manufacturer’s protocol. This procedure was performed twice to ensure an optimal purification of CD19-positive CD3-negative lymphocytes (B-cells) as assessed by flow cytometry analysis. The FITC-labeled anti-CD19 and the PE-labeled anti-CD3 antibodies were obtained from Biolegend (San Diego, CA, USA) and BD Pharmigen (San Diego, CA, USA), respectively. Purified B-cells were analyzed on an LSR-II flow cytometer (BD Biosciences, NJ, USA) prior to transfer (106 cells/mL, 37°C) into RPMI medium supplemented with 10% heat-inactivated FCS (Gibco®, Thermo Scientific, Carlsbad, CA, USA) and 1% penicillin/streptomycin antibiotics (“complete medium”). Macrophages were prepared from Pancoll-isolated PBMCs by overnight adhesion (in complete medium, 37°C) and further cultivation for 5 days. B-cells from HIV – infected individuals were purified in a Biosafety Level 3 (BSL3) laboratory as described above. For HIV production, Human Embryonic Kidney (HEK) 293T cells obtained from the American Type Culture Collection (ATCC; CRL-11268) were cultivated in Dulbecco’s modified Eagle’s medium (Gibco®, Thermo Scientific) in a BSL3 laboratory.
Virus production
The HIV-1-producing plasmid pNL4-3 contains the genome of the NL4-3 HIV-1 strain (GenBank AF324493.1). pNL4-3Denv was derived from pNL4.3 by a deletion of the env gene NheI-BsaBI region. Retroviruses were produced by calcium phosphate-mediated transfection of 293T cells as previously described 1. Aliquots were stored at – 80°C. P24 quantification was performed using HIV-1 P24 ELISA assays (PerkinElmer Life And Analytical Sciences, Rotkreuz, Switzerland) following the manufacturer’s instructions. All samples thus prepared were found to contain approximately 0.5-1 ng/µL of P24.The whole process was performed in a BSL3laboratory.
Determination of seric Tat concentration
Tat concentrations in sera from HIV-infected individuals were determined using both a dot blot assay and an ELISA assay. The detailed procedure and reagents is described in the Supplementary Information section.
Briefly, each serum was filtered through an Amicon® Ultra-0.5 mL centrifugal filter (30 kDa, Merck Millipore, Darmstadt, Germany), according to the manufacturer’s instructions. For dot blotting, 2µl samples were transferred onto a nitrocellulose membrane and dried at room temperature (RT) for 15 minutes. Following blocking (1 hr., RT with gentle rocking) in 5% (w/v)bovine serum albumin (BSA) in a Tris saline buffer (TBS, 200 mM Tris-HCl, 150 mM NaCl, pH 7.5) with 0.1% Tween, the blot was washed (5 min, RT) three times in 1% (w/v) BSA in TBS and 0.1% Tween followed by incubation (1 hour rocking, RT) with a mouse primary anti-Tat antibody (Santa Cruz Biotech, Inc., Santa Cruz, CA, USA, dilution 1:500). After three washes (each one 5 min, RT) in 1% (w/v) BSA in TBS and 0.1% Tween, the blot was further incubated (40 min, RT) with a highly absorbed horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Sigma Aldrich, St Louis, MO, USA, dilution 1:1000). Blots were developed using Immobilon Western Chemiluminescent HRP substrate (Merck Millipore) and an ImageQuant Las 4000 Mini (GE Healthcare, Little Chalfont Bucks, UK). A Tat titration curve was prepared for reference using a purified recombinant Tat protein obtained from the NIH AIDS Research and Reference Reagent Program, USA. The Tat quantifications were confirmed using an ELISA assay. Briefly, MaxiSorp® 96- wells plates (NuncTM, Thermo Scientific) were coated overnight (rocking, 4°C) with 50 µL per well of a 1 μg/mL mouse monoclonal antibody (sc-65916 Santa Cruz Biotech) directed to aminoacids 71-81 in the Tat protein sequence. After three washes in 200 μl PBST (1X PBS, 0.05% Tween 20) added per well and blocking in 200 μl per well with 5% BSA in PBST (1 hr., gentle rocking, RT), a new wash was performed before 50 μL undiluted patients’ samples were added to each well. After two hours incubation (gentle rocking, RT) and three washes in PBST, a goat anti-Tat polyclonal antibody (sc-17439 Santa Cruz Biotech) recognizing aminoacids 15-25 in the Tat sequence was added at a concentration of 1 μg/mL in 0.25% BSA/PBST (50 μL per well) followed by 1 hr. incubation at RT. After 3 washes in PBST, the plate was treated with horseradish peroxidase (HRP)-conjugated rabbit anti-goat antibody (Santa Cruz Biotech) at 4μg/mL in 0.25% BSA/PBST (50 μL per well, 1hr, RT). The plate was further washed three times with PBST, incubated (15 min, RT) in 50 μL per well of freshly prepared o-Phenylenediamine dihydrochloride (OPD substrate, Sigma Aldrich) in a citric buffer (final concentration 0.68 mg/mL) with addition of H2O2 (final concentration, 0.06%). The reaction was stopped by addition of 25μl 1M H2SO4 per well followed by chemiluminescence detection at 490 nm using the Opsys MR (Dynex, Denkendorf, Germany) plate reader. Samples and Tat dilutions for standard curves were in triplicates for every condition.
Tat immunodepletion
Tat was immunodepleted from the HIV-containing supernatant using a goat anti-Tat-HIV-1 polyclonal antibody (sc-17439, Santa Cruz Biotech) and Dynabeads Protein G (Life Technologies, Thermo Scientific) according to the protocol provided by the producer with the following modifications. The antibody was diluted 1:200 in 1X PBS and incubated with magnetic beads (1.5 µg, 10 min with rotation, RT), followed by washing in 1X PBS. The virus-containing supernatant was incubated again with bead-coupled antibodies (1 hr. with rotation, 4°C). Beads were precipitated using a magnet. The entire procedure was conducted in a BSL3 laboratory.
B-cell incubation
B-cells plated at 106 per mL were mixed with a HIV-1 virus-containing supernatant prepared as described above to a final Tat concentration of approximately 14 ng/mL. For experiments with purified Tat, we used a recombinant Tat protein produced by Ablinc and obtained through the NIH AIDS Research and Reagent Program. According to the manufacturer the Tat protein was >95% pure after purification by heparin-affinity chromatography and reverse phase HPLC which removed endotoxins2. For analysis of Tat dose dependent effects, the protein was used at concentrations ranging from 10 to 250 ng/mL. TatC22 is a mutant Tat protein with a 22CysGly whose transactivation function has been shown to be completely abrogated 3,4. Produced by Abcam (Cambridge, UK), the TatC22 recombinant protein was used at 250 ng/mL, a concentration also selectedfrom the dose response curve for experiments wild type Tat. Prior to 3D-FISH, some Tat incubations of B-cells were performed in the presence of the MRE11 inhibitor mirin (Calbiochem) or the RAG inhibitor raltegravir (Merck Millipore, USA, obtained through the NIH AIDS Research and Reference Reagent program, USA) used at 25 μM and 5.1 nM, respectively. H2O2 treatment of B-cells was performed in 50 µM H2O2 (Sigma-Aldrich) in complete medium (5 min, RT) followed by replacement with fresh complete medium.
3D FISH
“RainbowFISH” probes used for hybridization and staining of human chromosome loci 8q24 (MYC gene locus), 14q32 (IGH gene locus), 11q13 (CCND1 gene locus) and 11p15 (β-globin gene locus) were from genomic libraries provided by Empire Genomics (Buffalo, NY, USA). A “SureFISH” probe (Agilent, Santa Clara, CA, USA) was used for 19p13.3 (CIRBP gene locus). The MYC, CCND1 and β-globin probes were labeled with SpectrumOrange, the IGH and CIRBP probes, with SpectrumGreen. 3D FISH experiments were performed as previously described18.
Microscope image acquisition and analysis
Images were acquired using a TCS SP8 confocal microscope (Leica Microsystems, Berlin, Germany) with a 63X oil immersion objective. Z-stacks were acquired using a frame size of 1024×1024, and 0.5 µm z-steps, with sequential multitrack scanning using the 405, 488, 543, 633 nm laser wavelengths. Radial position and colocalization of gene loci were ascertained using the Bitplane® Imaris (Zurich, Switzerland) program when the distance between the center of one red signal was measured at a distance of 1 µm or less from the center of a green signal. For analysis of MYC-γH2AX, MYC-RAG1 or MYC-53BP1 colocalizations, the LEICA Application Suite X (Leica Microsystems) software was used.
Immunofluorescence
Purified B-cells were seeded on poly L-lysine-coated 15×15 mm coverslips (20 min, RT). After adhesion, B-cells were washed with 0.3X PBS and fixed in 4% paraformaldehyde in 0.3X PBS (10 min, RT) followed by 3 washes in 1X PBS. After blocking with 0.5% BSA (Euromedex, Souffelweyersheim, France) in 1X PBS (40 min, RT), cells were immunofluorescently stained with the following rabbit reagents: anti-RNA pol II antibody (Active Motif, La Hulpe, Belgium, dilution 1:500), anti-Histone H2AX phospho-Ser139 antibody (Active Motif, dilution 1:500), anti-53BP1 antibody (Abcam, dilution 1:500)) and RAG1 antibody (Novus Biologicals, Littleton, CO, USA, 1:200). Incubation with the primary antibodies (2 hrs., RT) was followed by 3 washes in 1X PBS and by incubation (1 hr., RT) with a secondary antibody conjugated to Alexa Fluor-633 or -488 (Life Technologies, dilution 1:200). For immuno-3D FISH, cells were first treated for 3D FISH as described above followed by indirect immunofluorescence staining before final mounting and analysis.
RT-qPCR
For quantitative evaluation of transcription levels, total RNA was extracted from purified B-cells using theNucleoSpin® RNA II kit according to the manufacturer's recommendations (Macherey-Nagel, Oensingen, Switzerland). Total RNA (1 µg) was primed in a 20 µL reaction mixture with an oligo(dT) primer and reverse transcriptase (Thermo Scientific). The obtained cDNA was amplified using specific primers and the FastStart Universal SYBR Green Master mix (RoX/Roche, Basel, Switzerland). Expression of target genes was analyzed using the 2-ΔΔCt method with normalization with GAPDH and comparison of expression between variously treated cells. For quantification, expression levels were set to 1 in controls. The following primer sequences were used:RAG1, forward 5’- TGGATCTTTACCTGAAGATG-3’, reverse 5’-CTTGGCTTTCCAGAGAGTCC-3’; MRE11, forward 5’-CCAGAGAAGCCTCTTGTACG-3’, reverse 5’-TTCCACCTCTTCGACCTCTTC-3’; MYCforward 5’-AGGAGGTGGCTGGAAACTTGT-3’; reverse 5’-CGCTATGCTGGATTTTGCTGCA-3’; GAPDH forward 5’-CTGCACCACCAACTGCTTAG-3’, reverse 5’-AGGTCCACCACTGACACGTT-3’.
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2 Dudley A, McKinstry W, Thomas D, Best J, Jenkins A. Removal of endotoxin by reverse phase HPLC abolishes anti-endothelial cell activity of bacterially expressed plasminogen kringle 5. Biotechniques 2003; 35: 724–732.
3 Garcia JA, Harrich D, Pearson L, Mitsuyasu R, Gaynor RB. Functional domains required for tat-induced transcriptional activation of the HIV-1 long terminal repeat. EMBO J 1988; 7: 3143–3147.
4 Rossi C, Balboni PG, Betti M, Marconi PC, Bozzini R, Grossi MP. Inhibition of HIV-1 replication by a Tat transdominant negative mutant in human peripheral blood lymphocytes from healthy donors and HIV-1-infected patients. Gene Ther 1997; 4: 1261–1269.