Reagents and antibodies
The following antibodies were used: rabbit polyclonal anti-human UNG2 clone PU59 and rabbit anti-mouse UNG2 clone PUMA have been previously described 30,31. The latter was used to immunoprecipitate mouse UNG2 which cannot be directly detected by PU59 antibody. Anti-hUNG2 rabbit polyclonal NBP1-49985 was from Novus Biologicals; anti-Vpr mixture was made with equal amounts of goat polyclonal antibody (sc-17493) from Santa Cruz Biotechnology, rabbit polyclonal antibody #709 and #11836 (both from NIH AIDS reagent program) ; anti-a tubulin mouse monoclonal antibody (T5168) was from Sigma; goat polyclonal anti-p24 (4999-9007) was from AbD Serotec; Secondary HRP-conjugated antibodies were from Jackson Laboratory. UGI inhibitor was purchased from New England Biolabs. MG132 was from Sigma Aldrich.
The pHR-Vpr (wt and mutants), pHR-GFP, pCMVDR8.2DVpr, pCMV-VSV-G constructs (see Figure 1c and Figure 6a) were kindly provided by Vicente Planelles (University of Utah School of Medicine, Salt Lake City, USA). pCMV HA-Vpr construct was generated by cloning a PCR-derived HA-Vpr amplicon derived from pNL4.3 Vpr as template in pcDNA3.1. Plasmids encoding for viral molecular clones pNL4.3 and pNL4.3ΔVpr (from W.C. Greene, Gladstone Institutes San Francisco, USA) were used to produce HIV-1 and HIV-1 DVpr viruses, respectively.
Mice were maintained in our animal facilities (BISCEm (Research Facility for Integrative Biology, Health, Chemistry and Environment), Limoges) approved by French Ministry of Agriculture (N° A8708505), under specific-pathogen-free conditions. Mice were housed and procedures were conducted in agreement with European Directive 2010/63/EU on animals used for scientific purposes applied in France as stipulated in the “Décret n°2013-118 du 1er Février 2013 relatif à la protection des animaux utilisés à des fins scientifiques”. All methods in the current study were carried out in accordance with relevant guidelines and regulations and all experimental protocols were approved by French institutions. The experimental protocols were specifically approved by Comité Régional Ethique Animale du Limousin n°33 and authorized by Ministère de l’Éducation Nationale, de l'Enseignement Supérieur et de la Recherche / Plateforme Autorisation-Projet website Recherche.gouv.fr (APAFIS #16216-2018072015045506 v3).
CH12F3-2 (from T. Honjo, Kyoto, Japan), and Daudi B-cells were maintained in RPMI 1640 complete medium. HEK 293T cells were maintained in DMEM complete medium. Complete media were supplemented with penicillin-streptomycin (Lonza) and 10% heat-inactivated fetal bovine serum (FBS) (Cambrex). Primary B-cells were prepared from 5- to 6-week old C57BL/6 mice (Charles River, USA) or from AID-/- mice (formally Aicda-/-) 32,33 provided by François Huetz (Pasteur Institute, Paris), as follows. Erythrocyte-depleted mouse spleen cells were cultured in RPMI medium supplemented with 10% FBS, sodium pyruvate, nonessential amino acids, 50 µM β-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Mature resting B-cells were isolated by depletion of CD43-expressing B-cells (Myltenyi Biotec).
In a 6-well plate format, HEK 293T cells (3x105 cells per well) were transduced with lentiviral vectors driving (Vpr+) or not (Vpr-) the expression of Vpr (produced as outlined in Fig 1c) at a M.O.I. of 10.,Efficient integration and expression was visualized 72 hours later by the expression of the GFP reporter by both VLPs (Figure 6a). Cells were then washed and co-cultured with Daudi B-cells (2x106 cells per well) suspended on top of transduced HEK 293T layers in cell culture Millicell hanging inserts with hydrophilic 0.4 µm PTFE membranes (MerckMillipore) (see Figure 6b). Daudi cells were immobilized on poly(L-lysine)-coated cover slips 72 hours later, formalin-fixed and labeled with DAPI and anti-Vpr antibodies to visualize nuclei and Vpr, respectively. In parallel, whole cell extracts were prepared from Daudi and HEK 293T cells and UNG activity was measured (see below in Uracil DNA glycosylase assays section) and Vpr immunoprecipitation. Briefly, cells were lysed in 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1mM MgCl2, 1% Triton X-100 and protease inhibitors (Complete, Roche). Fifty μg of total protein was kept as input and the remaining cell lysate (150 µg) was incubated with antibodies for 2h at 4°C and with Protein A/G Magnetic Beads (Thermo Fisher) overnight at 4°C. The beads/immune complexes were washed 6 times with 1 mL lysis buffer and released from the beads by boiling in 1x Laemmli buffer. UNG2 and Vpr contents were analyzed in input and Vpr IP samples, respectively. The same 6 well format was used to perform the co-culture experiment with MAGIC5B producer cells infected with either HIV-1 wt or HIV-1DVpr and Daudi as bystander cells maintained in cell inserts (Figure 6e).
Daudi cells were adsorbed on polyLysine-treated slides and fixed with Formalin (Sigma) containing 1% Triton-X-100. Cells were labeled with anti-Vpr antibody followed by labeling with a mixture of Alexa488-conjugated donkey anti-goat and anti-rabbit IgG (H+L) antibodies (Life Technologies). Nuclei were stained with DAPI staining solution from Sigma Aldrich. Cells were mounted on glass slides covered with anti-fade medium (Hardset Vectashield). Two-color images were obtained with a light microscope, Leica DC250 (Leica) with a Plan Apo 63 × /1.32-0.6 oil-immersion objective lens. Digital images were processed with the ImageJ software (NIH Image). Percentages of Vpr+ Daudi B-cells were determined by using the Cell Counting plugin of ImageJ. A minimum of 750 cells were counted per 6-well plate.
Cell cycle and apoptosis
Cells were collected, washed twice and resuspended in ice cold PBS. Then the cells were fixed with 70% EtOH for 1 hour on ice. Cells were pelleted for 5 min at 1,500 rpm, washed twice with PBS and resuspended in a solution containing 50 µg/ml propidium iodide, 0.1 mg/ml RNase A, 0.1% Triton X-100 in PBS. After 30 min incubation at 4°C in the dark, DNA content was determined by flow cytometry using an EPICS PROFILE XL4C cytofluorometer (Coulter). Appropriate gating was applied to discriminate singlets and aggregated cells. Curve fitting decomposition was done with FCS Express (De Novo Software). For apoptosis evaluation, cells were incubated in PBS containing CaCl2 (0.33g/L) and labeled with Annexin V-FITC (Immunotools) for 15 min at room temperature and analyzed by flow cytometry. Cell viability was performed by using the MTT viability assay Cell TiterTM (Promega).
Viral stock production
Viral stocks were produced by calcium phosphate transfection of HEK 293T cells. Briefly, cells were transfected with the proviral DNA constructs for 8 h, washed with pre-warmed DMEM-10% SVF medium. Viral supernatants were collected 48 h post-transfection, filtered and frozen in aliquots at -80°C. HA-Vpr delivering VLPs were purified by centrifugation through 20% sucrose cushion at 25,000 rpm for 2.5 h at 4°C in a Sw32Ti rotor (Beckman Coulter). The pellets were resuspended in PBS, aliquoted and stored at −80°C. The Vpr content of purified VLPs was checked by western blot analysis (Figure 2a). Viral stocks were titered using HIV-1 p24 Enzyme-linked immunosorbent assay (ELISA) kit (Ingen) and focus forming assay (FFA) following the expression of GFP from the lentiviral construct (Figure 1c and 2a). Viral stocks of wt or DVpr HIV-1 (NL4-3) were produced as described above in HEK 293T cells and titered as above. VSV-G pseudotyped wt HIV-1 viral stocks were produced using co-transfection of HEK 293T cells with a NL4-3 Denv derivative and pCMV-VSV-G construct.
Proteins were separated by 12.5% SDS–PAGE gels, transferred to a PVDF membrane (Millipore) and immunoblotted with the appropriate primary and horseradish peroxidase–conjugated secondary antibodies. Immune complexes were revealed by Enhanced Chemiluminescence (Thermo Scientific) and recorded with the Gene Gnome imaging system (SynGene). Band intensities were quantified using ImageJ software.
Uracil DNA glycosylase assays
The fluorescent probe PEG-U9 consisted of a 5’-FAM-GCACUUAAGAAUUG-PEG-CAAUUGUUAAGUGC-3’DAB oligonucleotide synthesized by Eurogentec 22. Uracil DNA glycosylase activity was assayed in a reaction buffer containing 20 mM Tris-HCl pH 8.0, 50 mM KCl, 0.2 mM MgCl2, 0.05% Brij-35. The mixture was incubated with PEG-U9 (100 nM) for 30 minutes at 37°C. Fluorescence was recorded using a Tecan Infinite 200 fluorimeter.
Genome uracil content quantification
Genomic DNA was extracted from 3x106 cells using DNAzol (Invitrogen) according to manufacturer’s recommendations. Pre-existing abasic sites (AP sites) in DNA were protected by incubation with methoxyamine (200mM) for 2 hours at 37°C. Then 4 µg DNA was incubated in the presence of 0.2 U recombinant E. coli UDG (New England Biolabs) for 15 min at 37°C. Apurinic/apyrimidinic sites (AP) generated by excision of uracil residues contained in DNA were titrated using the OxiSelect oxidative DNA Damage ELISA kit (Euromedex). Alternatively, genomic uracil content was determined by LC/MS/MS method 34. Briefly, DNA was isolated by phenol:chloroform:isoamyl extraction, treated with alkaline phosphatase to remove free deoxyribonucleotides, and then enzymatically hydrolyzed to deoxyribonucleosides. Deoxyuridine (dU) was then separated from deoxycytidine (dC) by HPLC fractionation using a reverse-phase column with embedded weak acidic ion-pairing groups (2.1 mm x 150 mm, 5 μm, Primesep 200, SIELC technologies), using a water/acetonitrile gradient containing 0.1% formic acid. The dU fraction was finally analyzed by ESI-LC/MS/MS using a reverse phase column (2.1 mm x 150 mm, 3.5 µm, Zorbax SB-C18, Agilent Technologies), using a water/methanol gradient containing 0.1% formic acid on an API5000 triple quadrupole mass spectrometer (Applied Biosystems) in positive ionization mode.
Class Switch DNA Recombination
CH12F3 cells were transduced with VLP HA-Vpr or DVpr. Twenty-four hours later, transduced cells were stimulated to undergo IgM-to-IgA switching with 5 ng/ml mouse recombinant IL4 (Peprotech), 200 ng/ml anti-CD40 monoclonal antibody (clone HM40-3, eBiosciences) and 1ng/mL hTGF-β (R&D Systems) for 3 days. After labeling cells with anti-B220-APC (clone RA3-6B2, eBiosciences) and anti-IgA-PE (Southern Biotechnology), CSR efficiencies (% of IgGA+ B220+ cells) were evaluated by flow cytometry. Primary mature mouse B-cells were transduced with Vpr-delivering VLPs or control VLPs, and stimulated 24 hours later to undergo IgM-to-IgG1 switching with 40 μg/mL LPS (Salmonella typhimurium, R&D Systems) and 50 ng/mL IL-4 (PeproTech) for 3 days. After labeling with anti B220-APC (clone RA3-6B2, eBiosciences) and anti-IgG1-PE (clone A85-1, BD Biosciences), CSR efficiencies (% of IgG1+ B220+ cells) were evaluated by flow cytometry. Data were acquired on a BD LSRII Fortessa cytometer and analyzed with the BD FACSDiva 6.1.3 software. Cell proliferation was measured with standard MTT/formazan colorimetric assay at 550 nm 35.
Statistical analysis were performed using either SZanalyze software (http://www.ezanalyze.com; Poynton (2007) Version 3.0), Boston MA, USA) by one-way analysis of variance (ANOVA), or with GraphPad Prism 5.04 (GraphPad software, La Jolla USA).