Plasmids

TASOR expression vectors pLenti-myc-FLAG, pLenti-TASOR-myc-FLAG were purchased from Origene. pLenti-TASOR-myc-DDK expresses a TASOR isoform of 1512 amino-acids (NCBI Reference Sequence: NP_001106207.1). TASOR-ΔPARP construct was obtained by deleting the DNA sequence corresponding to the 106-319aa using the CloneAmp™ HiFi PCR Premix (639298-Takarabio) and the following 5ʹ-3ʹ-oriented primers: F-CCAGGAAGTATGCAGTTGTGTCTTTTACTTACA, R-CTGCATACTTCCTGGGGATCTGAAAACTCC. pLentiCRISPRV2-sgTASOR-Cas9 was obtained by subcloning the following 5ʹ-3ʹ-oriented annealed primers: F-CACCGCTTTCCCAACTCGCATCCGT, R-AAACACGGATGCGAGTTGGGAAAGC), containing the sgRNAs targeting the first exon of TASOR, with the enzyme BsmBI. For complementation assays, pLenti-TASOR-DDK was made resistant to the guide by mutating the sgRNA-targeted TASOR sequence using the 5ʹ-CCAACAGACGCCTCGTGGGAGTCA-3ʹ primer. T819A and T819E TASOR mutants were produced by site-directed mutagenesis using the pLenti-TASOR-myc-FLAG construct as a template. The TASOR ORF was then subcloned into the pAS1B-HA vector. SAMDH1-Flag is expressed from pCDNA3.

HIV-2 (Ghana-1 strain) Vpx R42A was produced by site-directed mutagenesis using the pAS1B-HA-Vpx HIV-2 Ghana construct as a template.

Virus and VLP production

VSV-G pseudo-typed viruses and VLPs were produced in 293FT by the calcium-phosphate co-precipitation method. SIV3 + ΔVprΔVpx packaging vector was a gift from N. Landau and is described in [49]. VLPs (Vpx or Vpr expressed from pAS1B and incorporated into VLPs) were obtained from co-transfection of VSV-G plasmid, SIV3 + ΔVprΔVpx packaging vector and pAS1B-HA plasmid, empty or encoding for one viral protein (HIV-2 Ghana Vpx or Vpx R42A, HIV-1 Vpr or Vpr S79A). Cell culture medium was collected 48 h after transfection and filtered through 0.45 μm pore filters. Viral particles or VLPs were concentrated by sucrose gradient and ultracentrifugation. The incorporation of Vpx or Vpr into VLPs was assessed by Western-Blot.

Cell lines

Cell lines were regularly tested for mycoplasma contamination. Cells were cultured in media from ThermoFisher: DMEM (HeLa, 293FT), RPMI (Thp1 monocytes, Jurkat cells, JlatA1) containing 10% heat-inactivated fetal bovine serum (FBS, Eurobio), 1,000 units ml−1 penicillin, 1,000 µg ml−1 streptomycin. The J-Lat A1 cell line was a gift from Eric Verdin [33]. J-Lat A1 KO Control (CTL) and TASOR KD cells were generated by transfection of pLentiCRISPRV2-sgCtrl-Cas9 and pLentiCRISPRV2-sgTASOR-Cas9 respectively, with DMRIEC reagent. Transfected cells were cultured for 3 days prior to puromycin selection (1 µg/mL during 3 days). The J-Lat A1 TASOR KD cells were sorted by flow cytometry with the BD FACS ARIA3 cytometer of CYBIO platform (Institut Cochin) to separate three populations of cells: GFP-negative cells, GFP-positive-cells with low GFP expression (Dim) and GFP-positive cells with high GFP expression (Bright). Cells were then amplified for 2 weeks in culture before subsequent experiments.

HeLa LTR-ΔTAR-Luc cells were generated in the laboratory of Stephane Emiliani from the HeLa LTR-Luc cells described by du Chené et al. [50]. HeLa LTR-ΔTAR-Luc cells KD for TASOR expression were obtained by transitory transfection by the calcium phosphate method of the pLentiCRISPRV2-sgTASOR-Cas9 construct (control cells with pLentiCRISPRV2-sgCtrl-Cas9), without further selection step.

Nocodazole treatment and release

For nocodazole release experiments, proliferating HeLa cells were first grown to about 60% confluency prior to addition of 330 nM nocodazole for 18 h. Subsequently, cells were washed twice with PBS and released into fresh medium.

siRNA treatment

siRNA transfections were performed with DharmaFECT1 (Dharmacon, GE Lifesciences). The final concentration for all siRNA was 100 nM. The following siRNAs were purchased from Sigma Aldrich: siCDK1: SASI_Hs02_00325516; siCDK2: SASI_Hs02_00349201. The non-targeting control siRNAs (MISSION siRNA Universal Negative Control #1, SIC001) were purchased from Sigma Aldrich.

Luciferase activity assay

Cells were washed twice with PBS then lysed directly in wells using 1 × cell culture lysis reagent (Promega). Cell lysates were clarified by centrifugation, luciferase activity was measured using a luciferase assay system (Promega) and a TECAN multimode reader Infinite F200 Pro.

Flow cytometry analyses

Reactivation assay: J-Lat A1 cells were collected and resuspended in PBS-EDTA (0.5 mM). Data were collected and analyzed with a BD Accuri C6 cytometer and software CFlow Plus. At least 10,000 events in P1 were collected, the GFP-positive population was determined using a GFP-negative population arbitrary and the same gate was maintained for all conditions. Analysis was performed on the whole GFP-positive population.

Cell cycle assay: J-Lat A1 cells were seeded into 12 well plates, then transduced with VLPs for 48 h. The cells were then treated with 330 nM Nocodazole for 18 h prior fixation in 70% ethanol. Following treatment for 30 min at 37 °C with 0.2 mg/ml RNase A and 50 µg/ml propidium iodide in buffer H (20 mM HEPES, 160 mM NaCl, 1 mM EGTA), cells were analyzed for their DNA content using the LSR Fortessa cell analyzer (BD Biosciences). At least 10,000 GFP-positive cells were analyzed for their distribution in the different phases of cell cycle.

Primary CD4 (activation Noco, ETP)

PBMCs from the blood of anonymous donors (obtained in accordance with the ethical guidelines of the Institut Cochin, Paris and Etablissement Français du Sang) were isolated by Ficoll (GE Healthcare) density-gradient separation. CD4 + T cells were isolated by positive selection (using magnetic CD4 human MicroBeads from Miltenyi Biotec).

Cells were activated with CD3/CD28 agonists (T Cell transact) and stimulated with human IL-2 (50u/mL) for 3 days. Activated CD4 T cells were then treated with DMSO, 330 nM nocodazole or 10 mM ETP for 18 h. Cells were then washed twice with PBS and lysed with RIPA buffer. TASOR phosphorylation was then monitored by WB.

Immunofluorescence

Hela cells were transfected with TASOR-FLAG (WT or mutant). 48 h post transfection, cells were fixed with 4% PFA for 15 min and then permeabilized with PBS-Triton 0.1% for 10 min at room temperature. A saturation step was performed for 1 h with PBS BSA 3%. After PBS-mediated washes, cells were incubated with an 1/500 anti-TASOR (HPA006735-Merck) for 1 h at RT. Cells were then washed three times with PBS, then anti-rabbit Alexa 594 was added at a dilution of 1/1000 for 1 h at room temperature in a dark chamber. DNA was stained with Hoechst 33258 (382061-Sigma). After three PBS-mediated washes, the coverslips were mounted on slides with the use of ProLong™ Diamond Antifade Mountant (P36970-ThermoFisher). Imaging was performed with a Spinning-Disk LEICA confocal microscope from the IMAG’IC core facility at Institut Cochin.

Anti phospho-TASOR antibody

Anti-phospho-TASOR antibody was engineered by Genscript by immunization of rabbits using the following phosphorylated peptide (p-peptide): 815-LNS(pT)PDKKDYEQPTC-830.

Immunoprecipitation, Western blot procedures and antibodies

For TASOR-FLAG and SAMHD1-FLAG immunoprecipitations: HeLa/293 T cells grown in 10 cm dishes were transfected with pLenti-FLAG or with pLenti-TASOR-FLAG or SAMHD1-FLAG plasmids with CaCl2. 48 h after transfection, cells were lysed in 500 µl RIPA buffer (50 mM Tris–HCl pH7.5, 150 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 0.5% Triton X100) containing an anti-protease cocktail (A32965, ThermoFisher). Cell lysates were clarified by centrifugation and a minimum of 500 µg of lysate was incubated with pre-washed anti-FLAG-coupled Dynabeads (Invitrogen) at 4 °C, under overnight rotation. After three washes in RIPA buffer, immunocomplexes were eluted with Laemmli buffer 1 × and were separated by SDS–PAGE. The following antibodies, with their respective dilutions in 5% skimmed milk in PBS-Tween 0.1%, were used: anti-Flag M2 (F1804-200UG, lot SLCD3990, Merck) 1/1000; anti-TASOR (HPA006735, lots A106822, C119001, Merck) 1/1000 – for IF assays 1/500, anti-MPP8 (HPA040035, lot R38302, Merck) 1/1000; anti-MORC2 (PA5-51172, ThermoFisher) 1/1000; anti-Actin (AC40, A3853, Merck) 1/1000; anti-CDK1 (Cdc2p34(17), sc-54, Santacruz), anti-CDK2 (M2, sc-163, Santacruz), anti-Cyclin A (H-432, sc-751, Santacruz), anti-Cyclin B1 (H-433, sc-752, Santacruz), anti-SAMHD1 (SAB1400478, Sigma), anti-phospho-SAMHD1 (D702M, 89930, Cell signalling), anti-GAPDH (6C5, SC- 32233, Santa Cruz). All secondary antibodies anti-mouse (31430, lot VF297958, ThermoFisher) and anti-rabbit (31460, lots VC297287, UK293475 ThermoFisher) were used at a 1/20000 dilution before reaction with Immobilon Forte Western HRP substrate (WBLUF0100, Merck Millipore).