Computational protocol: CD81 association with SAMHD1 enhances HIV-1 reverse transcription by increasing dNTP levels

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[…] Cells were fixed in 2% paraformaldehyde (PFA; Electron Microscopy Sciences), permeabilized using the BD Cytofix/Cytoperm kit when observing intracellular proteins, and stained with primary antibodies followed by species-matching secondary antibodies (Invitrogen). Primary antibodies were: anti-CCR5 (Santa Cruz), anti-SAMHD1 (polyclonal antibody; Sigma), anti-CD82 (TS82b, kindly provided by Dr. E. Rubinstein, Villejuif, France), and antibodies produced in our laboratory (anti-CD9 (VJ1/20), anti-CD151 (LIA1/1), anti-CD63 (Tea3/18), and anti-CD4 (HP2/6)), , . Data was acquired with a FACSCantoII flow cytometer (BD), and analysed with BD FACSDIVA (BD) or FlowJo (FlowJo LLC) softwares. [...] Cells were adhered onto poly-L-lysine (PLL; Sigma), CD4, CD81 or CD9 monoclonal antibodies (10μg/ml) for 2h; or onto fibronectin (FN)-coated coverslips (20μg/ml, Sigma) for 2, 4 or 18h at 37ºC, fixed in 4% PFA, permeabilized with PBS 0.1%, 0.5% or 1% Triton X-100 for 5min or 30min, stained with primary antibodies followed by species-matching secondary antibodies coupled to Alexa Fluor fluorochromes (Invitrogen), and mounted in Prolong antifading medium (Invitrogen). Primary antibodies were: anti-CD81 (5A6), anti-SAMHD1 (monoclonal or polyclonal antibodies; Sigma), anti-EEA1, CD63 and LaminA (Santa Cruz), anti-HGS/HRS (Abcam), and Alexa-647-labeled anti-LAMP-1 (BioLegend). Duolink in situ proximity ligation assay (Sigma) was performed following the instructions provided with the kit. Primary antibodies were: SAMHD1 (polyclonal antibody), CD81 (5A6), ERM (90.3) or CD147 (VJ1/9; produced in our laboratory).Confocal images were obtained with a Zeiss LSM700 confocal scanning laser unit attached to an inverted epifluorescence microscope (Observer.Z1) fitted with a Pan APO Chromat 63X/1.4 oil immersion objective, using ZEN 2009 acquisition software (Carl Zeiss Microscopy GmbH). Images were analysed with ImageJ (NIH) software, using the Particle Analysis plugin. Measurement of Pearson’s coefficient and % of co-localized SAMHD1 and EEA1 was performed using Imaris (Bitplane), analysing three-dimensional stack confocal microscopy images. The SAMHD1 nuclear signal was excluded from the analysis. […]

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