Generating and Titrating Retroviral Vectors

293T and TE671 cells were maintained in DMEM (Invitrogen) and U937 cells in RPMI +[L]-Glutamine (GIBCO), each supplemented with 10% heat inactivated foetal calf serum (Biosera) and 1% penicillin/streptomycin (Sigma). U937 cells stably expressing SAMHD1 variants were prepared by transduction with MoMLV-based Puromycin-expressing VLPs, followed by selection with puromycin at 10 μg/mL. MoMLV-based YFP-expressing VLPs were made by cotransfecting 293T cells with pVSV-G (gifted from D. Lindemann), pKB4 [37 (link)] and pLGateway_SAMHD1IRESYFP (wild-type or mutants), harvesting 48 hr post-transfection. HIV-1GFP was produced by cotransfection of pVSV-G, p8.91 and pCSGW [38 (link)]. MoMLV-based Puromycin-expressing VLPs were made by cotransfection of pKB4, pVSV-G and pCMS28_SAMHD1 (wild-type or mutants). VLPs were titred on TE671 or 293T cells for normalisation prior to infection. Viruses with mutations in Reverse Transcriptase were normalised by HIV-1 p24 ELISA (Perkin Elmer).