Sub-cellular localization of incoming HIV-1 in quiescent CD4+ T cells. A. Incoming HIV-1 CA localizes at the centrosome in infected human primary quiescent CD4+ T cells. Quiescent CD4+ T cells (0.5 × 106 cells) were spinoculated with the NL4.3 strain of HIV-1 (moi = 1) as described . The NL4.3 viral stock was obtained from 24-h harvests of supernatant from 293T cells transduced with a plasmid encoding the full-length viral genome and was titrated by limiting dilution MAGI assay . At the indicated time points, infected and control cells were fixed in 4% PFA (15 min, 4°C), permeabilized with ice-cold methanol (5 min, 4°C) and stained with antibodies against HIV-1 CA protein (A25, Hybridolabs, Pasteur) and γ-tubulin (Abcam), a marker for the centrosome. Nuclei were stained with DAPI and images were acquired on a laser-scanning confocal microscope (LSM510 Meta; Carl Zeiss) equipped with an Axiovert 200 M inverted microscope, using a Plan Apo 63/1.4-N oil immersion objective. Co-localization between CA and γ-tubulin staining was observed in 58% to 75% of CA-positive cells. B) HIV-1 infection did not significantly alter the activation status of quiescent CD4+ T cells. Surface expression of T cell activation markers (CD25 and HLA-DR) was monitored by flow cytometry. C) Pericentriolar distribution of incoming HIV-1 CA in quiescent CD4+ T cells transduced with a VSVg-pseudotyped HIV-1 based lentivector carrying the GFP transgene. The lentivector stock was produced by co-transfected with an HIV-derived packaging construct, the VSVg-expressor vector and the plasmid vector (psPAX2, pMD2.G and pWPI, respectively, a gift from D. Trono), as described . The titre of the lentivector stocks was determined by measuring the percentage of GFP positive cells 48 h following transduction of 293T cells by flow cytometry. Transduced and control quiescent CD4+ T cells were immunostained and visualized as described above. Co-localization between CA and γ-tubulin staining was observed in 60% to 82% of CA-positive cells.