HIV-1 Nef triggers the formation of podosome rosettes, cell adhesion, and matrix degradation. (A) Immunofluorescence (IF) images of HIV-1–infected hMDMs on glass. HIV-1 p24 (red) and Alexa Fluor 488-coupled phalloidin (F-actin in green). (Upper) Arrowheads show individual podosomes in a noninfected cell and arrows show a podosome rosette in a mononucleated infected cells (p24 positive). (Lower) Arrows show a podosome rosette/belt in an infected MGC. (Right) Percentage of cells forming podosome rosettes is shown. Mean ± SD, n = 4. Only p24-positive cells were counted for HIV-1, HIV-1Δnef, and MGCs. (B) hMDMs were infected or not with HIV-1 or HIV-1Δnef and seeded on glass or on fibronectin. The percentage of adhesive cells after 2 hours was measured and reported as 100% for controls. Mean ± SD, n = 4. (C) (Upper) Scanning electron microscopy images of a noninfected macrophage and an infected MGC on top of Matrigel matrices after 24 hours of culture. Arrowhead: hole in Matrigel reflecting the matrix proteolytic activity of infected cells. (Lower) hMDMs were infected with HIV-1 or HIV-1Δnef and seeded on glass coverslips coated with gelatin fluorescein isothiocyanate (blue) for 24 hours. p24 (red) and F-actin (green). White arrowhead: noninfected macrophage; red arrowhead: infected macrophage/MGC degrading the ECM. (D) Quantification of matrix degradation is shown: number of cells degrading the matrix and degraded area/cell area. Mean ± SD, n = 4 (10 fields [×40] per condition). (E-F) hMDMs were transduced with a lentiviral vector expressing Nef_SF2-GFP or GFP (control). (E) F-actin staining (red) of a Nef_SF2-GFP expressing cell (green) on glass with podosome rosettes (arrow) and quantification of the number of cells forming podosome rosettes. Mean ± SD, n = 5. (F) Quantification of the number of cells degrading Alexa Fluor 594-conjugated fibrinogen. Mean ± SD, n = 3. Scale bars, 10 μm.