LEDGF/p75 is incorporated in HIV-1 particles. (A) Illustration of virus production and purification protocols. (B) Distribution of exosomes and HIV fractions: Exosomes as assessed by acetylcholinesterase (AChE) activity and HIV-1 as detected by p24 ELISA were effectively separated by velocity gradient purification (mean values ± standard deviations; n = 6). (C) Western blot analysis of individual fractions, pooled fractions 8–10 (delineated with a broken line) and producer cell lysate. (D) Negative staining electron microscopy (EM) of HIV fractions 8–10 purified in a Iodixanol step gradient with or without subtilisin treatment. Staining was performed with 0.5 phosphortungstic acid pH 7.2. (scale bar = 500 nm) (E) Western blot analysis of purified virus: Untreated virus is shown in lane 1, subtilisin treated virus in lane 2, subtilisin treated virus produced in presence of ritonavir (RTV) (added 6 hours post transfection) in lane 3 and subtilisin treated virus with RTV added during virus production and purification in lane 4. Immunoblots for IN and p24 are included as control, and indicate equal loading of the samples. Full length LEDGF/p75 is indicated by an arrowhead. Lanes 1–2 were run on a 12.5% gel, lanes 3–4 on a 4-12% gel with appropriate MW markers.