Post-entry effect of semen exosomes on HIV infectivity. Intracellular HIV-1NL4.3 (A) p24 ELISA and (B) RT activity in TZM-bl cells 3 h post-challenge. Data ± SD are plotted as fold change of vehicle set at 1. (C) Cell free supernatant progeny of HIV-1NL4-3 produced from TZM-bl 24 h post-challenge depicting levels of p24 Gag as assessed by p24 ELISA. (D) Intravirion RT activity of HIV-1 progeny produced from TZM-bl 24 h post-infection. Data ± SD are expressed as fold change of vehicle set at 1 for all graphs. (E) Intravirion Gag RNA expression in cell-free supernatant progeny of HIV-1NL4-3 produced from TZM-bl reporter cells 24 h post-challenge with HIV-1 pre-incubated with vehicle or SE. Total progeny viral RNA was isolated from cell-free supernatant, converted to cDNA and examined for HIV-1 Gag expression by RT-qPCR. Results were normalized for GAPDH. Data ± SD are expressed as fold change of vehicle set at 1. (F) Secondary infectivity of HIV-1 viral progeny isolated from cell free supernatant of TZM-bl reporter cells 24 h post-challenge with HIV-1 pre incubated with vehicle or SE. Input was normalized for p24, added to fresh TZM-bl reporter cells and infectivity was evaluated by luminescence output 24 h post-challenge. Data ± SD are shown as % secondary infectivity with progeny generated in the presence of vehicle set at 100%. For panels A-F, ns = not significant, **P < 0.02, Student's t test was used for all samples. (G and H) Immunoblot using 30 μg of viral protein (progeny) or cellular protein from TZM-bl cells challenged for 24 h with HIV-1NL4-3 ±SE and probed with antibody against HIV-1 RT or p24 Gag to detect (G) intravirion or (H) intracellular HIV-1 RT and p24 Gag proteins.