Figure 4. Exosomes containing CD4 reduce HIV-1 infection.

(A) Exosomes derived from A2.01, A3.01 GFP and A3.01 GFP/Nef cells were purified from 100 mL cell culture supernatants by ultracentrifugation. Proteins in exosome and cell lysates were analyzed by Western blot with antibodies to CD4, Alix and EEA1 (an early endosome protein absent in exosomes). An asterisk indicates a nonspecific band recognized by the anti-Alix antibody. (B) Aliquots of HIV-1 Luc⁺ particles (equivalent to 8 ng of p24) were incubated for 1 h with exosomes isolated from A2.01 or A3.01 GFP cell supernatants. The final concentrations of exosomes protein used in the assays are indicated. These HIV-1-exosome preparations were then used to infect MT4 cells. Cells infected with reporter viruses pre-incubated with exosome-free cell culture media were used as positive control of infection. The infectivity levels were evaluated by luciferase activity in MT4 cell lysates, and expressed as percentage of the levels in cells infected with HIV-1 Luc⁺ in the absence of exosomes (control), with the highest level of luciferase activity in control conditions set to 100%. The data represent the means ± standard deviations from three independent experiments. (C) Infection assays were repeated in MT4 cells using HIV-1 Luc⁺ particles pre-incubated with exosomes from A3.01 GFP and A3.01 Nef/GFP cells, as described above. Bar graph represents luciferase activity in MT4 cells infected with HIV-1 Luc⁺ in the presence of A3.01 GFP and A3.01 Nef/GFP exosomes, relative to levels in MT4 cells infected with HIV-1 Luc⁺ in the absence of exosomes (100%). The data represent the means ± standard deviations from three independent experiments. (D) Infection assays were performed in MT4 cells using a VSV-G pseudotyped MSCV that encodes GFP. Aliquots of VSV-G-MSCV(GFP) particles were incubated for 1 h with exosomes isolated from A3.01 GFP cell supernatants. The final concentrations of total exosome proteins used in the assays are indicated. The MSCV-exosome preparations were used to infect MT4 cells and cells infected with reporter viruses pre-incubated with exosome-free cell culture media were used as positive control of infection. The infectivity levels were evaluated by intensity of GFP fluorescence in cells by FACS analysis. Bar graph represent the levels of GFP fluorescence (median values from FACS histogram plots) from cells transduced with VSV-G-MSCV(GFP) pre-incubated with CD4⁺ exosomes, relative to cells transduced with VSV-G-MSCV(GFP) in the absence of exosomes (100%). P-values were calculated using the Student's t-test: * P<0.05; **, P<0.005, and ***, p<0.0005; NS, not significant.