No Nef detection in crude exosomes and no Nef transfer through exosomes from Nef-expressing Jurkat.

<p><b>A.</b> Crude exosomes were isolated from GFP- and Nef.GFP-expressing Jurkat, as well as its parental Jurkat by sequential centrifugation. Whole cell lysates (WCE) were also prepared from the cells. Both crude exosomes and WCE were analyzed by Western blotting, followed by direct visualization of the blots at a wavelength of 488 nm or antibody staining and ECL. Samples were subjected to incubation at 37°C for 30 min before SDS-PAGE to facilitate membranous protein detection and GFP visualization. * Non-specific bands. <b>B.</b> Fresh Jurkat (1 x 10⁵) were incubated with each of crude exosomes prepared above for 24 hr. The cells were then washed with PBS for multiple times and analyzed for GFP by flow cytometry. Crude exosomes from CD81.GFP-expressing cells were included as a positive control. The data were representative of two independent experiments.</p

Nef and CD81 localization in intracellular and extracellular vesicles.

<p>293T (5 X 10⁴) were plated in a 24-well plate and transfected with GFP, Nef.GFP, or CD81.GFP plasmid. Twenty-four hours post transfection, the cells were re-plated on top of polylysine-treated coverslip in a 24-well plate. Cells were fixed after 24 hr and processed for immunostaining using an anti-CD81 antibody, followed by Alexa Fluor 555-conjugated goat anti-mouse secondary antibody, which allows detection of both endogenous and exogenous CD81 expression and localization using a rhodamine filter under confocal microscope (60X objective). GFP tagged protein expression and localization were detected using a FITC filter. <b>A.</b> CD81 Nef co-localization; <b>B.</b> Extracellular vesicle-like structures (with extended exposure time). The micrographs were representative of images from multiple fields of three independent experiments.</p

Nef detection in the AChE+ fractions from Nef-transfected 293T.

<p>293T (2 x 10⁶) were plated in a 10 cm plate and transfected with GFP (<b>A-C</b>), Nef.GFP (<b>A, D & E</b>), or CD81.GFP (<b>F-H</b>). Transfected cells were then cultured in exosome-free medium for 3 days. Culture medium was collected and pooled (about 70 ml total) for crude exosomes (500 μl) as described above, while cells were harvested for cell lysates for whole cell extracts (WCE). WCE and crude exosomes were analyzed by Western blotting using TSG101 and cytochrome C (cyto C) antibody (<b>A</b>). GFP and Nef.GFP and CD81.GFP were visualized at a wavelength of 488 nm (<b>A & F</b>). The data were representative of three independent experiments. Then the crude exosomes were subjected to the OptiPrep gradient centrifugation and fractionated. Aliquots of each fraction were used for AChE activity assay (24 μl, <b>B, D & G</b>). The remaining fractions were diluted in 4 ml PBS and spun at 100,000 <i>g</i>, 70 min. The pellets were lysed in the RIPA buffer followed by Western blotting using indicated antibodies (<b>C, E & H</b>). WCE (100 μg) were included as controls (<b>C, E & H</b>). The AChE activities were mean ± SD of triplicates; the data were representative of three independent experiments.</p

Detection of CD81.GFP in the AChE+ fractions derived the crude exosomes treated with RIPA buffer.

<p>293T (2 x 10⁶) were plated in a 10 cm plate and transfected with CD81.GFP. Transfected cells were cultured in exosome-free medium for 3 days. Culture medium was collected, pooled (about 30 ml) and used to isolate crude exosomes as described above. The crude exosome pellet was lysed in 100 μl RIPA buffer, subjected to 3 rounds of freezing and thawing on dry ice, and diluted in 400 μl PBS, followed by the same 6%-18% OptiPrep gradient centrifugation. Aliquots of each fraction were used for AChE activity assay (24 μl, <b>A</b>). The remaining fractions (426 μl) were diluted in 4 ml PBS and spun by 100,000 <i>g</i>, 70 min. The pellets were lysed in the RIPA buffer, followed by Western blotting using an anti-TSG101, or CD81.GFP antibody. The AChE activities were mean ± SD of triplicates; the data were representative of three independent experiments.</p

No Nef uptake into 293T by microscopic imaging.

<p>293T (2 x 10⁶) were plated at a 10 cm plate, transfected with GFP, CD81.GFP, or Nef.GFP plasmid, and cultured for 16 hr, followed by direct microscopic imaging with a FITC filter or under the bright field (a 10X objective) (<b>A</b>). Transfected cells were then cultured in exosome-free medium for 3 days. Crude exosomes from the culture medium (40 ml) were prepared as described above, suspended in exosome-free medium, added onto fresh 293T in a polylysine-treated glass bottom dish, and incubated for 3 hr (<b>B</b>) and 12 hr (<b>C</b>). At the end of each incubation, images of the target cells were taken with a FITC filter or under the bright field (a 100X objective). The micrographs were representative of images from multiple fields of two independent experiments.</p

HIV-1 Nef transfer from HIV-infected cells to bystander cells.

<p>Jurkat were infected with HIV-1 NL4-3 (Wt) or <i>nef</i>-deleted NL4-3 (ΔNef) (<b>A & B</b>), HXB2 (Wt) or Nef mutated HXB2 (G2A) (<b>E&F</b>). When the infection reached 50%, determined by p24 staining, the cells were double stained using a rabbit anti-HIV-1 Nef antibody with Alexa anti-rabbit 488 and PE-conjugated mouse anti-p24 antibody. The cells were analyzed by flow cytometry for Nef staining fluorescence intensity in p24- cells (<b>A & E</b>), and the percentage of Nef+p24- cells (<b>B & F</b>). MT4 were infected with NLGi (Wt) or <i>nef</i>-deleted NLGi (ΔNef) (<b>C & D</b>). Co-culture of infected MT4 with Jurkat at 1:1 ratio was performed when MT4 infection reached 80% determined by GFP expression. The cells were collected 48 h post co-culture and stained using a mouse anti-Nef antibody (1539) with Alexa anti-mouse 647. The cells were analyzed by flow cytometry for Nef staining fluorescence intensity in GFP- cells (<b>C</b>), and the percentage of Nef+GFP- cells (<b>D</b>). The data were mean ± SD of triplicates and representative of three independent experiments. *, <i>p</i> < 0.05 **, <i>p</i> < 0.01. <b>G.</b> Jurkat were infected with HIV-1 NL4-3 Wt or ΔNef. When the infection efficiency reached more than 90%, the cells were co-cultured with Jurkat at a ratio of 1:1 but at a cell density of 1 x 10⁶, 2 x 10⁶, or 4 x 10⁶ cells /ml for 16 hr. At the end of the co-culture, the cells were double stained using rabbit anti-HIV-1 Nef antibody with Alexa anti-rabbit 488 and PE-conjugated mouse anti-p24 antibody. The cells were analyzed by flow cytometry for Nef+p24- cells. The data were mean ± SD of triplicates and representative of three independent experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01.</p

No Nef uptake into Jurkat by flow cytometry.

<p>293T cells were transfected with cDNA3 (Mock), GFP, Nef.GFP, or CD81-GFP plasmid and cultured in exosome-free medium for 3 days. Cell culture media were collected and removed of cell debris (Supernatants), or used to prepare crude exosomes (Exosomes) as described above. Jurkat (1 x 10⁵) were incubated with 100 μl crude exosomes (<b>A</b>) or 100 μl supernatants (<b>B</b>) for 3, 12, 24, or 48 hr. The cells were then washed with PBS multiple times and analyzed by flow cytometry. The data were representative of four independent experiments.</p

Intercellular HIV-1 Nef transfer.

<p>Nef.GFP-expressing Jurkat (0.5 x 10⁶) were co-cultured with 0.5 x 10⁶ of SP-DilC-labeled Jurkat (<b>A,</b> top panels), SP-DilC-labeled THP-1 (<b>B</b>), or HPA (<b>C</b>) in a volume of 200 μl medium in a 24-well plate (i.e., at a cell density of 0.5 x 10⁶/ml) for 16 hr. GFP-expressing Jurkat were used as a control (<b>A,</b> bottom panels). HPA were identified by GFAP staining, and DAPI staining was also performed to discern HPA from Nef.GFP-expressing Jurkat by the size of the nuclei (<b>C</b>). Nef transfer from Nef.GFP-expressing Jurkat to Jurkat (<b>A</b>), THP-1 (<b>B</b>) and HPA (<b>C</b>) was shown by arrows.</p