FIG 2.
Intestinal mucosal mast cell-mediated HIV-1 capture. (A) Detection of HIV-1 VLP binding on mast cells by flow cytometry. VLPs containing Gag-GFP were pulsed with mast cells at 4°C, and VLPs/ΔEnv were used as the control to monitor nonspecific binding. Trypsin treatment was used to remove surface-bound viruses. (B) HIV-1 VLP association with cells was observed by confocal microscopy. (C) Binding of replication-competent HIV-1 AD8 on mast cells as visualized by TEM. Arrows indicate viruses. (D) Binding of gp120 on mast cells. Purified mast cells were cultured with recombinant gp120 glycoproteins for 1 h at 4°C and then fixed for immunostaining and detected by flow cytometry. (E) Expression of HIV-1 attachment factors as detected by immunostaining with specific antibodies and flow cytometry. (F) Colocalization of HIV VLPs with DC-SIGN, HSPG, or α4β7 integrin. Purified mast cells were incubated with HIV-Gag-GFP/JRFL VLPs (40 ng p24gag) for 1 h at 4°C and then seeded onto poly-l-lysine-coated microscope slides. Cells were fixed and immunostained with specific antibodies against human DC-SIGN, HSPG, α4, or β7, followed by secondary Alexa 546-labeled goat anti-mouse IgG antibodies. Nuclei were stained with DAPI, and cells were observed by confocal microscopy.