Penetration of HIV-1 in early and late endosomes, and vacuoles.

(A) HIV-1SF33 was added to the AP surface of polarized tonsil epithelial cells, and one set of cells was fixed after 30 min. (B) Another set of cells were cultured for 6 days after removing uninternalized virions. Cells were coimmunostained for HIV-1 p24 (green) and EEA1, LBPA, LAMP1 and rabankyrin-5 (all red), which are markers for early endosomes, MVBs, lysosomes, and vacuoles, respectively. (A, bottom panels). Cells were stained with mouse and rabbit isotype control antibodies. Cells were analyzed by Nikon Eclipse E400 fluorescence microscope. Yellow indicates colocalization of HIV-1 p24 with endosome markers. Nuclei are counterstained with DAPI (blue). Similar data were obtained in three independent experiments. (C) HIV-1SF33 (200 ng/ml p24) was added to the AP surface of polarized tonsil cells grown in Transwell inserts with a 24-mm diameter. One set of cells were cultured for 30 min, and another set of cells were cultured for 6 days. Cells were dissociated with trypsin, and homogenized and vesicular fractions were separated in sucrose gradients. Each fraction was examined for EEA1, LAMP1, rabankyrin, LBPA, and HIV-1 p24 by Western blotting. Similar data were reproduced in two independent experiments. Immunoblots were performed at least twice and representative results are shown.