Transcytosis of HTLV-1 virions across the epithelial cell monolayer. (A) Experimental setting to study the viral passage after coculture. Caco-2 cells were cultured on Transwell filters before the addition of HTLV-1–infected or uninfected lymphocytes (48-hour cultures) to the apical compartment. (B) Temperature dependence of the viral p19 transport across the monolayer. Cocultures of differentiated Caco-2 cells with HTLV-1–infected lymphocytes were incubated at either 4°C or 37°C. Determination of the amount of p19 protein in the basal compartment of the Transwell devices after 2 hours of contact with lymphocytes was by ELISA assay. Left column, coculture of Caco-2 cells/uninfected lymphocytes; middle column, coculture of Caco-2 cells/infected lymphocytes pretreated with EDTA (positive control); right column, coculture of Caco-2 cells/infected lymphocytes. Results are from 3 different experiments performed at 37°C (left) or 4°C (right). *P < .05. (C-E) Immunogold staining for HTLV-1 p24 protein in the cytoplasm of Caco-2 cells cocultured with infected lymphocytes by transmission electron microscopy. After 2 hours of contact between Caco-2 cells and HTLV-1–infected lymphocytes, cell monolayers on Transwell filters were fixed by ultrarapid cooling, sectioned, and immunogold labeled for the p24 capsid protein. (C-D) Immunoreactivity of HTLV-1 p24 Gag protein in specific clusters of gold particles (black arrow in C) in the cytoplasm of Caco-2 cells cocultured with infected lymphocytes. (E) No such clusters of gold particles were detected in the cytoplasm of Caco-2 cells cocultured with uninfected lymphocytes. Scale bar indicates 500 nm in panels C and E and 50 nm in panel D. Observation was with a JEOL 1010 transmission electron microscope operating at 80 kV. Image acquisition was with a Nikon CCD camera.