Assessment of viral passage across a tight human epithelial barrier. (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 to the apical compartment. After 48 hours of coculture, basal medium was harvested for p19 measurement by ELISA (B), p24 measurement by Western blot (C), or ultracentrifuged. After resuspension, the pellet was analyzed for the presence of infectious virions by incubation with 293-LTR-GFP cells by GFP emission assay (D-F) or p24 immunoreactivity (G) of these cells. (B) The p19 viral protein content in the basal compartment was determined by an ELISA assay after a 48-hour coculture of Caco-2 cells with HTLV-1–infected lymphocytes on Transwell filters (middle column). A significant amount of p19 protein was detected compared with epithelial cells cocultured with uninfected lymphocytes (left column). *P < .05. Right column shows the positive controls, which were treated with EDTA. Mean values from 3 experiments are shown. (C) Western blot detecting p24 protein in the basal compartment after ultracentrifugation (lane a, coculture with uninfected lymphocytes; lane b, coculture with epithelial cells/HTLV-1–infected lymphocytes; lane c, coculture with HTLV-1–infected lymphocytes treated with EDTA). The whole-cell lysate of infected lymphocytes is shown in lane d. (D-G) Presence of infectious virions in the basolateral chamber. Medium of the basal compartment of a coculture (Caco-2/HTLV-1–infected lymphocytes for 48 hours) was ultracentrifuged and incubated for 5 days on 293T-LTR-GFP cells after assessment of GFP gene expression to detect transactivation by the viral Tax protein. (D) Transactivation of the viral promoter LTR by infectious virions contained in the basal compartment after coculture with HTLV-1–infected lymphocytes. € EDTA-treated positive control. (F) Negative control with the medium derived from a coculture with CEM-uninfected lymphocytes. (G) Detection of p24 immunoreactivity and syncytium formation in 293-LTR-GFP cells incubated with ultracentrifuged pellet from the basal compartment of Caco-2 coculture with HTLV-1–infected lymphocytes, as in panel D. For panels D-F, original magnification was 200×; for panel G, 400×. Observation was with a Nikon Microphot FxA (D-F) or a Leica DMRB (E) microscope equipped for fluorescence.