FIG. 6.

Intracellular trafficking of Cy3-labeled Ad vectors in A549 cells. The kinetics of Cy3-labeled Ad vectors are shown in red, and colocalization with cathepsin B-containing late endosomal or lysosomal cellular compartments is shown in green. Cells were pulse-infected with Cy3-Ads for 15 min at 37°C and allowed to further incubate for the indicated periods. Then cells were fixed, permeabilized, and stained with anti-cathepsin B-specific primary antibody. The staining of primary antibody was detected with Alexa-488-conjugated secondary antibody (green), and images of cells were taken on a Leica confocal microscope. To determine colocalization of virus with cathepsin B-positive intracellular compartments, images of the same areas were acquired in red and green channels consecutively. CAR-interacting Ad5L and Ad5ΔRGD vectors do not colocalize with cathepsin B-positive compartments until 2 h postvirus infection, while CD46-interacting Ad5/35L and Ad5/35ΔRGD vectors colocalize with these cellular compartments as early as 30 min postvirus infection. Note that vectors with the penton RGD motif deletion are distributed more widely throughout the cytoplasm than RGD motif-possessing variants at earlier times postinfection. Representative fields are shown. Magnification, ×200.