HAdV-37 enters human corneal fibroblasts through caveolin-1 containing, membrane associated vesicles.

Human corneal fibroblasts were fixed in 2 % paraformaldehyde and the fixed, dehydrated cell pellet embedded and sectioned at 70-90 nm. Specimens were stained with uranyl acetate and Sato’s lead stain, and viewed by transmission electron microscopy. A. Photo-electron micrograph of cell membrane demonstrates multiple flask shaped vesicles resembling caveolae (1), and the formation of a cavesome-like structures (2). B-D. After 30 min of absorption at 4°C, and 30 min incubation at 37°C, virus can be seen within similar structures (3), and in some cases within what appear to be early or late fusions of caveolae to form caveosomes (4). Immunoelectron microscopy for caveolin-1 (E, F) was performed on cells infected as above, but treated with 2.3 M sucrose in PBS for 15 min, frozen, and sectioned at -120° C at 50-80 nm thickness. After immunostaining, protein-A nanogold was visualized by electron microscopy. (E) Uninfected cells stained for caveolin-1 demonstrate binding of 10 nm gold particles to flask-shaped vesicles. (F) After virus infection, virus (arrows) can be seen within vesicles also associated with gold particles. Scale bar = 500 nm.