Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein

Immunofluorescent Localization and Electron Microscopy: COS 7 cells which had been grown to semi-confluency on glass coverslips (Coming Glass Works, Coming, NY), in 35-ram dishes, were trans-feLLed in Tris-buffered saline as described above, washed with chloroquine for 3 h, and incubated with DME containing serum. At 47 h after removal of DNA/DEAE-dextran, cells were washed in PBS and fixed for 45 min at 22"C in 2% formaldehyde, freshly prepared from paraformaldehyde, and then buffered in 0.05 M phosphate, pH 7.5. The coverslips were rinsed in PBS for 20 rain, then soaked in I% Triton X-100 in PBS for 20 rain to permeabilize the cells. After two 10-rain rinses in PBS, the coverslips were incubated for 1 h at 37"C in a solution containing polyclonal rabbit anti-VP7 diluted 1:400 in PBS and rbodamine conjugated to wheat germ agglutinin (R-WGA) (Vector Laboratories, Inc., Burlingame, CA) diluted 1:300 or 1:400. The cells were then rinsed exhaustively in PBS and incubated in a 1:300 dilution of secondary goat anti-rabbit IgG conjugated to fluoreseein (Cappel Laboratories, Cochranville, PA) for 45 min at 37°(L Cells were photographed in the same plane of focus, with a Zeiss Ili RS photomicroscope, using appropriate filters for fluorescein or rhodamine.