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

Cell Growth, Transfection, Tunicamycin Treatment, and Radiolabeling: The RRI strain of Escherichia coli was used for the propagation of all plasmid DNA used for transfections. After standard bacterial lysis procedures (29) , DNA was isolated and purified by cesium chlorideethidium bromide ultracentrifugation followed by precipitation and resuspension in water. The procedure for transfection of COS 7 cells (18) follows that of published procedures (34) with several modifications. COS 7 cells were grown on 100-mm dishes in Dulbecco's modified Eagle's medium (DME) (Gibco Laboratories, Grand Island, NY), containing 5% each of calf and fetal calf serum, 100 U/ml penicillin, 100 ~g/ml streptomycin (Gibco Laboratories) and 2 mM L-glutamine. Monolayers that were 60-80% confluent were washed and transfected in Tris-buffered saline (19) . DNA (l 5-30 ~g/ml) was added to each plate followed by the addition of DEAE-dextran (Pharmacia Fine Chemicals, Piscataway, NJ; 2 × l06 daltons; 500 ug/ml) (19) . After 1.5-2 h at 37"C, the Tris-buffered saline solution was removed and DME, containing serum as above and 100 uM chloroquine (Sigma Chemical Co., St. Louis, MO), was added to the cells. After incubation for 3 h at 37"C, DME without chloroquine but containing serum and additions as above was added. At 45 h after DNA/ DEAE-dextran removal, the cells were incubated at 37"C for l h in DME salts lacking serum and methionine but supplemented with all other amino acids and l mg/ml glucose. Transfected cells were then labeled for 2.5 or 4 h at 37"C on a rocker platform in the above medium to which L-[35Slmethionine at a concentration of 150 uCi/ml was added. At the end of the labeling period, the medium was collected and non-adherent cells were pelleted by centrifugation in an Eppendorf centrifuge for l0 min. Supernatants were removed and analyzed for expressed secreted material.