FIG. 5.

Expression of 89.6G glycosylation mutants from VSV recombinants. Approximately 5 × 10⁵ BHK cells on 35-mm-diameter plates were infected with wt or recombinant VSVs at an MOI of 10 for 1 h in 0.5 ml of DMEM. The medium was then replaced with 2 ml of DMEM supplemented with 5% FBS, and cells were incubated for an additional 7 h at 37°C. Cells were washed twice with methionine-free medium and labeled in 0.5 ml of methionine-free medium containing 100 μCi of [³⁵S]methionine. After 1 h, cells were washed with PBS and lysed with 0.5 ml of detergent solution. Lysates were transferred to 1.5-ml Eppendorf tubes and centrifuged at 10,000 rpm in an Eppendorf microcentrifuge for 2 min, and supernatants were transferred to new tubes. About 10 μl of each sample was analyzed by SDS-8% PAGE, followed by exposure to a PhosphorImager screen (Molecular Dynamics). The positions of two VSV proteins (L and G) as well as HIV Env G are indicated. MW, molecular weight (in thousands).