FIG 8.

Differential signaling is required for steady-state DG turnover and DG-mediated viral entry. (A) Inhibition of rLCMV-LASVGP infection with selected inhibitors. A549 cells were pretreated with the Dyngo 4a, EIPA, jasplakinoline, IPA3, and EMD 1214063 at the indicated concentrations for 30 min, followed by infection with the rLCMV-LASVGP (LASV), rLCMV-VSVG (VSV), or AdV5 expressing GFP (AdV5) at 200 PFU/well in the presence of drug. After 1 h, cells were washed three times with medium containing 20 mM ammonium chloride, followed by 16 h of incubation in the presence of the lysosomotropic agent. Infection of rLCMV-LASVGP and rLCMV-VSVG was detected by IFA and AdV5-GFP by direct fluorescence as described in the legend to Fig. 1D. Data are means ± SD (n = 3). (B) Pulse-chase assay of cell surface DG in the presence of inhibitors. Intact monolayers of A549 cells were pretreated with the indicated drugs (Dyngo 4A, EIPA, EMD 1214063, and IPA3 [40 µM each] and jasplakinoline [2 µM]), chilled on ice, and subjected to cell surface biotinylation with NHS-X-biotin as described in the legend to Fig. 6A. Biotinylated proteins were precipitated from 90% of lysates with agarose beads, whereas 10% of lysates underwent total protein extraction. Beta-DG was detected in Western blots as described in the legend to Fig. 1B. One representative example of three independent experiments is shown.