FIG 7.

Engagement of rLCMV-LASVGP affects receptor internalization. (A) Schematic of the virus-induced receptor internalization assay. For details, please see text. (B) Virus engagement affects receptor internalization. Monolayers of A549 cells were subjected to cell surface biotinylation with the thiol-cleavable membrane-impermeable reagent sulfo-NHS-SS-biotin in the cold. Labeled cells were then incubated with biotin-SS-labeled rLCMV-LASVGP (50 particles/cell) or a mock virus preparation for 1 h in the cold as described in the legend to Fig. 5D, followed by removal of unbound virus. Cells were rapidly shifted to 37°C at the indicated time points, chilled on ice, and treated with TCEP (+TCEP) or reaction buffer only (−TCEP). Residual TCEP was carefully quenched, and cells were lysed. Cleared lysates were subjected to IP with MAb VIA4 matrix (α-DG) or MAb 83.6 to GP2 as indicated. Biotinylated α-DG and LASV GP2 were detected with streptavidin-HRP in Western blots under nonreducing conditions using ECL. The anti-GP2 blots in the presence and absence of TCEP were exposed for 2 min and 10 s, respectively. The anti-α-DG blot with TCEP was exposed for 5 min, and the anti-α-DG blot without TCEP was exposed for 30 s. One representative example of three independent experiments is shown.