FIG 5.

Kinetics of Axl-mediated viral endosomal escape. (A) Detection of functional DG and Axl in HEK293H and HT-1080 cells by Western blotting as described for Fig. 1A, including the LARGE-deficient cell line HeLa. Please note the residual signal for functionally glycosylated α-DG in HeLa cells, which was absent in HT-1080. (B) Verification of DG- and Axl-mediated entry of rLCMV-LASVGP into HT-1080 and HEK293H cells, respectively. The indicated cells were blocked with MAb IIH6 to glycosylated α-DG (100 μg/ml), polyclonal Ab goat anti-Axl (20 μg/ml), and control antibodies (Ctrl) for 2 h in the cold as described for Fig. 3E. Cells were then incubated with rLCMV-LASVGP at 100 PFU/well (HEK293H) and 300 PFU/well (HT-1080) for 2 h in the cold in the presence of antibodies. Cells were washed and kept in complete medium for 1 h at 37°C; then 20 mM ammonium chloride was added, the cells were cultured for 16 h and fixed, and infection was detected as described for Fig. 3B. Data are means ± SD (n = 3). (C) Endosomal escape of virus. rLCMV-LASVGP at 100 PFU/well (HEK293H) and 300 PFU/well (HT-1080) was attached to monolayers of the indicated cells in the cold for 2 h in complete medium. Unbound virus was removed, and cells were rapidly shifted to 37°C. At the indicated time points, 20 mM ammonium chloride was added and left throughout the experiment. After 16 h, infection was assessed by IFA as described for Fig. 3B. Given are means ± SD (n = 3). (D) Blocking of viral infection of HT-1080 and HEK293H cells with heparin. rLCMV-LASVGP (LASV) and rVSVΔG-VSVG (VSV) were diluted in complete medium and pretreated with the indicated concentrations of heparin for 1 h in the cold, followed by infection of cell monolayers for 1 h at 37°C in the presence of the inhibitor. After 1 h, complete medium containing 20 mM ammonium chloride was added, followed by 16 h of incubation and detection of infection by IFA as described for panel C. Data are means ± SD (n = 3).