FIG 4.

Axl-mediated LASV entry is serum dependent and requires the PS of the viral envelope. (A) Schema of the entry assay. For details, please see the text. LE, late endosome. (B) The entry of rLCMV-LASVGP into HT-1080 cells is serum dependent. Purified rLCMV-LASVGP (300 PFU/ml), rLCMV-VSVG (200 PFU/well), and rVSVΔG-TCRVGP (100 PFU/well) were pretreated with increasing concentrations of serum and added to HT-1080 cells cultured in 96-well plates for 2 h in the cold. Cells were washed 3 times and incubated with complete medium containing 10% (wt/vol) FBS at 37°C. After 1 h, complete medium containing 20 mM ammonium chloride was added, followed by 16 h of incubation in the presence of the lysosomotropic agent. Infection was detected by IFA as described for Fig. 3B. Data are means ± SD (n = 3). (C) Blocking of infection of HT-1080 cells with ANX-V. Purified rLCMV-LASVGP and rLCMV-VSVG were diluted in DMEM and pretreated with the indicated concentrations of ANX-V for 2 h in the cold. The virus–ANX-V mixture was then diluted 1:10 in complete medium containing 10% (wt/vol) FBS, resulting in a virus concentration of 300 PFU/well, and then added to HT-1080 cells for 1 h at 37°C. 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 Fig. 3B. Data are means ± SD (n = 3).