## 3 Results
### Axl Is Required for LASV Entry into Human Cells
(**A**) Surface expression of glycosylated dystroglycan (DG) and Axl RTK in HUVEC cells. Live, nonpermeabilized cells were stained at 4 \({}^{\circ}\)C with specific antibodies to sugar chains of DG or Axl RTK and analyzed by fluorescence-activated cell sorting (FACS); (**B**) Surface expression of Axl RTK in A549 cells treated with R428. Cells were incubated with 2 \(\mu\)M of R428 for 1.5 h, and then fixed and stained with the specific anti-Axl RTK antibody; (**C**) Schematic experimental setting of the entry assay; (**D**) Dose-response curves in entry assays for R428 inhibition of rLCMV/LASV\({}_{\text{GP}}\) infection in A549, HT-1080, and HUVEC cells. Error bars represent standard deviations (\(n=3\)); (**E**) FACS analysis of Axl RTK surface expression in parental and 293T with Axl-constitutive expression; (**F**) Relative entry of vesicular stomatitis virus (VSV)-based pseudotype decorated with EBOV\({}_{\text{GP}}\) (VSV\(\Delta\)/EBOOV\({}_{\text{GP}}\)), rLCMV/LASV\({}_{\text{GP}}\), and of AdV5 in Axl-transfected 293T cells. Error bars represent standard deviations (\(n=3\)). Asterisk (*) denotes \(p\)-value \(<0.01\) (Student’s \(t\)-test); (**G**) Depletion of Axl RTK from A549 cells. Two days post siRNA transfection, expression of Axl RTK was detected by Western blot, using \(\alpha\)-tubulin (Tub) as the loading control. Efficiency of Axl RTK depletion was assessed by densitometry using ImageJ J. Numbers beneath the blot indicate the percentages of the signal ratio of Axl/\(\alpha\)-tubulin; (**H**) Infection of Axl-siRNA-depleted A549 cells. Cells transfected with siRNA-Axl or siRNA-sc (scramble, negative control) were infected with AdV5-EGFP, VSV\(\Delta\)G-EBOV\({}_{\text{GP}}\), or rLCMV/LASV\({}_{\text{GP}}\). Error bars represent standard deviations (\(n=3\)). Asterisk (*) denotes \(p\)-value \(<0.01\) (Student’s \(t\)-test). To further confirm a role for Axl RTK in LASV infection, we performed infections in 293T cells, which have undetectable endogenous Axl RTK levels, and in 293T cells that constitutively overexpress Axl RTK (293T/Axl) (Figure 1E). To control the functional overexpression of Axl, we used Ebola virus (EBOV), whose infection is known to be promoted by Axl RTK [48; 49; 50]. Given the high biosecurity required for handling replication-competent EBOV, we used a vesicular stomatitis virus (VSV)-based pseudotype platform, in which the original VSVG had been exchanged with an EGFP reporter to allow monitoring of viral infection, and the heterologous EBOVGP was provided from an expression vector. This VSV-based pseudotype platform has been shown in previous studies to be a reliable surrogate for entry of several viruses, including filoviruses [89]. Axl RTK overexpression in 293T cells resulted in a two-fold increase (\(p<0.01\)) of rLCMV/LASVGP infection and a six-fold increase in VSVAG-EBOVGP transduction (Figure 1F and Figure S1F). In contrast, adenovirus 5 (AdV5) infection remained unaffected by Axl RTK expression in 293T cells. Furthermore, depletion of Axl RTK in A549 cells resulted in an approximately two-fold reduction of rLCMV/LASVGP (\(p<0.01\)) and VSVAG-EBOVGP relative infection and transduction, respectively, while AdV5 infection remained unaffected (Figure 1G,H and Figure S1G). In summary, our data provide evidence that LASV entry partially depends on Axl RTK expression.