## 3 Results
### LASV Colocalizes with Glycosylated DG within the Plasma Membrane
In summary, R428 inhibitory effects on rLCMV-LASVGP productive infection are not due to an increased IFN-I response. HGFR is a RTK that is implicated in the entry of LASV and other viruses into human epithelial cells [27; 59]; however, its exact role during viral entry is currently unknown. To extend our previous studies, we determined the contribution of HGF to LASV cell entry. We compared rLCMV/LASVGP infections in A549 cells, which express Axl and HGFR RTKs in the context of functional DG, with infections in HT-1080 cells, which likewise express Axl and HGFR RTKs but lack functional glycosylated DG (Figure S1A,B and Figure 5A). As described for the entry assay (Figure 1C), we preincubated A549 and HT-1080 cells, for 30 min, at 37 \({}^{\circ}\)C, with increasing concentrations of EMD which is a potent HGFR kinase inhibitor with an IC\({}_{50}\) of 3 nM [65], which did not affect HGFR expression (Figure 5B). Then, cells were cooled down to 4 \({}^{\circ}\)C, infected with rLCMV-LASVGP in the presence of the drug, and shifted to 37 \({}^{\circ}\)C, for 1 h. The results showed that inhibition of HGFR by EMD reduced rLCMV/LASVGP entry in a dose-dependent manner in both A549 cells (IC\({}_{50}\) = 8.5 uM and CC\({}_{50}\) > 90 uM) and HT-1080 cells (IC\({}_{50}\) = 3.7 uM and CC\({}_{50}\) > 90 uM) (Figure 5C and Figure S4A), in the absence of overall cytotoxicity (Figure S2B). Figure 4: Axl RTK does not affect the interferon-I response. (**A**) A549 or (**B**) HT-1080 cells were pretreated with DMSO or 2 uM R428, 30 min before viral infection, and rLCMV/LASVGP (MOI 0.01 PFU/cell), LCMV Arm 53b (MOI 0.01 PFU/cell), or VSV (MOI 1 PFU/cell) were added in presence of DMSO or 2 uM R428. After infection, 20 mM ammonium chloride were added to the media to prevent multiple rounds of replication. Total RNA was extracted 16 h after infection, reverse transcribed, and qPCR performed to determine interferon \(\beta\) (INF \(\beta\)) expression levels. Error bars represent standard deviations (\(n\) = 6). Axl and hepatocyte growth factor receptor cooperate during LASV entry. Figure 5: Axl and hepatocyte growth factor receptor (HGFR) RTKs cooperate during LASV entry. (**A**) HGFR surface expression in A549 and HT-1080 cells detected by FACS analysis; (**B**) HGFR surface expression upon EMD1204063 (EMD) administration. A549 cells were incubated for 1.5 h with EMD (20 \(\upmu\)M), and then fixed and stained for HGFR detection with specific antibody; (**C**) Dose dependent entry inhibition of rLCMV/LASV\({}_{\text{GP}}\) by EMD in A549 and HT-1080 cells. Error bars represent standard  
deviations (\(n=3\)); (**D**) Phosphorylation and expression of HGFR after HGF administration. Cells were incubated with HGFR for indicated times, lysed, and tested by WB for HGFR and phosphorylated HGFR (p-HGFR). Tubulin was used as loading control. Normalized ratios were calculated from signal ratios of (HGFR/tubulin)(p-HGFR/tubulin); (**E**) Time-of-addition assay of HGF. HGF (50 nM) or vehicle (PBS) were added to A549 cells at the indicated times relative to infection with rLCMV-VSVG or rLCMV/LASVGP. Error bars represent standard deviations (\(n=3\)). Asterisk (*) denotes significant differences (student's \(t\)-test; \(p<0.01\)); (**F**) HGFR RTK/NP colocalization. Precooled (4 \({}^{\circ}\)C) A549 cells were infected, for 2 h, in the cold with rLCMV/LASVGP (MOI 50 PFU/cell), and then shifted to 37 \({}^{\circ}\)C for indicated times. HGFR RTK was visualized in red and viral NP in green. Representative confocal images are shown. Scale bar represents 5 \(\upmu\)m. Percentages of double puncta (NP/HGFR RTK) values obtained from the analysis of 8 randomly selected cells per condition. Error bars represent standard deviations; (**G**) DG/HGFR RTK colocalization in mock-infected and rLCMV/LASVGP-infected cells. Live A549 cells were preincubated for 2 h in the cold with mock or rLCMV/LASVGP (MOI 50 PFU/cell), and rapidly shifted to 37 \({}^{\circ}\)C during indicated times. DG is visualized in red and Axl RTK in green.