## 3 Results
### LASV Colocalizes with Glycosylated DG within the Plasma Membrane
Next, we assessed the effect of EIPA, R428, and EMD on rLCMV/LASV\({}_{\text{GP}}\) cellular internalization. To that aim, A549 cells were incubated in the cold with the virus at high MOI (50 PFU/cell), and the temperature was subsequently shifted to 37 \({}^{\circ}\)C, for 40 min, to allow virus internalization in the presence of the aforementioned drugs. Then, viral NP was stained with a specific antibody. The RTK inhibitors, R428 and EMD, as well as EIPA, all prevented virus internalization, resulting in accumulation of viral particles at proximity of the cellular membrane (Figure 6B). Despite the different pattern in dextran-containing vesicles observed among R428, EMD, and EIPA, the three drugs lead to comparable patterns, with accumulation of virion-containing vesicles at the membrane proximity (Figure 6A,B). In summary, these results reveal that Axl and HGFR RTKs participate in virus internalization through macropinocytosis, and their inhibition consequently leads to inhibition of trafficking of virus-containing vesicles through the cytoplasm. \begin{table}
\begin{tabular}{c c c c} \hline \hline  & \multicolumn{3}{c}{**A549 Cells**} \\ \hline
**R428 Conc. (\(\mu\)M)** & **EMD Conc. (\(\mu\)M)** & **Effect** & **CI Value** \\ \hline
0.125 & 10.0 & 0.49 & 1.20910 \\ \hline
0.25 & 10.0 & 0.52 & 1.24309 \\ \hline
0.5 & 10.0 & 0.55 & 1.33485 \\ \hline
1.0 & 10.0 & 0.54 & 1.78463 \\ \hline
2.0 & 10.0 & 0.64 & 1.59548 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Drug interaction (R428/EMD) analysis in A549 cells. Analysis performed with CompuSyn [95]. CI values <1 means synergistic interaction, approximately 1 additive effect, and >1 antagonistic interaction. \begin{table}
\begin{tabular}{c c c c} \hline \hline  & \multicolumn{3}{c}{**HT-1080 Cells**} \\ \hline
**R428 Conc. (\(\mu\)M)** & **EMD Conc. (\(\mu\)M)** & **Effect** & **CI Value** \\ \hline
0.125 & 10.0 & 0.95 & 1.20408 \\ \hline
0.25 & 10.0 & 0.97 & 1.12084 \\ \hline
0.5 & 10.0 & 0.97 & 1.28517 \\ \hline
1.0 & 10.0 & 0.99 & 1.04369 \\ \hline
2.0 & 10.0 & 0.999 & 0.554 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Drug interaction (R428/EMD) analysis in HT-1080 cells. Analysis performed with CompuSyn [95]. CI values <1 means synergistic interaction, approximately 1 additive effect, and >1 antagonistic interactionAxl and HGFR participate in classical macropinocytosis. Figure 6: R428 and EMD inhibit macropinocytosis-related rLCMV/LASV\({}_{\text{GP}}\) internalization. (**A**) A549 cells were pretreated with DMSO, 40 uM EIPA, 5 uM R428, or 40 uM EMD, for 30 min. Subsequently, cells were incubated with Alexa Fluor 488 labeled dextran (250 uS/mL) in the presence of the inhibitors, for 40 min, at 37 \({}^{\circ}\)C, washed, fixed with PFA, and subjected to confocal microscopy. Note: EIPA treatment resulted in a faint blue fluorescence _throughout_ the cytosol. Representative fields for each condition are shown. Scale bar: 10 um; (**B**) Precooled A549 cells were incubated for 2 h, at 4 \({}^{\circ}\)C, with rLCMV/LASV\({}_{\text{GP}}\) (MOI 50 PFU/cell). Cells were washed and either fixed or shifted to 37 \({}^{\circ}\)C, for 45 min, in the presence of DMSO or inhibitors (same concentrations as in (**A**)), as indicated. Then, cells were fixed, stained for viral NP (113 antibody), and analyzed by confocal microscopy. Representative fields are shown for each condition (\(n=10\)). Scale bar corresponds to 10 μm. Interestingly, whereas Axl RTK inhibition by R428 decreased the number of dextran-labeled vesicles, HGFR inhibition by EMD resulted in an accumulation of vesicles in proximity to the plasma membrane (Figure 6A). In agreement with previous reports [27; 37], we also found in viral infection experiments that 18.6% (\(\pm\)7.7%) of total Alexa Fluor 488-conjugated, dextran-containing vesicles also contained rLCMV/LASV\({}_{\text{GP}}\) viral particles (Figure S5).