## 3 Results
### LASV Colocalizes with Glycosylated DG within the Plasma Membrane
Representative images collected by confocal microscopy are shown. Scale bar represents 5 \(\upmu\)m. Colocalization of DG and HGFR RTK was quantified by Pearson's coefficient in mock and infected cells after shifting to 37 \({}^{\circ}\)C, for 0, 2, 5, and 10 min. Error bars represent standard deviations of 10 randomly selected cells per condition. Scale bars represent 5 \(\upmu\)m; (**H**) Dose response inhibition of rLCMV/LASVGP relative entry into A549 and HT-1080 cells by R428, alone or in combination with EMD (10 \(\upmu\)M). Relative entry values were normalized to the infectivity in the absence of drug (DMSO). Error bars represent standard deviations (\(n=3\)). To analyze the role of HGFR from a different angle, we first stimulated HGFR in A549 cells with exogenous HGF (50 nM), which resulted in a quick and timely precise receptor activation (Figure 5D). Moreover, upon activation, HGFR underwent degradation, becoming undetectable 90 min after HGF addition. Then, we performed time-of-addition experiments, adding 50 nM HGF before, during, and after rLCMV/LASVGP infection. The addition of HGF before or after viral infection did not affected rLCMV/LASVGP relative infectivity, whereas its presence during viral entry increased rLCMV/LASVGP entry in approximately 40% (Figure 5E and Figure S4C). In contrast, infectivity of rLCMV-VSVG, which enters cells independently of HGFR [27], was not affected by the presence of HGF at any of the time points analyzed (Figure 5E and Figure S4C). Next, we sought to investigate if HGFR directly interacted with viral particles during infection. To that aim, precooled (4 \({}^{\circ}\)C) A549 cells were infected with rLCMV/LASVGP, for 2 h, at 4 \({}^{\circ}\)C, and shifted to 37 \({}^{\circ}\)C for indicated times. Then, cells were fixed at 0, 2, 5, or 10 min after infection, and stained with specific antibodies for HGFR and viral NP detection. Similar to previous visualization of Axl RTK and NP, HGFR did not increase colocalization with NP over time, after infection (Figure 5F). Moreover, we followed up HGFR/DG clustering upon viral infection in A549 cells and found that HGFR and DG did not increase their colocalization due to rLCMV/LASVGP infection (Figure 5G). To further assess the relative contributions of Axl and HGFR RTKs during LASV entry, in the absence and presence of functional DG, and a potential interaction between both receptors, we performed infections with rLCMV/LASVGP in the presence of EMD (10 \(\upmu\)M) and increasing concentrations of R428. The results showed that regardless of the presence or absence of functional DG EMD significantly enhanced the antiviral effect of R428 (Figure 5H). The antiviral effect of either R428 alone or in combination with 10 \(\upmu\)M of EMD was more pronounced in HT1080 than in A549 cells. To determine the nature of the interaction, antagonistic, additive or synergistic, between both RTK inhibitors, we analyzed out results with CompuSyn software [95]. The analysis revealed that in both cell lines, A549 and HT-1080 cells, R428, and EMD had an additive effect one to the other (CI values approximately 1) (Tables 1 and 2). LASV enters cells through a macropinocytosis-related pathway [27; 37]. Thus, given the evidence of involvement of Axl and HGFR RTKs in LASV entry, next, we addressed the relative contribution of both RTKs to classical macropinocytosis. In most cells, macropinocytosis occurs at basal levels and can be tuned through different stimuli, including activation of specific RTKs by growth factors [96; 97; 98]. RTK signaling results in activation of Rho GTPases and PAK1, leading to actin rearrangement and macropinocytic cup formation [98]. To assess the role of Axl and HGFR RTKs in macropinocytosis, we treated A549 cells with R428 or EMD, respectively, and monitored macropinosome formation and trafficking with the fluid marker Alexa Fluor 488-conjugated dextran [99]. In agreement with its mechanism of action, EIPA significantly reduced the number of dextran-labeled vesicles [100] (Figure 6A).