## 4 Discussion
Nevertheless, although we observed rLCMV-LASV\({}_{\text{GP}}\) colocalization with preformed DG clusters at the plasma membrane, virions did not seem to increase colocalization over time with Axl RTK. This is in agreement with previous observations of LASV infectivity reduction due to effective antibody perturbation targeting DG but not Axl RTK [80] and suggests, at best, transient, weak interactions between LASV\({}_{\text{GP}}\) and Axl RTK. Nevertheless, it could be conceivable that LASV induces DG clustering, followed by the recruitment of additional entry factors or co-receptors, for example, Axl RTK, to the initial site of attachment. Therefore, we assessed if LASV membrane attachment and subsequent internalization would increase DG-Axl colocalization, which was not the case. Nevertheless, it could be that the basal activity of Axl RTK is required for LASV virion trafficking through the cytoplasm, and thus its inhibition abolishes virion-containing vesicles transportation. This would be in agreement with the lack of colocalization of viral particles or DG with Axl RTK, as well as with all results that demonstrate Axl RTK participation in LASV entry. Moreover, as R428 inhibited R428 catalytic activity [83], it is suggested that this activity is specifically required for LASV entry. Previous and present findings have indicated that LASV entered target cells by a macropinocytosis-related pathway [27; 80] and that the inhibition of HGFR, a RTK involved in macropinocytosis [64], resulted in LASV entry reduction regardless the extent of DG glycosylation [27]. The reduced macropinosome trafficking induced by inhibition of Axl or HGFR RTKs suggested that R428 and EMD exerted their antiviral effects by interfering with cytoplasmic transport of virus-containing vesicles, after initial cellular attachment and internalization of LASV. The observed basal macropinosome and LASV virion-containing vesicle traffic observed in untreated cells, and its inhibition by R428 and EMD, further supported the hypothesis that Axl and HGFR RTKs basal signaling was required for LASV transportation. Furthermore, we found that both RTK inhibitors, EMD and R428, displayed modest additive antiviral effects, resulting in strong inhibition of rLCMV/LASV\({}_{\text{GP}}\) entry. LASV infection is associated with liver function impairment [8; 9]; therefore, although EMD is already in clinical trials for cancer therapy [84; 86], administration of EMD for LHF treatment should be finely controlled to minimize potential side effects on liver physiology. Nevertheless, the possible combinations with other antivirals could allow one to reduce EMD doses to those that are able to reduce LASV infection while minimizing the undesired effects at the hepatic function. Indeed, cocktail treatments with both RTKinhibitors and Rib revealed synergistic interactions, allowing reduction of drug doses and suggesting potential for further development of antiviral therapies. A previous study [35] found that Tim-1 and Axl RTK did not contribute to LASV pseudovirus transduction of 293T cells expressing highly glycosylated DG, which represented an apparent contradiction with our studies. The Axl RTK and Tim-1 which both belong to the TAM and TIM receptor families, respectively, thus, bind PS and can support viral entry by binding to the viral phospholipid membrane, depending on the relative GP/PS abundance on the virion surface and the virion architecture. Pseudoviruses are powerful tools in virology in which a virus lacking its envelope glycoprotein is used as a platform to generate pseudoviruses decorated with heterologous glycoproteins. Therefore, many factors affect pseudoviral production, including the expression level of ectopic GP, or the phospholipid composition of viral membranes, which can affect virus behavior, phenotype, and receptor usage.