## 3 Results
### Axl Participates in Early Steps of LASV Entry  
To further validate the participation of Axl RTK during the early steps of rLCMV/LASVGP infection, we administered R428 before, during, and after viral infection (time-of-addition assay, Figure 2A). To ensure viral infection synchronization, precooled (4\({}^{\circ}\)C) cells were infected with rLCMV-LASVGP and shifted to 37\({}^{\circ}\)C, for 1 h, followed by washing with a medium containing 20 mM NH4 Cl, and incubation for 16-20 h, at 37\({}^{\circ}\)C. Indicated inhibitors were added at different times relative to the moment of the infection (\(-0.5\), \(0\), \(+1\), \(+2\), or \(+4\) hpi), in order to discriminate between effects on the viral entry or viral transcription/replication, and kept throughout the experiment, until fixation and quantification by IFA. The NHE inhibitor 5-(\(N\)-ethyl-\(N\)-isopropyl)-amiloride (EIPA) was used as a control for LASV entry inhibition, and the replication inhibitor Rib was included as an inhibitor of post-entry steps of infection [26; 90]. Irrespective of the extent of DG glycosylation, R428 inhibited rLCMV/LASVGP infection in both cell lines when added before or during entry, similar to EIPA, whereas only mild antiviral effects were observed at later post-entry steps of infection (Figure 2B,C and Figure S2A,B and [37]).As expected from its mechanism of action, Rib exerted a potent antiviral effect in this experimental setup, but it did not reduce rLCMV/LASVGP entry when administered during entry steps (Figure S2C). Since LASV entry is a complex, multiprocess, we sought to discern possible roles of Axl RTK in virus attachment and endocytosis from later steps of viral entry. To this end, we employed an established virus uptake assay [55; 77; 91] to track virus attachment and internalization of virion particles. In this assay format, purified rLCMV/LASVGP was labeled with the thiol-cleavable reagent NHS-SS-biotin, resulting in a biotin label that was sensitive to reducing agents without affecting infectivity (Figure 2D). The thiol-sensitive biotin label could be cleaved with the membrane-impermeable reducing agent Tris(2-carboxyethyl)phosphine (TCEP). This resulted in specific cleavage of the biotin label from the virus exposed to the extracellular space, whereas the biotin label on internalized virus particles becomes resistant, and therefore provided a sensitive tool to measure viral internalization, which was able to distinguish viruses attached to \"open cups\" and in closed vesicles still at the membrane proximity. The results showed that treatment with R428 inhibited virus internalization (Figure 2E). In agreement with its mechanism of action, EIPA also efficiently prevented virus internalization. Pretreatment with either R428 or EIPA did not affect virus attachment, evidenced by similar signals for biotinylated GP2 detected in the absence of TCEP (Figure 2E). Figure 2: Axl RTK participates only in early steps of rLCMV/LASV\({}_{\rm GP}\) entry. (**A**) Schematic experimental setting of the time-of-addition assay. R428, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), or ribavirin (Rib) were added at the indicated times relative to infection. Ammonium chloride (20 mM) was added 1 h after infection (+1 hpi) to prevent reinfections; (**B**,**C**) Inhibition of rLCMV/LASV\({}_{\rm GP}\) infection in time-of-addition assays by R428 (0.5 uM), EIPA (20 uM), and Rib (100 uM) in A549 (**B**), and HUVEC cells (**C**); (**D**) Schematic experimental setting of the TCEP assay to monitor virus internalization, (**E**) Viral uptake in the presence of EIPA and R428 in A549 cells. After quenching of residual TCEP, cells were lysed, and viral GP isolated by IP with mAb 83.6 to LCMV GP2. Biotinylated GP2 was detected with streptavidin-HRP in Western blot under non-reducing conditions using enhanced chemiluminescence (ECL).