## 3 Results  
### Axl Is Required for LASV Entry into Human Cells  
Human epithelial cells of the respiratory tract are likely to be early targets during zoonotic transmission of LASV [8] and to express Axl RTK in the context of functionally glycosylated DG [27; 38]. Since functional studies of LASV entry into human epithelial cells would require significant amounts of homogeneous tissue culture material, work with primary human small airway epithelial cells (SAEC) is challenging. As recently reported, SAEC and the immortalized human alveolar epithelial cell line A549 showed similar levels of productive rLCMV/LASVGP infection [80], which correlated with similar expression patterns of functional glycosylated DG and the alternative receptors Axl RTK and T cell immunoglobulin and mucin domain (TIM)-1 [27; 37; 80]. Therefore, similar susceptibility and candidate receptor expression make A549 cells a suitable model for functional studies of human respiratory epithelial cells, and expression of fully glycosylated DG and Axl in A549 cells has been previously confirmed in our laboratory [37] (Figure S1A). LASV infection also perturbs endothelial function [81; 82] and human umbilical cord microvascular endothelial cells (HUVEC) display expression of Axl RTK, as well as under-glycoslated DG [37], as validated by our fluorescence-activated cell sorting (FACS) analysis (Figure 1A). The human fibrosarcoma cell line, HT-1080, which is susceptible to rLCMV/LASVGP infection, has previously been shown to robustly express Axl RTK but lacked functional DG [36; 37], as confirmed by our analyses (Figure S1B). To evaluate the role and relative contributions of Axl RTK in different functional DG contexts, we compared rLCMV/LASVGP infection in A549, HUVEC, and HT-1080 cells. Because we failed to transiently express in 293T cells both wild type Axl RTK and its variant bearing loss-of-function mutations in the intracellular domain (Figure S1C), we used the Axl RTK inhibitor R428 in A549, HT-1080, and HUVEC cells, which expressed endogenous Axl RTK [37]. R428 is the most specific available inhibitor of Axl RTK and strongly inhibits its phosphorylation (IC\({}_{50}\) < 30 nM). It is a reversible inhibitor and shows high specificity for Axl over other RTKs in cell-based assays [83; 84], without affecting Axl RTK expression (Figure 1B). Because of the potential compensatory effect or off-target effects, such as Tie-2, Ftl-1, Flt-3, Ret, or Abl [83], we chose a short time window of drug action, combined with drug washout, assuring micro-reversibility, and therefore minimized potential consequences of the R428 administration and allowed us to dissect the Axl RTK role during LASV entry [85] (Figure 1C). Moreover, R428 is currently in phase II clinical trials for human anticancer therapy and has a favorable toxicity profile, suggesting minimal off-target effects [84; 86]. For the entry assay (Figure 1C), cells were pretreated for 30 min, at 37 \({}^{\circ}\)C, with the indicated inhibitor, cooled down to 4 \({}^{\circ}\)C, infected with rLCMV-LASVGP, and shifted to 37 \({}^{\circ}\)C, for 1 h, in the presence of the drug. Then, the cell media was replaced with fresh media containing 20 mM of ammonium chloride (NH\({}_{4}\)Cl) to prevent subsequent replication rounds. When added to cells, ammonium chloride instantly raised the endosomal pH, preventing subsequent acidic-dependent endosomal escape of viruses, without causing overall cytotoxicity [87; 88]. After 16-20 h, cells were fixed, and infection was quantified by immunofluorescence assay (IFA) for LCMV NP. Our results showed that R428 reduced rLCMV/LASVGP infectivity in a dose-dependent manner in A549, HUVEC, and HT-1080 cells expressing functional Axl RTK (Figure 1D and Figure S1D). The IC\({}_{50}\) values for R428 were <1 uM in the cell types tested, resulting in a therapeutic index (TI = CC\({}_{50}\)/IC\({}_{50}\)) >40. None of the tested R428 concentrations exerted significant cellular toxicity (Figure S1E). Figure 1: Axl receptor tyrosine kinase (RTK) participates in LASV infection.