## 3 Results
### LASV Colocalizes with Glycosylated DG within the Plasma Membrane  
Our findings provided evidence for Axl RTK participation in LASV entry in the presence or absence of fully functional DG. Therefore, we investigated a possible clustering of LASV with DG or Axl RTK during viral entry. Using confocal microscopy, in agreement with previous reports, we found that rLCMV/LASV\({}_{\text{GP}}\) attached preferentially to DG clusters at the plasma membrane, which could serve as \"docking sites\" for free virus (Figure 3A). Nevertheless, rLCMV/LASV\({}_{\text{GP}}\) and Axl RTK colocalization did not increase over time upon viral infection (Figure 3B). Contrary to the spotted pattern observed in DG, it is noteworthy that Axl RTK signal was diffuse and extended along the viral membrane, rendering comparisons between DG/NP and Axl RTK/NP colocalizations challenging. Then, we sought to investigate if LASV could induce transient DG-Axl RTK clustering during viral entry. The results showed no differences in colocalization of DG and Axl as comparing with uninfected and with infected cells at different time points after infection (Figure 3C). Axl RTK participates in infection of several RNA viruses [48; 51; 93; 93]. Interestingly, Axl signaling seems to contribute to virus internalization, and can also promotes later steps of viral multiplication [51; 93; 52]. Virus-induced activation of Axl RTK can promote replication by antagonizing IFN-I signaling, as illustrated by the flaviviruses dengue, West Nile, and Zika [93; 94; 52]. To address whether or not the observed contribution of Axl RTK to LASV increased entry was mediated by suppression or the IFN-I response, we performed infections in the absence or presence of R428 in immunocompetent A549 cells and monitored IFN\(\beta\) mRNA levels, at 16 h post infection. The results showed that the presence of R428 did not affect IFN\(\beta\) expression triggered by infection with LCMV, rLCMV-LASV\({}_{\text{GP}}\), or VSV (Figure 4A and Figure S3). Figure 3: Colocalization of rLCMV/LASV\({}_{\rm GP}\) viral particles, DG, and Axl RTK. (**A**) DG/nucleotide(NP) and (**B**) Axl RTK/NP colocalization. Precooled (4 \({}^{\circ}\)C) A549 cells were infected, for 2 h, in the cold with rLCMV/LASV\({}_{\rm GP}\) (MOI 50 PFU/cell), shifted to 37 \({}^{\circ}\)C for indicated times, and fixed. DG (A) or Axl RTK (B) were visualized in red and viral NP in green (**A**,**B**). Representative confocal images are shown. Scale bar represents 5 \(\upmu\)m. Double puncta percentages were obtained from the analysis of 20 (**A**) and 8 (**B**) randomly selected cells per condition. Error bars represent standard error; (**C**) DG/Axl RTK colocalization in mock-infected and rLCMV/LASV\({}_{\rm GP}\)-infected cells. Live A549 cells were preincubated, for 2 h, in the cold with mock or rLCMV/LASV\({}_{\rm GP}\) (MOI 50 PFU/cell), and rapidly shifted to 37 \({}^{\circ}\)C for indicated times. DG is visualized in red and Axl RTK in green. Representative images collected by confocal microscopy are shown. Scale bar represents 5 \(\upmu\)m. Colocalization of DG and Axl 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. Axl does not contribute to productive LASV entry via Interferon-I suppression. To further investigate if Axl RTK signaling could suppress the IFN-I response during LASV infection in the absence of glycosylated DG, we performed rLCMV-LASVGP infections with or without R428 in HT-1080 cells, lacking functional DG (Figure 1A). R428 did not affect IFN\(\beta\) expression upon OW mammarenaviruses or VSV control infections (Figure 4B and Figure S3). Nevertheless, in uninfected HT-1080 cells, the administration of R428 slightly increased the IFN\(\beta\) mRNA levels, suggesting a potential alteration of the innate immune response in the absence of other stimuli.