Fig 2: AXL kinase activity is required for ZIKV replication in human glioblastoma cell lines(A and B) Timeline of post-exposure R428 treatment. LN-229 and U-87 MG cells were first infected at MOI 1 for 90 min. After 5 h the cells were treated with indicated concentrations of R428 or vehicle for 24 h (A). The percentage of infected cells were quantified by flow cytometry (B). (C) Cell viability was evaluated after R428 (0.1, 0.3, and 1 µM) or vehicle treatment in LN-229, U-87 MG, and A-172 glioblastoma cell lines. At 32 h, the WST-1 assay was used to determine cell viability. The percentage of viable cells was calculated after normalizing to the DMSO (vehicle) infected cells. In (B), data shown are means ± SD and are representative of three independent experiments. Significance was calculated using one-way ANOVA (n.s., not significant; ***p < 0.001, ****p < 0.0001).

Fig 3: AXL-expressing cell lines are permissive to ZIKV (strain MR-766) infection(A) Relative protein expression level of AXL in human glioblastoma (LN-229, U-87 MG, U-251 MG, and A-172) and breast cancer (MDA-468) cell lines. (B) The human glioblastoma and breast cancer cell lines were challenged with ZIKV at MOI 1. At 24, 48, and 72 hpi, the percentage of infected cells were quantified by flow cytometry. (C) AXL-expressing glioblastoma (LN-229 and U-87 MG) and AXL-negative breast cancer (MDA-468) cell lines were infected with increasing MOI (0.01, 0.1, 1, 5, and 10) for 24 h, and the percentage of infected cells were quantified by flow cytometry. (D) Cell lines were challenged with ZIKV at MOI 1 for 24, 48, and 72 h. The relative expression of ZIKV NS3 RNA was quantified by qRT-PCR at each time point. (E) ZIKV titer (PFU/mL) comparison between cell lines in supernatants by plaque assay at 48 and 72 hpi infected at MOI 1. (F) Immunofluorescence staining for ZIKV in glioblastoma and breast cancer cell lines at 72 hpi at MOI 10. Images were acquired through an Olympus IX83 microscope. ZIKV, green; DAPI, blue. Scale bars, 50 µm. In (C), (D), and (E) data shown are means ± SD of averages of two biological replicates performed in triplicate. Significance was calculated using two-way ANOVA (n.s., not significant; **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig 4: Melanoma cells in vivo show anti-tumor effect and apoptosis following combined inhibition of AXL and BRAF. Subcutaneous Melmet 1 tumors were treated with BRAFi (12.5 mg/kg), AXLi (50 mg/kg), the combination or vehicle (DMSO/methylcellulose) twice/day for 14 days. The tumor volume over time, presented as relative to the volume at treatment initiation (a) and the levels of indicated protein related to treatment efficacy (b) and apoptosis (c) in two tumors from each treatment group at day 1 and 10 are presented (cropped gels). (a) Average ± SEM, n = 8, ***p = 0.001.

Fig 5: R428 and EMD inhibit macropinocytosis-related rLCMV/LASVGP internalization. (A) A549 cells were pretreated with DMSO, 40 µM EIPA, 5 µM R428, or 40 µM EMD, for 30 min. Subsequently, cells were incubated with Alexa Fluor 488 labeled dextran (250 µg/mL) in the presence of the inhibitors, for 40 min, at 37 °C, washed, fixed with PFA, and subjected to confocal microscopy. Note: EIPA treatment resulted in a faint blue fluorescence throughout the cytosol. Representative fields for each condition are shown. Scale bar: 10 µm; (B) Precooled A549 cells were incubated for 2 h, at 4 °C, with rLCMV/LASVGP (MOI 50 PFU/cell). Cells were washed and either fixed or shifted to 37 °C, for 45 min, in the presence of DMSO or inhibitors (same concentrations as in (A)), as indicated. Then, cells were fixed, stained for viral NP (113 antibody), and analyzed by confocal microscopy. Representative fields are shown for each condition (n = 10). Scale bar corresponds to 10 µm. Interestingly, whereas Axl RTK inhibition by R428 decreased the number of dextran-labeled vesicles, HGFR inhibition by EMD resulted in an accumulation of vesicles in proximity to the plasma membrane (Figure 6A). In agreement with previous reports [27,37], we also found in viral infection experiments that 18.6% (±7.7%) of total Alexa Fluor 488-conjugated, dextran-containing vesicles also contained rLCMV/LASVGP viral particles (Figure S5).