Fig 2: Inhibition of AAK1 kinase activity decreases RABV infection in vitro. (A–F) Cells were pretreated with different concentrations of sunitinib or DMSO for 1 h at 37 °C before infection with ERA-mCherry at an MOI of 0.01. At 48 h post-infection, a high-content quantitative image-based analysis was used to measure the relative infection ratio (normalized to NT siRNA-treated cells) of ERA-mCherry in HEK-293 cells (A), SK cells (C), and mPN cells (E). The viability of HEK-293 cells (B), SK cells (D), and mPN cells (F) treated with the indicated concentrations of sunitinib was determined using CellTiter-Glo reagent. Data are relative fluorescence values normalized to the level of the NT control. A one-way ANOVA was used for the statistical analysis. **, p < 0.01, ***, p < 0.001, ****, p < 0.0001; n.s., not significant. (G) HEK293 cells were treated with sunitinib (2µM) or DMSO and then infected with ERA-mCherry (MOI = 0.01). Viral titers were determined at the indicated time points. Multiple t-tests were used for the statistical analysis, *, p < 0.05. (H) Sunitinib counteracted the effect of AAK1 overexpression on RABV infection. HEK293 cells were transfected with pCAGGS-AAK1 and pCAGGS. At 48 h post-transfection, cells were treated with sunitinib and subsequently infected with ERA-mCherry at an MOI of 0.1. Viral titers in the supernatant were determined at 48 h post-infection and were normalized to that of the “infection only” group. Tukey’s multiple comparisons test was used to analyze the statistical difference. ****, p < 0.0001; n.s., not significant.

Fig 3: AAK1 functions at the early stage of RABV infection. HEK293 cells (A) and SK cells (B) were treated with sunitinib (3 µM) at the indicated time points before or after ERA-mCherry infection. Viral titers in the supernatant were determined at 24 h post-infection. A one-way ANOVA was used for the statistical analysis. ****, p < 0.0001. (C) HEK-293 cells were treated with sunitinib (3 µM) or DMSO for 1 h at 37 °C, and then incubated with ERA-mCherry at an MOI of 2 for 2 h at 4 °C. After removing unbound viruses by washing, total cellular RNA was extracted and subjected to qPCR to quantify cell-bound RABV RNA. A t-test was used for the statistical analysis. n.s., not significant. (D) Representative images showing tyramide signal amplification immunofluorescence staining of ERA-N-mCherry (red), AP2 (green), and Rab5 (purple) in sunitinib-treated or DMSO-treated cells (I). Co-localization of RABV with Rab5 or AP2 was observed and quantified in 15 to 20 randomly chosen cells from each sample. Statistical results represent the ratio of RABV:Rab5 co-localization normalized to RABV: AP2 co-localization (II). A t-test was used for the statistical analysis. *, p < 0.05. Bars, 8 or 10 µm.