## 3 Results
### Attachment Limits GP-Driven Entry into SH-SYSY Cells  
To further characterize the step of filovirus infection impaired in SH-SYSY cells, we studied filovirus GP-specific attachment using recombinant soluble EBOV GP1 fused to human Fc or a human Fc alone [24] (Figure 8A). We chose HepG2 cells as a control, since they express the GP-binding C-type lectin ASGP-R and bind to filovirus GP [28; 68], and compared them with susceptible HEK293T and resistant SH-SYSY cells. EBOV GP1-Fc bound most efficiently to HepG2 cells and to HEK293T cells with lower efficiency. However, it did not bind to SH-SYSY cells, suggesting that the latter lacks any attachment factor for EBOV GP. Figure 7: Surface expression does not explain susceptibility to filovirus infection. (**A–D**) Axl and TIM-1 cell surface expression. Cells were surface stained with saturating concentrations of specific abs and their correspondent isotype controls and fluorescent signal quantified by flow cytometry. Protein expression profiles of (**A**) Axl and (**C**) TIM-1 in different cell lines are shown as histograms of a representative experiment. (**B–D**) Cell surface expression of Axl and TIM-1 in terms of average delta mean fluorescence intensity (\(\Delta\)MFI) from three independent stainings. \(\Delta\)MFI was calculated by subtracting the geometric mean intensity values of the isotype control from the specific staining values. SD is shown as error bars. Figure 8: Impaired filovirus attachment on SH-SYSY cells can be overcome by surface factor overexpression. (**A**) Binding of EBOV GP-Fc fusion protein to cell surface. EBOV-GP1 and human Fc (100 nM) were incubated for 1.5 h with the indicated cell lines and detected with a secondary ab against the human Fc fragment. MFI signal was recorded for Fc or EBOV GP1-Fc and subtracted from secondary ab MFI. Bars represent mean of two biological replicates performed in duplicate (\(n=4\)) and symbols represent each individual value. Error bars represent the SD. (**B**) Overexpression of several cell surface factors and their role in filoviral GP-dependent entry. Susceptible HEK293T, resistant SH-SYSY WT or SH-SYSY cells genetically engineered to individually express different entry host factors were transduced with NoEnv, EBOV, MARV, and VSV-G luciferase-encoding pseudoparticles. Mock infection was performed to control background signal. Enhanced susceptibility to filovirus infection was calculated as the fold change difference over SH-SYSY WT cells by dividing the RLU values of 100 \(\upmu\)l lysed cells of each engineered cell line and HEK293T cells with the SH-SYSY WT RLU values. Fold change differences between SH-SYSY WT and HEK293T or engineered SH-SYSY cell lines were used for statistical analysis. Graph is the representation of 3 independent transductions done in triplicate (\(n=9\)). Error bars depicts SD. For the analysis of significance in all 3 graphs a multiple \(t\) test with a Holm-Sidak multiple comparison correction method was conducted. \(P\)-value significance is shown as: n.s. \(P>0.05\); \({}^{\star}P\leq 0.05\); \({}^{\star}\pi\)\(P\leq 0.01\); \({}^{\star}\pi\)\(P\leq 0.001\); \({}^{\star}\pi\)\(P\leq 0.0001\). Diverse Cell Surface Factors Can Overcome the Block to Filovirus GP-Driven Cell Entry in SH-SYSY Cells  
We next asked whether ectopic expression of surface factors that promote filovirus attachment could rescue GP-driven entry into SH-SYSY cells (Figure 8B). We chose to overexpress Axl and TIM-1 proteins because their expression was either very low or absent in SH-SYSY cells, but substantial in a subset of susceptible cell lines (Figure 7A-D, Table S2). Furthermore, we overexpressed Mer tyrosine kinase since it was expressed well in HEK293T and SW13 susceptible cell lines, which had clustered in the transcriptome analysis with the resistant cell lines (Figure 6B, Table S2). Finally, we overexpressed DC-SIGN as one Ca\({}^{2+}\)-dependent C-type lectin and NPC1 as negative control.