## 3 Results
### Intracellular Filovirus Entry Factors are Expressed and Functional in SH-SYSY Cells  
To further investigate the resistance phenotype of SH-SYSY cells, we analyzed protein expression (Figure 5A,C,E) and functionality (Figure 5B,D,F) of the intracellular proteins operating during the endolysosomal phase of filovirus infection [39; 40; 41]. Cathepsin L was expressed to a similar extend in all cell lines, while expression of procathepsin B (44 kDa) and the active cathepsin B isoforms (27 kDa and 24 kDa) varied among cell lines but did not correlate with susceptibility to filovirus infection (Figure 5A). For instance, SH-SYSY cells expressed the 27 kDa active isoform at a level comparable to that of susceptible HPMEC cells. In addition, we measured substrate-specific cleavage activity of cathepsin B and cathepsin L in the cell lysates (Figure 5B), but the relative cleavage activities did not correlate with susceptibility to filovirus GP-driven transduction. Notably, the resistant SH-SYSY cells showed cathepsin B and cathepsin L substrate-specific cleavage activity, suggesting that GP-processing can occur on this cell line. Finally, specific inhibition of cathepsin B/cathepsin L with FYdmk [64] confirmed that filovirus GP-driven transduction into susceptible cell lines required cathepsin B and cathepsin L activity as expected (Figure S4A). Figure 4: SH-SYSY cells do not express a dominant entry restriction factor. Huh-7.5 or SH-SYSY cells stably expressing the tetracycline-inducible Tet On 3G transactivator protein were cocultured with HEK293T-H6 cells stably expressing transactivator-inducible ZsGreen1 green fluorescent protein for 24 h. Cells were chemically fused with PEG. One hour after fusion, cells were transduced with NoEnv, EBOV, MARV, or VSV-G pseudoparticles encoding mCherry for 6 h at 37 \({}^{\circ}\)C. Seventy-two hours post transduction, cells were fixed with 3% PFA and analyzed for heterokaryon formation (ZsGreen1 protein expression) and susceptibility to pseudoparticle infection (mCherry protein expression). (**A**) Confocal microscopy images of one representative experiment with SH-SYSY and HEK293T-H6 cells. Scale bars = 50 \(\upmu\)m (**B**) Quantification of transduced cells by flow cytometry. Left-hand side of graph represents transduction percentage of single cell line controls and right-hand side from heterokaryons (discriminated  
previously by gating on ZsGreen1 positive cells). Mean \(\pm\) SD. of three independent cell fusions and subsequent transduction experiments (\(n=3\)). Figure 5: Intracellular entry factors are expressed and functional in SH-SY5Y cells. Endogenous protein expression of (**A**) cathepsin B and L, (**C**) NPC1, and (**E**) TPC1 and 2. Cell lysates were analyzed for protein expression by western blotting with protein specific abs. \(\beta\)-tubulin (55 kDa) was used as internal control. (**B**) Cathepsin B and L activity assay. Cathepsins substrate-specific proteolytic cleavage was measured with a commercially available kit as described in methods. Assay limit of detection (LOD) represented as a dotted line. Experiments were conducted thrice with graph bars representing mean experimental value, individual values and SD. (**D**) Intracellular cholesterol accumulation. HEK293T (upper panels) or SH-SY5Y cells (bottom panels) were treated either with vehicle (left) or with 10 \(\upmu\)g/mL of the NPC1 inhibitor U18666A (right) for 24 h. Cells were fixed with 0.1% TX100 and stained for unesterified cholesterol using filipin. Images are representative of two independent experiments. Scale bar 200 \(\upmu\)m. (**F**) EGF intracellular accumulation. HEK293T (upper panels) or SH-SY5Y cells (bottom panels) were treated either with vehicle alone (left) or with the TPC1/2 inhibitor tetrandrine (2 \(\upmu\)g/mL) (right) for 24 h followed by incubation with EGF-Alexa Fluor 555 (red) for 30 min. Cells were fixed with 3% PFA, permeabilized with 0.1% TX100, stained with DAPI (blue) and analyzed by confocal microscopy.