## 3 Results
### Intracellular Filovirus Entry Factors are Expressed and Functional in SH-SYSY Cells
Scale bar 20 \(\upmu\)m.  
The bona fide receptor NPC1 was ubiquitously expressed but its relative expression did not correlate with filovirus susceptibility (Figure 5C). For instance, HPMEC, the cell line with the highest susceptibility to EBOV or MARV GP-mediated transduction (Figure 1A,B), displayed the lowest NPC1 expression. We also assessed NPC1 functionality in HEK293T or SH-SY5Y cells by inhibiting its cholesterol transport activity with U18666A [65] (Figure 5D). In untreated cells, cholesterol was distributed throughout the entire cells with the highest signal at the plasma membrane. In contrast and as reported before [66; 67], cholesterol accumulated more in the cell center in U18666A treated cells, most likely in late endosomes and endolysosomes, indicating that these cells expressed functional NPC1. Finally, U18666A treatment inhibited filovirus GP-driven transduction of susceptible cells (Figure S4B), confirming that entry into these cells is NPC1 dependent. In sum, the data show that SH-SY5Y cells express functional NPC1. Similarly, all cell lines expressed also the endosome traffic regulators TPC1 and TPC2 (Figure 5E). As for the other intracellular host factors, TPC1 relative expression was heterogeneous and did not correlate with susceptibility to transduction driven by the GPs of EBOV or MARV. On the other hand, TPC2 was expressed to a similar extent with SH-SY5Y cells displaying the lowest level of TPC2. To test whether TPC2 accounted for the resistance of SH-SY5Y cells to GP-driven transduction, susceptible HEK293T and resistant SH-SY5Y cells were treated with the inhibitor tetrandrine, which blocks TPC1/2 and results in epidermal growth factor (EGF) accumulation in LAMP-1 positive endosomes [39]. Both cell lines displayed larger and brighter organelles after drug treatment, indicating EGF accumulation and suggesting that also TPC1/2 were functional in SH-SY5Y cells. Finally, tetrandrine inhibited filovirus GP-driven transduction of all susceptible cell lines tested as expected (Figure S4C). Collectively, these results confirmed the essential functions of cathepsin B, cathepsin L, NPC1, TPC1, and TPC2 for filovirus infection, and demonstrated that neither an absence nor a dysfunction of these intracellular proteins can explain the resistance of SH-SY5Y cells to GP-mediated transduction or infection. Filovirus Resistance Is Neither Explained by SH-SY5Y Cells' Transcriptome nor by the Expression Profile of Filovirus Attachment Factors  
Besides these intracellular proteins essential for filovirus infection, the resistance of SH-SY5Y could also result from a lack of plasma membrane proteins implicated in filovirus entry. Since a long list of host factors seems to contribute to filovirus attachment (Table S1), we performed a global gene profiling analysis. We extracted total RNA from our panel of cell lines as well as from primary human hepatocytes (PHH) and quantified relative mRNA levels by microarrays. We used hierarchical clustering analyses to compare the respective gene expression profiles. Figure 6A represents an unbiased clustering analysis of all detected transcripts and Figure 6B depicts clustering of all filovirus cell surface or soluble extracellular entry factors described so far. The cell lines were grouped in two major clusters by an unbiased analysis (Figure 6A). This observation also hold true for the analysis considering only reported entry factors (Figure 6B), except that the Huh-7.5 cells were outside the cluster closest to PHH. Whereas one cluster contained most of the susceptible cell lines, the other enclosed all resistant cell lines, including SH-SY5Y, together with the susceptible HEK293T, SW13, and Huh-7.5 cell lines (unbiased analysis only). Thus, the gene expression profiles of the susceptible cell lines HEK293T and SW13 were more similar to resistant cell lines than to other susceptible lines.