The Glycoproteins of All Filovirus Species Use the Same Host Factors for Entry into Bat and Human Cells but Entry Efficiency Is Species Dependent

Hoffmann, Markus; González Hernández, Mariana; Berger, Elisabeth; Marzi, Andrea; Pöhlmann, Stefan

We employed inhibitors of cellular factors or processes known to be required for filovirus GP-driven entry into human cells to investigate whether filoviruses hijack the same factors/ process to enter bat cells. We observed a profound decrease in GP-mediated transduction when endosomal acidification (inhibited by ammonium chloride or bafilomycin A1), calciumdependent two-pore channels (inhibited by tetrandrine), cysteine proteases such as cathepsins and calpain (inhibited by E-64d and MDL28170) were blocked. Similarly, U18666A treatment, which induces an NPC1 knock-out phenotype, reduced entry into all cell lines tested, in line with the published finding that LLOV-GP requires NPC1 for entry into bat cell lines [38] . In general, no appreciable differences in the effectiveness of the inhibitors between human (HEK-293T) and fruit bat (EpoNi/22.1 and EidNi/41) cells were observed. A slightly less efficient block of GP-mediated entry into bat cells by agents interfering with endosomal acidification as well as the activity of TPCs and cysteine proteases might result from species specific differences in the drug targets. Additionally, it cannot be ruled out that the usage of more specific cysteine At 18 h post inoculation, the activity of virus-encoded firefly luciferase as an indicator for transduction efficiency was quantified and normalized against the values of the respective VC (x-fold changes). The results of a representative experiment carried out with quadruplicate samples are shown and were confirmed in an independent experiment, conducted with a separate pseudotype batch. The following inhibitor concentrations were used: Mannan (final concentration: 25 μg/ml), ammonium chloride (NH 4 + ; 50 mM), bafilomycin A1 (50 nM), tetrandrine (2 μM), E-64d (50 μM), MDL28170 (50 μM), camostat mesylate (100 μM), U18666A (20 μM). Error bars indicate SD. An unpaired student's t-test was used to test statistical significance (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).