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

For the generation of rhabdoviral pseudotypes bearing heterologous viral glycoproteins we employed a replication-deficient VSV vector. Instead of the genetic information for VSV-G, the vector contains two separate open reading frames coding for enhanced green fluorescent protein (EGFP) and firefly luciferase (fLuc), VSV Ã ΔG-fLuc [39, 42, 52] . The vector was propagated in a previously described VSV-G-expressing, transgenic cell line [54] . A detailed protocol for the production of pseudotyped VSV (VSVpp) can be found elsewhere [39] . In brief, BHK-21 cells were transfected with expression plasmids for VSV-G (positive control), the respective filovirus glycoproteins or empty plasmid (pCAGGS; negative control) using Lipofecta-mine2000 (Thermo Fisher Scientific) according to manufacturer's protocol. At 16 h post transfection, the cells were first inoculated with VSV Ã ΔG-Luc at a multiplicity of infection of 3, then incubated with a polyclonal anti-VSV serum to block the infectivity of residual input virus (both for 1 h at 37°C and 5% CO 2 ). Finally, the cells received fresh culture medium and were further incubated for 16-20 h, before the VSVpp-containing supernatants were collected, centrifuged (to remove cell debris) and aliquoted. Aliquots were stored at 4°C for a maximum of 7 days.