The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells

Hoffmann, Markus; Krüger, Nadine; Zmora, Pawel; Wrensch, Florian; Herrler, Georg; Pöhlmann, Stefan

We employed a replication-deficient VSV vector for pseudotyping that contains two separate open reading frames, coding for enhanced green fluorescent protein (EGFP) and firefly luciferase (fLuc), instead of the genetic information for VSV-G, VSV Ã ΔG-fLuc [28, 31, 35] . Propagation of VSV Ã ΔG-fLuc was performed in a previously described VSV-G-expressing, transgenic cell line [37] . Generation of VSV pseudotypes (VSVpp) was performed as follows: HEK-293T cells were transfected by calcium-phosphate precipitation with expression plasmids encoding viral surface proteins, VSV-G (positive control) , NiV-F/G, FLUAV-HA and/or NA and bat-FLUAV-HAL and/or NAL, or empty plasmid (pCAGGS) as negative control. In order to investigate the potential of human TTSPs to proteolytically activate batFLUAV-HAL for host cell entry, we additionally cotransfected the cells with expression plasmids for TMPRSS2, DESC-1 or MSPL. At 16 h post transfection, the cells were inoculated with VSV Ã ΔG-fLuc at a multiplicity of infection of 3 for 1 h at 37°C and 5% CO 2 . Subsequently, the cells were washed and incubated with an anti-VSV serum to neutralize residual input virus. Finally, the cells received fresh culture medium and were further incubated for 16-20 h, before the VSVpp-containing supernatants were collected, clarified from cell debris by centrifugation and aliquoted. Aliquots were stored at 4°C for a maximum of 7 days. For proteolytic activation of HA/HAL by trypsin, pseudotypes were incubated with bovine trypsin (Sigma-Aldrich; final concentration: 50 μg/ml) for 20 min at 37°C. Subsequently, trypsin was inactivated by addition of soybean trypsin inhibitor (Sigma-Aldrich; final concentration: 50 μg/ml).