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

To assess expression of HAL proteins, we transfected BHK-21 cells that were grown on coverslips with expression plasmids for HAL17-FLAG or HAL18-FLAG using the Lipofecta-mine2000 reagent (ThermoFisher Scientific) according to manufacturers' protocol. Cells transfected with an empty expression vector served as negative control. At 24 h post transfection, cells were fixed with 4% paraformaldehyde/PBS, permeabilized by incubation with 0.2 M Triton X-100/PBS (10 min at room temperature) and subsequently incubated with anti-FLAG (mouse, 1:1,000, Sigma-Aldrich) and Cy3-labeled anti-mouse (1:750, Sigma-Aldrich) antibodies. After each antibody incubation, the cells were washed three times with PBS and finally incubated with DAPI (Roth, 5 min/37°C) to stain the nuclei before the coverslips were mounted on glass slides using Mowiol (AppliChem) supplemented with anti-bleaching reagent (DABCO, Roth). Representative pictures were taken at a 10x magnification using a Nikon Eclipse Ti fluorescence microscope and the NIS Elements AR software (Nikon).