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

BatFLUAV-HAL cleavage was investigated by cotransfection of HEK-293T cells, which were grown in 6-well plates, with expression plasmids for batFLUAV-HAL and different TTSPs (TMPRSS2, DESC-1 or MSPL) or by incubation of HAL-expressing cells with trypsin (1, 5, 10, 50 μg/ml; 20 min/37°C) directly before cell lysates were produced. Cells cotransfected with empty plasmid and not subjected to trypsin treatment served as negative control. The 1918-HA served as positive control, since cleavage by TTSPs and trypsin has been previously shown [33] . At 24 h post transfection, the cells were washed with PBS, resuspended in 100 μl 2x SDS-containing lysis buffer (50 mM Tris [pH 6.8], 10% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.1% bromophenol blue, 1 mM EDTA) and boiled for 20 min at 96°C. To assess incorporation of HAL into VSVpp, 1 ml of the respective pseudotypes was loaded onto a 20% sucrose cushion in PBS and centrifuged at 17,000x g for 2 h at 4°C. After discarding the supernatant, the pelleted pseudotypes were mixed with 30 μl 2x SDS-containing lysis buffer and boiled for 20 min at 96°C. Finally, all samples were subjected to immunoblot analysis. For this, anti-FLAG (mouse, 1:1,000, Sigma-Aldrich), anti-FLUAV (goat, 1:1,000, Millipore), anti-VSV-M (mouse, 1:1,000, Kerafast), anti-VSV-G (I1, mouse hybridoma supernatant from CRL-2700, ATCC, 1:200) and anti-ß-actin (mouse, 1:1,000, Sigma-Aldrich) served as primary antibodies. Peroxidase-coupled anti-mouse (1:10,000, Dianova) and anti-goat (1:5,000, Dianova) antibodies served as secondary antibodies. Signal detection was carried out in a ChemoCam imager together with the ChemoStar professional software (both Intas) using a self-made chemiluminescence substrate (recipe available upon request).