Influenza HA Subtypes Demonstrate Divergent Phenotypes for Cleavage Activation and pH of Fusion: Implications for Host Range and Adaptation
Figure 8
HA-mediated fusion in the absence of trypsin, the presence of trypsin, or the presence of TMPRSS2, as measured using the luciferase reporter gene fusion assay.
Vero cells were transfected with 1.0 µg HA plasmid, 1.0 µg T7 luciferase plasmid, and, where indicated, 0.25 µg TMPRSS2 plasmid. At 16 hours post transfection, HA-expressing Vero cells were either left untreated or treated with TPCK-trypsin (5 µg/ml), and overlaid with BSR-T7/5 target cells. Cells were then treated with PBS or PBS that had been pH adjusted to 5.0 with citric acid, neutralized, and incubated at 37°C for 6 hr to allow for cell-to-cell fusion to occur. Cell populations were then harvested and the luciferase activity resulting from the fused cell populations was quantified and is expressed as the mean ± standard deviation of relative luminescence units (RLU).