Influenza HA Subtypes Demonstrate Divergent Phenotypes for Cleavage Activation and pH of Fusion: Implications for Host Range and Adaptation
Figure 6
The pH of fusion for a select representative group of HA proteins.
(A) Photomicrographs of syncytia formation assay. BHK cells were transfected with 1.0 µg HA plasmid. At 16 hrs post transfection, HA-expressing BHK cells were treated with TPCK-trypsin (5 µg/ml), followed by treatment with pH-adjusted PBS in 0.1 pH unit increments, neutralized, and incubated at 37°C for 2 hr. Cells were then washed with PBS, fixed, stained, and imaged. Photomicrographs corresponding to the last pH at which syncytia were observed and 0.1-pH unit higher are shown. (B) Luciferase reporter gene assay for fusion. Vero cells were co-transfected with 1.0 µg HA plasmid and 1.0 µg T7-Luciferase plasmid. At 16 hrs post transfection, HA-expressing Vero cells were treated with TPCK-trypsin (5 µg/ml), followed by treatment with C. soybean trypsin inhibitor (20 µg/ml). HA-expressing Vero cells were overlaid with BSR-T7/5 target cells that constitutively express T7 RNA polymerase. The two cell populations were incubated for 1 hr, at which time cell monolayers were pulsed with pH-adjusted PBS in 0.2 pH unit increments at the indicated pH for 5 min to trigger fusion, and then were neutralized. Cells were incubated for 6 hrs at 37°C to allow for cell-to-cell fusion to occur, which would mediate transfer of the T7-luciferase plasmid and expression of firefly luciferase. Luminescence was measured as an indicator of membrane fusion in cell lysates. The graphs show the mean background-adjusted relative luminescence (± standard deviation) as a function of pH obtained from three independent experiments.