Influenza HA Subtypes Demonstrate Divergent Phenotypes for Cleavage Activation and pH of Fusion: Implications for Host Range and Adaptation
Figure 2
Analysis of trypsin-mediated cleavage-activation of HA in the absence and presence of NA.
(A) BHK cells were transfected with 1.0 µg HA plasmid or co-transfected with 1.0 µg HA plasmid and 0.1 µg NA plasmid. At 16 hours post transfection, cells were starved for methionine and cysteine, and were pulse labeled with 25 µCi [35S]-methionine for 15 min, followed by a 3 hr chase period. Where indicated, radiolabeled, HA-expressing cell monolayers were treated with 5 µg/ml TPCK-Trypsin for 15 min. Total cellular HA protein was immunoprecipitated with a HA-specific antibody and resolved on 12% SDS-PAGs. The gels were fixed and dried, and radiolabeled proteins were visualized by phosphorimaging. Representative images of gels are shown with the HA subtype denoted at the top of each gel and the identity of each band is denoted on the left. (B) Quantitation of trypsin-mediated cleavage of HA in the absence of NA. Quantitative analysis was performed on three independent experiments. For each HA, the intensity of the bands corresponding to HA0 and HA2 were normalized to their respective methionine content and the percent cleavage (mean ± standard deviation) is expressed as a percentage of the total cellular HA, by the equation (HA2/(HA2+HA0))×100%. * denotes no detectable cleavage. (C) Quantitation of surface-expressed HA. BHK cells were transfected with 1.0 µg HA plasmid. At 16 hours post transfection, cells were starved for methionine and cysteine, and were pulse labeled with 25 µCi [35S]-methionine for 15 min, followed by a 3 hr chase period. Cell surface proteins were then biotinylated and total HA protein was immunoprecipitated with a HA-specific antibody, followed by immunoprecipitation with streptavidin, and resolved on 12% SDS-PAGs. The gels were fixed and dried, and radiolabeled proteins were visualized by phosphorimaging. Quantitative analysis of three independent experiments is shown. The percentage of surface-expressed HA was determined by dividing the amount of surface-expressed HA by the total HA. (D) Quantitation of trypsin-mediated cleavage of HA in the presence of NA. Quantitative analysis was performed as described in (B). * denotes no detectable cleavage.