Influenza HA Subtypes Demonstrate Divergent Phenotypes for Cleavage Activation and pH of Fusion: Implications for Host Range and Adaptation
Figure 5
Analysis of HA cleavage by human serine proteases, HAT and TMPRSS2.
(A) BHK cells were transfected with 1.0 µg HA plasmid and 0.25 µg of either HAT or TMPRSS2 expression 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. Total 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. The identity of each HA is denoted above the gel and the migration of HA0, HA1, and HA2 is denoted to the left of the gel. (B) Quantitation of HAT-mediated cleavage of HA. 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 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 TMPRSS2-mediated cleavage of HA. Quantitative analysis was performed on three independent experiments, as described in (B). The percent cleavage shown for the H5 and H5VN HAs was determined by subtracting the percent cleavage observed in the absence of trypsin from the percent cleavage observed in the presence of TMPRSS2.