Influenza HA Subtypes Demonstrate Divergent Phenotypes for Cleavage Activation and pH of Fusion: Implications for Host Range and Adaptation
Figure 4
Effect of co-expressing NA on cell surface expression of HA.
(A) BHK cells were transfected with 1.0 µg H13 plasmid only, or co-transfected with 0.1 µg, 0.5 µg, or 1.0 µg N6 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. The amount of NA plasmid is denoted at the top of each gel and the migration of HA0 is denoted to the left of the gel. T = Total HA, S = Surface expressed HA. The graph to the right of the gel shows the quantitative analysis of three independent experiments. The percentage of surface-expressed HA was determined by dividing the amount of surface-expressed HA by the total HA. (B) Experimental details are the same as in A, except that BHK or Vero cells were transfected with 1.0 µg H3, H5, H7, Japan, or Aichi HA plasmid only or in combination with 0.1 µg of cognate NA plasmid. The data shown represents the quantitative analysis of three independent experiments. Quantitation was performed exactly as stated in (A).