Figure 4. Amino acid residue V134 is a critical determinant for efficient H5pp production.

(A) H5pp was produced using H5Anh, H5Cam, their single or double mutants at position 133 and 134. NAvb was added 16 hr post transfection. Culture supernatant containing H5pp was harvested at 48 hr post transfection and luciferase activity in MDCK cells was measured at 72 hr post H5pp transduction. Results are shown as means ± SD (n = 3 independent experiments); **p<0.01 compared to their respective wild type HA by the unpaired Student's t-test. (B) Cell lysates at 48 hr post transfection were analyzed for HA protein expression using anti-FLAG antibody. GAPDH antibody was used as the loading control. H5pp produced in the supernatant were concentrated by ultracentrifugation and the H5pp pellets were analyzed by western blotting using anti-FLAG and anti-p24 antibodies. (C) HA protein expression at the cell surface was analyzed by immunofluoresent staining followed by flow cytometry as described in Material and Methods. Left and right histograms in the same graph depict cells transfected with pcDNA and H5-HA respectively. Results of mean fluorescence intensity (MFI) are presented as means ± SD (n = 5 independent experiments); **p<0.01; *p<0.02 by the unpaired Student's t-test.