Figure 4: Plaque size, NA activity, NA accumulation and substrate affinity of the BR/59/07 NA proteins with different amino acid substitutions.

(a) Plaque size of viruses carrying ten different NA amino acid substitutions, in the absence (white) or presence (grey) of the H275Y mutation. The ten amino acid residues were replaced with the corresponding NC99-like consensus residues. (N45H+E78K were substituted together, as both reside in the stalk region.) Data represent mean±s.e.m. diameter of ~10 randomly selected plaques. Dashed line indicates mean plaque diameter of wild-type NA^BR/59. (b) Enzyme activity of total NA^BR07 and NA^BR07-H275Y variant proteins with five different amino acid substitutions, expressed by equal quantities (0.5 μg) of the respective plasmids. Dotted and dashed lines indicate mean activity of NA^BR/59and NA^BR/59-H275Y, respectively. (c) Representative western blotting showing total accumulation of protein variants with substitutions; the variants were expressed by equal quantities (0.5 μg) of the respective plasmids. Black and grey indicate wt and H275Y-mutant NA, respectively. Panels show detection by anti-HA-tag (upper), anti-β-actin (centre) and anti-GFP (lower). (d) NA Km values of rgBR/59/07 viruses carrying the respective NA amino acid substitutions in the absence and presence of the H275Y mutation. Dotted and dashed lines indicate the values of NA^BR/59and NA^BR/59-H275Y, respectively. All graphs represent mean±s.e.m. of three to five times independent experiments. *P<0.05, two-tailed t-test, versus NA^BR/59; ^†P<0.05, two-tailed t-test, versus NA^BR/59-H275Y.