Figure 2: NA protein phenotypes of seasonal H1N1 influenza viruses of the NC99 and BR07 lineages.

(a) Neuraminidase enzyme activity of total NA proteins expressed by equal quantities (0.5 μg) of the respective plasmids. Rep, representative NA protein of the indicated lineage; Int, intermediate NA protein of the indicated lineage, fully or partially conforming to the ten consensus lineage residues. The residue at the NA protein 275 position (wild-type (wt) or H275Y) is indicated in parentheses. NA-H275Y is shown when the H275Y was introduced by mutagenesis. Data were normalized to NA^BR07 protein. Dotted and dashed lines indicate the activity level of lineage-representative NA^GA/17 and NA^BR/59 proteins, respectively. (b) Representative western blotting showing total accumulation of NA protein variants expressed by equal quantities (0.5 μg) of the respective plasmids. Black and grey indicate wt and H275Y-mutant NA proteins, respectively. Panels show detection by anti-HA-tag (upper), anti-β-actin (middle) and anti-GFP (lower). (c) Representative flow cytometric plots of cell surface accumulation of NA protein variants expressed by equal quantities (0.5 μg) of respective plasmids. The pairs differed only at position 275, with the exception of the last panel, the BR07-like NAs. (d) Km values of the NA proteins. The dotted and dashed lines indicate the value of lineage-representative NA^GA/17 and NA^BR/59, respectively. All graphs show mean±s.e.m. from 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 its counterpart wt NA at left side.