The H7N1 NS1 protein stimulates Src activity through its SH3-binding domain.
a. In vitro Src kinase reactions were carried out in the absence or presence of the Src peptide (white bar) or increasing amounts of F3 (light grey bars), F6Δ221–230 (black bars) or human RIL proteins (dark grey bars) as substrates. Activity is expressed as relative to the reaction with the Src peptide substrate (100%). Values are the means of duplicate experiments. Error bars represent ±SD. b. Dose-dependent increase of the affinity of Src for its peptide substrate as a function of NS1 concentrations. Apparent peptide affinity values determined in the absence (Kmpep) or in the presence (KmpepNS1) of NS1, were used to calculate the relative affinity increase (1-Kmpep/KmpepNS1). Values are the means of calculations from three independent determinations of the affinity constants. Error bars are ±SD. c. Stimulation of Src activity, expressed as the increase in the ratio of enzyme velocity in the presence (vRIL) and in the absence (v) of increasing amounts of RIL alone (circles) or in the presence of fixed doses of F4Δ225–230 (triangles), or F6Δ221–230 (squares) NS1 proteins. Values are the means of three independent replicates. Error bars are ±SD. d. Sequence alignment of the SHB domains 1 and 2 of different influenza viruses. Conserved residues with respect to the consensus are shaded in grey, non-synonimous substitutions in conserved residues are in boxed in black. e. Stimulation of Src catalytic efficiency (Vm/Kmpep) by H7N1 F3 NS1, H5N1 NS1, or the H3N2 2007 epidemic human isolates VR9220 and VR9240 NS1. Each bar represents the mean value of three independent determinations of the Vm/Kmpep. Error bars are ± S.D. f. In vitro Abl kinase reactions were performed in the presence of the specific Abl peptide substrate and in the absence (white bars) or in the presence of increasing amounts of F3 (grey bars), H5N1 NS1 (black bars) or human influenza H3N2 NS1 (light rey bars) proteins. Experiments were in triplicate. Error bars represent ±SD.