FIG 3.

Impact of naturally occurring NS1 variants on p85β binding. (A) Crystal structure of the NS1 effector domain (silver) highlighting naturally occurring variants among the 20 p85β contact residues (PDB accession number 3L4Q). Each residue is labeled, and the pie charts indicate the prevalence of various amino acids at each position among ∼24,000 IAV NS1 sequences obtained from the NIAID Influenza Research Database. (B) (Top) Western blot analysis of anti-V5 immunoprecipitates from lysates of 293T cells expressing V5-GST or various V5-NS1 single amino acid mutants together with FLAG-p85β. Proteins of interest were detected using specific antibodies. Input samples were similarly processed. Band intensity was quantified using a Li-Cor Odyssey scanner. (Bottom) The mean numbers of relative units from three independent experiments are plotted. Error bars represent standard deviations (SDs). Statistical significance was determined by the Student t test (*, P < 0.004545).