Fig 5.

HDX-MS characterization of NP protein from whole IAV after transient exposure to acidic conditions. (A) H/D exchange percentages were measured for NP protein from whole IAV samples held at neutral conditions (pH 7) for 30 s. Samples were then incubated in D₂O for 30 s, quenched, and digested prior to HDX-MS analysis. Data points indicate the mean percentage of H/D exchange ± SD for each peptide plotted using its central amino acid (n = 3, Uniprot: P03466). Continuous lines indicate regions of continuous peptide coverage by MS. Protein domains of interest are indicated by gray shading and white lines; nNLS, nonconventional-conventional nuclear localization signal; RBL, RNA binding loop; cNLS, conventional nuclear localization signal; NAS, nuclear accumulation signal; TL, tail loop. (B) Whole-virus samples were held at neutral pH or adjusted to pH 4, and then neutralized back to pH 7 after 10 or 30 s prior to processing for HDX-MS. Data presented as residual plots showing the difference in H/D exchange percentages induced by exposure to pH 4 when compared to corresponding pH 7 control samples. Individual data points indicate the mean percentage of H/D exchange difference for triplicate samples. (C) Crystal structure of A/WSN/33 NP (PDB: 2IQH), showing regions of coverage by HDX-MS. White indicates regions of no peptide coverage by HDX-MS. Head and body domains, along with RNA binding loop and tail loop of NP, are annotated based on Ye et al. (54).