Nonstructural Protein 1 of Influenza A (NS1A) Demonstrates Strain-Specific dsRNA Binding Capabilities

Figure 1. Dimeric NS1A. N-terminal RBD in green, LR in salmon, and ED in light blue with the key W187 residue shown. The unresolved disordered ED C-terminal tail shown as a blue dashed line. Second monomer unit set transparent for clarity and regions of interest are labeled. PDB# 4OPH.

Figure 2. Binding affinity of Brevig Mission RBD for dsRNA. (A, B) Paired fluorescence polarization data for binding affinity of His-GST-RBD (Kd = 125 ± 9 nM) and fEMSA assay showing increased dsRNA binding with increasing His-GST-RBD concentration (Kd = 180 ± 42 nM). Data was plotted in OriginPro 2021b and fit to a logistic curve to obtain binding affinity. (C, D) Paired fluorescence polarization data for binding affinity of His-RBD (Kd = 83 ± 11 nM and 2160 ± 460 nM) and fEMSA assay showing increased dsRNA binding with increasing His-RBD concentration (Kd = 55 ± 16 nM). Data was plotted in OriginPro 2021b and fit to a biphasic (FP) or logistic (fEMSA) curve to obtain binding affinity. Leftmost lane is RNA only. Free dsRNA is depicted by blue helix on the right, with RBD dimer-dsRNA complexes shifted above, where C1 and C2 are distinct complexes while C3′ presents as a streak, indicating less stable complex formation. Data shown are representative of three independent experiments while values are presented as averages with standard deviations. fEMSA fitting data shown in Figure S28.

Figure 3. Multiple sequence alignment of commonly studied NS1A strains. Sequences were obtained from NCBI and aligned using BioEdit. RBD region highlighted in green, linker region highlighted in pink, ED highlighted in blue. Alpha helices indicated by red tubes; beta sheets indicated by blue arrows. Nonconserved residues highlighted in red. Residues selected for mutation for strain comparison are indicated by purple circles, while “control” mutants are indicated by yellow circles. The commonly deleted C-terminal tail residues are boxed in black.

Figure 4. (A) Crystal structure of RBD monomer highlighting mutated residues. (B) Comparison of dsRNA binding affinity of RBD mutants as compared with the His-GST-RBD WT; analyzed via two-tailed student-t test. Error bars represent SD of triplicate runs. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Figure 5. (A) Crystal structure of ED monomer highlighting mutated residues. (B) Comparison of dsRNA binding affinity of ED constructs as compared with the His-GST-ED WT; analyzed via two-tailed student-t test. Error bars represent SD of triplicate runs. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.