Fig. 4: PB1-K578 is ubiquitinated and interacts with a loop in the PB2 N-terminus.

a A549 cells were co-transfected with strep-tagged PB1 or PB1-K578A and HA-tagged UB. UB-modified PB1 subunits were strep-purified using denaturing conditions and analyzed by western blot. For de-ubiquitination, bound PB1 constructs were treated with USP2. Co-precipitated UB-HA levels were quantified and presented as the mean fold change of WT (±SEM), n = 4 independent biological replicates. P values compared to WT from Welch’s corrected two-tailed t-test are indicated. b, c Illustration of PB1-K578 in the 3D structures of the WSN polymerase (vRNA-bound conformation; b) and the 3D structure of the symmetric dimer of the H3N2 polymerase (PDB: 6QNW; c), showing the K578 containing PB1-helix (cyan), the flexible loop in the PB2 N-terminus (yellow) harboring PB2-E72 and the helix in the PA C-terminus that participates in dimer formation. Distances between PB1-K578 and PB2-E72 are indicated in Angstrom (Å). Amino acids involved in the dimer interface are indicated. Created with ChimeraX. d Mean lifetime of ionic interactions with PB2-E72 for 100 ns simulations, n = 4 independent biological replicates for each condition (±SEM). P values < 0.05 compared to WT from Welch’s corrected two-tailed t-test are indicated. e, f RMSD distribution of PB2-E72 (e) and PB2-Q73 (f) for WT, K578R and K578A simulations. Each 100 ns simulation generated 1000 RMSD values leading to a total number of 4000 values for each condition. Equal bin sizes with 0.5 Å steps were used for of each histogram. g RMSF values of PB2 residues (±SEM). Significance of mean differences was analyzed by Welch corrected two-sided t-test, n = 4 independent experiments. P values are summarized in Supplementary Table 2. Source data are provided as a Source data file.