Differential Behaviours and Preferential Bindings of Influenza Nucleoproteins on Importins-α

Influenza viruses are negative single-stranded RNA viruses with nuclear transcription and replication. They enter the nucleus by using the cellular importin-α/-β nuclear import machinery. Influenza nucleoproteins from influenza A, B, C and D viruses possess a nuclear localization signal (NLS) localized on an intrinsically disordered extremity (NP_TAIL). In this paper, using size exclusion chromatography (SEC), SEC-multi-angle laser light scattering (SEC-MALLS) analysis, surface plasmon resonance (SPR) and fluorescence anisotropy, we provide the first comparative study designed to dissect the interaction between the four NP_TAILs and four importins-α identified as partners. All interactions between NP_TAILs and importins-α have high association and dissociation rates and present a distinct and specific behaviour. D/NP_TAIL interacts strongly with all importins-α while B/NP_TAIL shows weak affinity for importins-α. A/NP_TAIL and C/NP_TAIL present preferential importin-α partners. Mutations in B/NP_TAIL and D/NP_TAIL show a loss of importin-α binding, confirming key NLS residues. Taken together, our results provide essential highlights of this complex translocation mechanism.