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-alpha/-beta 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 (NPTAIL). 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-alpha identified as partners. All interactions between NP(TAILs)and importins-alpha have high association and dissociation rates and present a distinct and specific behaviour. D/NP(TAIL)interacts strongly with all importins-alpha while B/NP(TAIL)shows weak affinity for importins-alpha. A/NP(TAIL)and C/NP(TAIL)present preferential importin-alpha partners. Mutations in B/NP(TAIL)and D/NP(TAIL)show a loss of importin-alpha binding, confirming key NLS residues. Taken together, our results provide essential highlights of this complex translocation mechanism.