Nucleoprotein phosphorylation site(Y385) mutation confers temperature sensitivity to influenza A virus due to impaired nucleoprotein oligomerization at a lower temperature

Mutations in viral proteins can lead to the cold adaption of influenza A virus and the cold-adapted virus is an important vaccination instrument. Here, we identify a novel strain of influenza A virus with cold sensitivity conferred by a mutation at a phosphorylation site within the nucleoprotein(NP). The highly conserved tyrosine 385 residue(Y385) of NP was identified as a phosphorylation site by mass spectrometry. The constructive NP phosphorylation mimicked by Y385 E mutation was fatal for virus replication, while the continuous Y385 dephosphorylation mimicked by Y385 F mutation had little impact on virus replication in vitro. Notably, the Y385 F virus showed much lower replicative capacity in turbinates of mice compared with the wild type virus. Moreover, the replication of Y385 F virus was significantly reduced in both A549 and MDCK cells grown at 33°C, when compared to that at 37°C. These results indicated that the Y385 F mutation led to cold sensitivity of virus. We further found that the cold sensitivity of Y385 F virus could be attributed to diminished NP oligomerization rather than any changes in intracellular localization. Taken together, these findings suggest that the phosphorylation of NP may be a critical factor that regulates the temperature sensitivity of influenza A virus.