The extended C-terminal region of influenza C nucleoprotein is important for nuclear import and RNP activity.

The influenza C virus (ICV) is a human-pathogenic agent, and the infections are frequently identified in children. Compared to influenza A and B viruses, the nucleoprotein of ICV (NPC) has an extended C-terminal region of which the functional significance is ill defined. We observed that the nuclear localization signals (NLSs) found on the nucleoproteins of influenza A and B virus subtypes are absent at corresponding positions on ICV. Instead, we found that a long bipartite nuclear localization signal resides at the extended C-terminal region, spanning from R513 to K549. Our experimental data determined that the KKMK motif within this region plays important roles in both nuclear import and polymerase activity. Similar to the influenza A viruses, NPC also binds to multiple human importin alpha isoforms. Taken together, our results enhance the understanding of the virus-host interaction of the influenza C virus. IMPORTANCE As a member of the Orthomyxoviridae family, the polymerase complex of the influenza C virus structurally resembles its influenza A and influenza B virus counterparts, but the nucleoprotein differs by possessing an extra C-terminal region. We have characterized this region in view of nuclear import and interaction with the importin alpha protein family. Our results demonstrate the functional significance of a previously uncharacterized region on Orthomyxoviridae nucleoprotein (NP). Based on this work, we propose that importin alpha binding to influenza C virus NP is regulated by a long bipartite nuclear localization signal. Since the sequence of influenza D virus NP shares high homology to that of the influenza C virus, this work will also shed light on how influenza D virus NP functions.