Role of N-terminus of Nucleoprotein in Influenza RNA expression

The influenza viral ribonucleoprotein complexes (vRNPs) are responsible for viral RNA synthesis. Each vRNP is comprised of one vRNA segment, the viral RNA dependent RNA polymerase complex (RdRP), and multiple copies of nucleoprotein (NP). NP serves as scaffold in formation of vRNPs, but also regulates vRNP activity. The N-terminus of NP contains a nonconventional nuclear localization signal (NLS1) essential for initial vRNP nuclear import, but also interacts with host RNA helicases to enhance viral RNA replication in the nucleus. NP contains at least one additional NLS sequence, with bioinformatics revealing a third NLS in some NP proteins. To examine the role of the N-terminus of NP aside from its vRNP nuclear localization activity, we constructed N-terminal 20 amino acid deletion mutants with or without the addition of the conventional NLS from SV-40 T-antigen, termed del20NLS-NP and del20-NP. In the context of reconstituted vRNPs, both exhibit nuclear localization, consistent with NLS1 being utilized for vRNP localization but not NP localization and vRNP formation in the nucleus. Furthermore, both demonstrate decreased vRNP RNA synthesis activity, exacerbated as the vRNA template is lengthened, consistent with a lack of interaction with host RNA helicases. Interestingly, del20-NP vRNP activity is less severe than del20NLS-NP, suggesting perturbations of the N-terminus disrupt vRNP activity. Our results support the N-terminal region of NP as a flexible interaction domain important not only for initial vRNP nuclear localization at the start of infection, but also efficient viral gene expression during virus replication and support pursuing NP as an antiviral target