Targeting influenza virus hemagglutinin to Xcr1+ dendritic cells in the absence of receptor-mediated endocytosis enhances protective antibody responses

DNA polymerase (pol ) requires nuclear localization to fulfil its DNA repair function. Although its small size has been interpreted to imply the absence of a need for active nuclear import, sequence and structural analysis suggests that a monopartite nuclear localization signal (NLS) may reside in the Nterminal lyase domain. Binding of this domain to Importin 1 (Imp1) was confirmed by gel filtration and NMR studies. Affinity was quantified by fluorescence polarization analysis of a fluorescein-tagged peptide corresponding to pol residues 2–13. These studies indicate high affinity binding, characterized by a low micromolar Kd, that is selective for the murine Importin 1 (mImp1) minor site, with the Kd strengthening to ∼140 nM for the full lyase domain (residues 2–87). A further reduction in Kd obtains in binding studies with human Importin 5 (hImp5), which in some cases has been demonstrated to bind small domains connected to the NLS. The role of this NLS was confirmed by fluorescent imaging of wild-type and NLS-mutated pol (R4S,K5S) in mouse embryonic fibroblasts lacking endogenous pol . Together these data demonstrate that pol contains a specific NLS sequence in the N-terminal lyase domain that promotes transport of the protein independent of its interaction partners. Active nuclear uptake allows development of a nuclear/cytosolic concentration gradient against a background of passive diffusion.