['The influenza A virus replicates in the nucleus of its host cell. Thus, entry of the influenza genome into the cell nucleus is necessary for establishing infection. The genome of the influenza A virus consists of eight single-stranded, negative-sense RNA molecules, individually packed with several copies of the viral nucleoprotein (NP) into ribonucleoprotein particles (vRNPs). These vRNPs are large, rod-shaped complexes containing a core of NP, around which the RNA is helically wrapped. The vRNPs are the entities that enter the nucleus, and their nuclear import must be mediated by nuclear localization sequences (NLSs) exposed on the vRNPs. NP contains at least two putative NLSs, one at the N-terminus (NLS1) and one in the middle (NLS2) of the protein. These NP NLSs have been shown to mediate the nuclear import of recombinant NP molecules. However, it remains to be determined which NLS mediates the nuclear import of influenza vRNP complexes.', 'To directly track the nuclear import of the influenza A genome, we developed an experimental assay based on digitonin-permeabilized cells and fluorescently-labeled vRNPs isolated from the influenza A virus. We used this assay to determine the contribution of the two proposed NLSs on NP to the nuclear import of influenza vRNP complexes. Peptides that mimic each of the two NLSs on NP were used to compete with vRNPs for their nuclear import receptors. In addition, antibodies against the two NP NLSs were used to block the NLSs on the vRNP complexes, and thereby inhibit vRNP nuclear import. Both peptide competition and antibody inhibition of either sequence resulted in decreased nuclear accumulation of vRNPs. The two sequences act independently of each other, as inhibition of only one of the two NLSs still resulted in significant, though diminished, nuclear import of vRNPs. Furthermore, when both sequences were blocked, vRNP nuclear import was almost completely inhibited. Antibody inhibition studies further showed that NLS1 on NP is the main contributor to the nuclear import of vRNPs.'] Is nuclear import of influenza A viral ribonucleoprotein complexes mediated by two nuclear localization sequences on viral nucleoprotein?