Influenza A virus is a negative-stranded RNA virus with 8 genomic segments coding for at least 11 proteins . Gene segment 5 encodes the 498-amino acids (aa) long nucleoprotein (NP) that has been shown to be a multifunctional protein with critical roles during various stages of the viral life cycle. The viral RNA tightly bind to NP and polymerase subunit proteins, PB1, PB2 and PA, resulting in the formation of viral ribonucleoproteins (vRNPs) a prerequisite for successful transcription and replication [1–5]. The primary role of NP as an RNA-binding protein and thus its role as a structural protein contributing towards the formation of the RNP complex within the virion is clearly evident. However, it is the series of events following infection that delineates the major function of NP, which mainly constitute importing of the vRNP complex into nucleus, then exporting it back to cytoplasm, and finally preventing their reentry into nucleus [1–3, 6–10]. Interaction of NP with several other viral and host proteins is critical for this nuclear import and export [1, 11–13].
Nuclear import of NP is regulated by two nuclear localization signals (NLS), a non-conventional NLS (nNLS) and a classical NLS (cNLS) present within the NP [3, 8, 10, 14, 15]. A nuclear import signal in a protein is characterized by one (monopartite) or two (bipartite) short stretches of basic amino acids [16–18]. The monopartite signals comprise of two types: 4 residue pattern (called "pat4") or the 7 residue pattern (called "pat7"). Pat4 NLS comprises of either a stretch of 4 basic amino acids (K or R) or 3 basic amino acids (K or R) with the fourth aa being either H or P. Pat7 is a pattern starting with P and followed within 3 residues by a basic segment containing 3 K/R residues out of 4. Thus, based on the above classification the nNLS of NP (3S xGTKRSY xxM13, important residues highlighted in bold) is aptly termed non-conventional NLS as it does not fall under both pat4 or pat7 type of signals [13, 14]. The second NLS of NP (cNLS) on the other hand though, has been classified as a bipartite signal (198KR GINDRNFWRGENGRK TR216, important residues highlighted in bold) and is located near the middle of the polypeptide [15–18]. Deletion and mutation analysis of both the nNLS and cNLS reveal that these signals are essential for nuclear import, viral mRNA synthesis, vRNA transcription, replication and nucleolar accumulation [3, 7, 8, 10, 13–15]. Interestingly, a mutant NP lacking both of these NLSs was still transported to the nucleus suggesting the existence of at least one additional NLS between the cNLS and C-terminus region of NP [8, 14].
We report here a third novel overlapping bipartite NLS (obpNLS) in the NP of WS/33L strain located between the nNLS and cNLS regions identified by bioinformatic analyses. Our analysis revealed a single amino acid change (M to R) at position 105 resulted in converting a 32-aa stretch into two obpNLS (90KK TGGPIYRRVDGK WRR106 and 105RR ELILYDKEEIRR IWR121, residues constituting bpNLS are in bold). Using full-length and deletion constructs of NP in fusion with GFP, we present experimental evidence that the obpNLS is indeed a stand alone functional transport signal and supports efficient translocation of the chimeric protein to the nucleus. Furthermore, comparative analysis of 500 NP sequences revealed that 9 influenza virus strains contain NP with similar aa change (R instead of M) at position 105 with 100% homology to the obpNLS region, suggesting the authenticity of the identified amino acid change as a natural variant.