title: Application of bioinformatics-coupled experimental analysis reveals a new transport-competent nuclear localization signal in the nucleoprotein of influenza A virus strain.
abstract: BACKGROUND
  Two nuclear localization sequences (NLS) in influenza A virus nucleoprotein (NP) have been demonstrated to be critical for nuclear import of NP and viral ribonucleoprotein complexes. However, a deletion mutant lacking these two signals was still able to localize to the nucleus suggesting the presence of yet another (a third) potential NLS in the NP protein. In order to identify the nature of this potential NLS signal in the NP of a WS/33L influenza virus A strain, we utilized the tools of bioinformatics coupled with functional experimental analyses in the present study.
  
  
  RESULTS
  Comparison of the deduced aa sequence of NP of WS/33L strain with the published WS/33 NP sequences revealed that a single amino acid (aa) change (Met to Arg) at position 105 results in converting the flanking regions (between aa position 90-121, a 32-residue stretch) into two classical overlapping bipartite NLS (obpNLS). GenBank search revealed that 9 out of 500 published NP sequences contain a similar Arg at position 105 (instead of Met) with a 100% homology to the obpNLS region. Various NP-green fluorescent protein (GFP) fusion constructs with and without the signal (obpNLS-Arg105) were utilized to understand the functional nature of this signal. We analyzed the transport competency of the expressed chimeric proteins in terms of their cellular localization by confocal immunofluorescence assay. Our analysis revealed that all NP-GFP constructs containing the wild-type (R105) sequence localized predominantly to the nucleus. Constructs lacking the obpNLS or constructs with reverse mutation (R105 to M105) on the other hand exhibited predominant cytoplasmic localization pattern. Interestingly, when the 32 aa obpNLS was fused with an unrelated viral protein (rotavirus NSP6) that has been known to be cytoplasmic protein, the chimeric protein (obpNLS-NSP6) was efficiently transported into the nucleus, indicating an efficient nuclear transport function of the 32-residue obpNLS in the NP of WS/33L strain of influenza A virus.
  
  
  CONCLUSIONS
  This report while not only establishing a new NLS in the influenza A virus strain, it also reinforces the idea that proper application of bioinformatics-coupled experimental analysis serves as a powerful tool in identifying new functional signals in proteins of interest.
authors.affiliation: Section of Cell Biology, Laboratory of Cellular Hematology, Division of Hematology, Office of Blood Research and Review, Center for Biologics Evaluation and Research (F,D,A,) Bethesda, MD 20892, USA. krishna.ketha@fda.hhs.gov
authors.forename: Krishna Mohan V
authors.initials: KM
authors.lastname: Ketha
authors.forename: Chintamani D
authors.initials: CD
authors.lastname: Atreya
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism* / Q000821:virology; D019295:Computational Biology; D003593:Cytoplasm / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D009980:Influenza A virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism*; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction; D014758:Viral Core Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: C048842:NP protein, Influenza A virus; D019913:Nuclear Localization Signals; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; D049452:Green Fluorescent Proteins