Scientific Publications by FDA Staff
BMC Cell Biol 2008 Apr 28;9(1):22
Application of Bioinformatics-Coupled Experimental Analysis reveals a new Transport-Competent Nuclear Localization Signal in the Nucleoprotein of an Influenza A Virus strain.
Ketha KM, Atreya CD
Ketha, KMV (reprint author), US FDA, Ctr Biol Evaluat & Res, Div Hematol,Off Blood Res & Review, Sect Cell Biol,Lab Cellular Hematol, Bethesda, MD 20892 USA US FDA, Ctr Biol Evaluat & Res, Div Hematol,Off Blood Res & Review, Sect Cell Biol,Lab Cellular Hematol, Bethesda, MD 20892 USA
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-Arg 105) 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, suggesting the nuclear transport function of the 32-residue obpNSL in the NP of WS/33L strain of influenza virus A. CONCLUSION: This report while not only establishing a new NLS in the influenza virus A 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.
|Category: Journal Article|
|PubMed ID: #18442378|
|PubMed Central ID: #PMC2386121|
|Includes FDA Authors from Scientific Area(s): Biologics|
|Entry Created: 2011-10-04||Entry Last Modified: 2012-08-29|