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.

Conclusion: 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.

Figure 1

Amino acid (aa) comparison of WS/33 NP sequences. Sequence generated from WS/33L in the present study is compared with 5 other WS/33 sequences represented as WS-A [includes sequences EMBL:Q1K9H2, EMBL:AAG41965 and EMBL:ABF21292] and WS-B [includes sequences GenBank:AAA43452 and GenBank:P15682]. NP sequence of the 1918 pandemic flu strain [GenBank: ] is also included for comparison. Identical amino acids are represented by "-"; Phylogenetically important regions (PIR) are underlined; phylogenetically important positions (PIP) are indicated by an asterisk.

Figure 2

Schematic representation of various domains of the NP. Numbers indicate amino acid positions. nNLS: non-conventional nuclear localization signal; cNLS: classical nuclear localization signal; NAS/CRS: nuclear accumulation signal/cytoplasmic retention signal; obpNLS: overlapping bipartite nuclear localization signal; bpNLS: bipartite nuclear localization signal. Critical amino acids contributing to a bpNLS motif are represented by two basic aa-rich domains (in bold) and the spacer 10 aa are underlined. Also note that the cNLS contains a slightly longer (12 aa) spacer region and thereby does not strictly qualify as a classical bpNLS by the PSORT program.

Figure 3

Schematic representation of NP-full-length and deletion constructs. Various NP-FL and deletion clones were made between the nNLS and cNLS regions. GFP is represented by chequered-box; NLS regions are indicated by darkly-shaded box; nuclear accumulation signal (NAS) is indicated by striped-box; rotavirus NSP6 is represented by an empty, unshaded box. Critical amino acid (R or M) at position 105 is indicated by larger font size and an asterisk. Cellular localization of each construct is indicated by N (nuclear), N>C (nuclear greater than cytoplasm), C>N (cytoplasm greater than nuclear), and C (cytoplasmic).

Figure 4

Immunoblot analysis of NP-GFP constructs. All NP-GFP plasmid constructs and the empty vector C2-GFP (control) were transfected into COS-7L cells and cell lysates were analyzed to confirm expression of the fusion proteins. Molecular weight markers (in kDa) are indicated on the left. The caspase-cleaved N-terminal fragment of NP-L is indicated by an asterisk (*).

Figure 5

Confocal immunofluorescence analysis of various NP constructs containing the obpNLS. COS-7L cells transfected with the various NP-GFP constructs were fixed and mounted with propidium iodide-containing medium to indicate nuclear staining. NP-L exhibits a typical punctuate, nuclear localization pattern. obpNLS+NC, obpNLS+N-ter and obpNLS+C-ter all localize to the nucleus.

Figure 6

Expression analysis of bpNLS-1 and bpNLS-2. bpNLS-1R and bpNLS-2R (with R¹⁰⁵) localizes predominantly to the nucleus. Note that the bpNLS-1M (with M¹⁰⁵) exhibits a predominantly cytoplasmic localization pattern but also localizes to the nucleus as well. Arrows indicate a dividing cell with two daughter nuclei and the bpNLS-1M demonstrating an exclusive cytoplasmic localization. bpNLS-2M (with M¹⁰⁵) localizes to the cytoplasm.

Figure 7

Expression analysis of construct lacking obpNLS and construct with obpNLS in fusion with a known cytoplasmic protein. NPΔ obpNLS, transfected in COS-7L cells, exhibits a characteristic cytoplasmic localization pattern. C2-GFP, used as a vector control, localizes to the cytoplasm. Rotavirus non structural protein 6 (NSP6) fused with GFP (GFP-NSP6) exhibits a typical punctate, cytoplasmic localization pattern. However, note that the obpNLS+NSP6 (obpNLS fused to the N-terminus of NSP6) exhibit an exclusive nuclear localization pattern. Bar represents 20 μm.