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. 2007 Jun 4:4:49.
doi: 10.1186/1743-422X-4-49.

Nuclear import of influenza A viral ribonucleoprotein complexes is mediated by two nuclear localization sequences on viral nucleoprotein

Winco W H Wu  1 , Ying-Hua B Sun, Nelly Panté
Affiliations

Nuclear import of influenza A viral ribonucleoprotein complexes is mediated by two nuclear localization sequences on viral nucleoprotein

Winco W H Wu et al. Virol J. .

Abstract

Background: 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.

Results: 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.

Conclusion: Our results demonstrate that both NLS1 and NLS2 on NP can mediate the nuclear uptake of influenza A vRNPs.

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Figures

Figure 1
Figure 1
Fractionation profile of the purification of vRNP complexes from influenza A by glycerol gradient centrifugation. Fractions were collected from the top (lane 1) to the bottom (lane 16) of a glycerol gradient and analyzed via reducing SDS-PAGE containing 10% polyacrylamide. The gel was stained with Coomassie blue. The arrows indicate the mobility of the influenza NP and M1 proteins. The positions of the molecular weight standards (in kDa) are indicated to the left.
Figure 2
Figure 2
Biotinylation of influenza vRNPs. (A) Northern blot of biotinylated influenza vRNA in the vRNP complexes. The biotinylated vRNPs were subjected through urea gel electrophoresis, transferred onto nitrocellulose, and detected by blotting with streptavidin alkaline phosphatase. Shown are the results of the same blot exposed at two different times. As a control, biotin maleimide only was run on the gel. Urea gel electrophoresis of the same biotinylated influenza vRNA shown in A. (B) The vRNA within the vRNP complex was visualized directly by staining the gels with SYBR Safe. The arrows denote the positions of the influenza vRNA. Two different concentrations of the vRNA are shown. The positions of various sizes of RNA molecular standards (in bp) are shown on the left. (C) Electron microscopy visualization of specific binding of streptavidin-gold (10-nm diameter) to biotinylated influenza vRNPs. Gold particles exclusively associated with one end of the vRNPs.
Figure 3
Figure 3
Nuclear import of fluorescein-labeled influenza vRNPs. Fluorescein-labeled influenza vRNPs are competent for nuclear import. Nuclear import assays were carried out in digitonin-treated HeLa cells, and cells were visualized by confocal microscopy. Representative images of three independent experiments are shown. (A) Control experiment with a 70 kDa dextran fluorescently-labeled with Texas Red to verify that the plasma membrane, but not the nuclear envelope, is permeabilized by digitonin. (B) Cy3-labeled BSA carrying a classical NLS (Cy3-NLS-BSA) and fluorescein-labeled influenza vRNP complexes (fluorescein-vRNP) were assayed in the digitonin permeabilized HeLa cells. Nuclear import assays were carried out in import buffer alone (- energy - cytosol) or in the presence of exogenous cytosol and an energy-regenerating system (+ energy + cytosol). (C) Bar diagram of the ratio of nuclear-to-cytoplasmic fluorescence for the experimental conditions shown in A and B. Each bar graph shows the mean value and standard error from 100–110 individual cells.
Figure 4
Figure 4
Competition of influenza vRNP nuclear import with peptides against NP NLSs. Peptides carrying the NLSs of influenza NP compete for nuclear import of influenza vRNPs. (A) Fluorescein-labeled influenza vRNPs were assayed in digitonin-permeabilized HeLa cells in the presence of cytosol, an energy-regenerating system, and the absence or presence of different peptides. Cells were visualized by confocal microscopy, and representative images of three independent experiments are shown. (B) Bar diagram of the ratio of nuclear-to-cytoplasmic fluorescence for the experimental conditions shown in A. Each bar graph shows the mean value and standard error from 100–110 individual cells.
Figure 5
Figure 5
Inhibition of influenza vRNP nuclear import with antibodies against NP NLSs. Antibodies against the NLSs of influenza NP inhibit the nuclear import of influenza vRNPs. (A)Fluorescein-labeled influenza vRNPs were assayed in the digitonin-permeabilized HeLa cells in the presence of cytosol, an energy-regenerating system, and the absence or presence of different antibodies. Cells were visualized by confocal microscopy, and representative images of three independent experiments are shown. (B) Bar diagram of the ratio of nuclear-to-cytoplasmic fluorescence for the experimental conditions shown in A. Each bar graph shows the mean value and standard error from 100–110 individual cells.
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