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Nuclear import of influenza A viral ribonucleoprotein complexes is mediated by two nuclear localization… Wu, Winco W; Sun, Ying-Hua B; Panté, Nelly Jun 4, 2007

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ralssBioMed CentVirology JournalOpen AcceResearchNuclear import of influenza A viral ribonucleoprotein complexes is mediated by two nuclear localization sequences on viral nucleoproteinWinco WH Wu, Ying-Hua B Sun and Nelly Panté*Address: Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, BC, V6T 1Z4, CanadaEmail: Winco WH Wu - winco@zoology.ubc.ca; Ying-Hua B Sun - yhbettysun@gmail.com; Nelly Panté* - pante@zoology.ubc.ca* Corresponding author    AbstractBackground: The influenza A virus replicates in the nucleus of its host cell. Thus, entry of theinfluenza genome into the cell nucleus is necessary for establishing infection. The genome of theinfluenza A virus consists of eight single-stranded, negative-sense RNA molecules, individuallypacked 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 theRNA is helically wrapped. The vRNPs are the entities that enter the nucleus, and their nuclearimport must be mediated by nuclear localization sequences (NLSs) exposed on the vRNPs. NPcontains 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 recombinantNP molecules. However, it remains to be determined which NLS mediates the nuclear import ofinfluenza vRNP complexes.Results: To directly track the nuclear import of the influenza A genome, we developed anexperimental assay based on digitonin-permeabilized cells and fluorescently-labeled vRNPs isolatedfrom the influenza A virus. We used this assay to determine the contribution of the two proposedNLSs on NP to the nuclear import of influenza vRNP complexes. Peptides that mimic each of thetwo 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, andthereby inhibit vRNP nuclear import. Both peptide competition and antibody inhibition of eithersequence resulted in decreased nuclear accumulation of vRNPs. The two sequences actindependently 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 showedthat 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 nuclearuptake of influenza A vRNPs.Published: 4 June 2007Virology Journal 2007, 4:49 doi:10.1186/1743-422X-4-49Received: 5 February 2007Accepted: 4 June 2007This article is available from: http://www.virologyj.com/content/4/1/49© 2007 Wu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 12(page number not for citation purposes)Virology Journal 2007, 4:49 http://www.virologyj.com/content/4/1/49BackgroundAs part of its replication cycle, the genome of the influ-enza A virus must enter the nucleus of its host cell. Theinfluenza A virus genome consists of eight single-stranded, negative-sense viral RNA (vRNA) molecules ofvarying sizes that are individually packed and stabilizedby multiple copies of nucleoprotein (NP; ~56 kDa) intoviral ribonucleoprotein (vRNP) complexes. NP forms acore around which the vRNA is helically wrapped [1].Approximately 24 nucleotides associate with each NP[2,3]. Thus, given that each vRNA is about 890–2,341nucleotides long (reviewed in [4]), each influenza vRNPhas 37–97 copies of NP. The crystal structure of oligo-meric NP has recently been solved and revealed a possibleRNA-binding groove made up of a large number of basicresidues [5]. In addition to NP, each vRNP also contains asingle copy of a trimeric RNA polymerase complex. In thevirus, these vRNPs are enclosed by the viral envelope, andare organized into a distinct pattern with seven vRNPs ina circle surrounding one vRNP at the center [6]. Duringcell entry, the influenza virion containing these incomingvRNP complexes is internalized into an endosomal com-partment by either clathrin- or caveolae-dependent mech-anisms [7,8]. The acidic environment of the endosomethen triggers the fusion of the viral envelope with theendosomal membrane to release the vRNPs into the cyto-plasm. The vRNPs are then transported in the cytoplasmby diffusion [9], and gain access to the nuclear importmachinery of the cell (nuclear pore complexes (NPCs)and soluble nuclear import receptors). After reaching theNPCs, the vRNPs are then imported into the nucleus in anenergy-dependent manner [9-11]. The nuclear import ofincoming vRNPs allows for subsequent genomic replica-tion; nuclear transcription and cytoplasmic synthesis ofnew viral proteins; nuclear import of newly-synthesizedNP and RNA polymerases; and nuclear assembly andexport of newly-synthesized vRNP complexes (reviewedin [12-15]).Two mechanisms for nuclear import exist [16,17]: Passivediffusion occurs for molecules less than 9 nm in diameter,or proteins smaller than 40 kDa. Facilitated translocation,on the other hand, can accommodate molecules up to 39nm in diameter [18]. This mechanism is highly selectiveand requires the energy from GTP hydrolysis by the smallGTPase Ran [19,20]. In addition, facilitated translocationrequires a signal residing on the imported molecule (orcargo), and soluble cytoplasmic receptors that recognizethe signal and carry the cargo through the NPC. A majorbreakthrough in the study of nuclear import has been therecent identification of several signals and transport recep-tors mediating different nuclear transport pathways. Thefirst-identified and best-studied nuclear import signal iszation sequences (NLSs) [21-23], and is now referred to asthe classical NLS or cNLS. The receptor for the cNLS con-sists of two proteins, importin α and importin β. The cNLSis recognized by importin α, which acts as an adapterbetween the cNLS-bearing protein and importin β, thesubunit which interacts directly with the NPC [24,25]. Inaddition, many other NLSs have been now identified(reviewed in [26]), and they bind different transportreceptors that belong to an increasing family of proteinsrelated to the importins.Since influenza vRNPs enter the nucleus through the NPCvia facilitated translocation [10], one or more exposedNLSs on the vRNPs must be responsible for mediatingtheir nuclear import. Although all four proteins of influ-enza vRNPs (NP and the three RNA polymerases) carryNLSs, NP is believed to be the protein responsible for thenuclear import of influenza vRNPs. This hypothesis isbased on experimental data that have demonstrated thatNP mediates the nuclear import of in vitro-assembled NP-RNA complexes [27,28]. NP contains at least two NLSs:one at the N-terminus of NP (residues 1–13; NLS1)[29,30], and one in the middle of NP (residues 198–216;NLS2) [31]. Both sequences function as NLSs when fusedto cytoplasmic proteins [30,31]. However deletion ofboth NLS1 and NLS2 still results in nuclear accumulationof NP, suggesting the presence of a third NLS [32], whoseprecise location is yet to be mapped. NLS1 has been stud-ied in some detail and it is known that it binds to impor-tin α1 and α5 [30], two of the six human homologues ofimportin αs currently identified (α1, α3, α4, α5, α6, α7)(reviewed in [33,34]). NLS2 is less well characterized, andwas originally proposed to be a classical bipartite NLS.However, the recently-solved crystal structure of NP sug-gests that the two clusters of basic amino acids in NLS2 aretoo close to be recognized by importin α as a bipartite NLS[5]. Because mutations of the second cluster of basicamino acids (positions 213, 214 and 216) of NLS2 arecritical to the nuclear import of recombinant NP [31], it ispossible that this region may act as a monopartite NLS.The specific importin α homologue that recognizes NLS2remains to be identified. However, since full length NPbinds to importin α1, α3, and α5 [35], any of thesehomologues may recognize NLS2.The contribution of NLS1 and NLS2 to the nuclear importof NP is controversial because they have been shown tomediate the nuclear import of recombinant NP in somestudies but not in others [27,29,31,32]. Because NP formssmall oligomers in equilibrium with monomers [5,36], itis unclear whether the NLSs on NP function during theoligomeric or the monomeric state of NP, and whetherthe different tags fused to recombinant NP in those stud-Page 2 of 12(page number not for citation purposes)characterized by one (monopartite) or two (bipartite)short stretches of basic amino acids, called nuclear locali-ies may have interfered with the NLSs, or with the oli-gomerization of NP and consequent exposure of the NLSs.Virology Journal 2007, 4:49 http://www.virologyj.com/content/4/1/49It is also unknown from these studies whether newly-syn-thesized NP enters the nucleus as oligomers or as mono-mers.While the contribution of the NLSs to the nuclear importof newly-synthesized NP is controversial, even less isknown about the specific NLS(s) that target incominginfluenza virus-derived vRNP complexes to the nucleus. Ithas recently been shown that the nuclear import of invitro-assembled NP-RNA complexes was mediated byNLS1 on NP [27]. However these studies used recom-binant NP with GFP fused to its C-terminus, which mayhave interfered with the assembly of NP because accord-ing to the crystal structure of NP, oligomerization requiresa tail loop close to the C-terminus (residues 402–428) [5].Thus these in vitro-assembled RNPs may differ structurallyfrom influenza-assembled vRNP complexes within mam-malian cells. More recently, a reverse genetics approach,essentially involving the co-transfection of recombinantRNA and NP under the control of non-influenza promot-ers, has also noted an important role of NLS1 in mediat-ing influenza RNA nuclear import [37]. Compared to invitro-assembled RNPs or recombinant RNA and NP, how-ever, it is possible that naturally-occurring, influenzavirus-derived vRNPs may not actually utilize NLS1, ormay also require NLS2, on NP for proper nuclear import.In this study, we address these key questions by studyingthe nuclear import of vRNP complexes isolated from nat-urally-occurring influenza A virions.ResultsPurification and biotinylation of influenza vRNP complexesTo study the nuclear import of the influenza genome itwas important to first establish a method to purify andfluorescently label the influenza vRNPs in their nativestate. To purify the influenza vRNPs we used the well-established protocol of Kemler et al. [38], which consistsof disrupting the influenza virions and then separating thereleased vRNPs from the other components of the virus byvelocity sedimentation on a glycerol gradient. To mimic asclosely as possible the conditions that influenza vRNPsundergo during cell infection after being internalized intoendosomes, the vRNPs were purified under acidic condi-tions (pH 5.5). Fig. 1 represents a typical distribution pro-file of the proteins in the fractions. As previously shown[38], this procedure yields fractions containing vRNPsdevoid of M1 and other influenza viral proteins, as judgedby the presence of only the NP protein by Coomassie bluestaining of the gel. Since M1 has been found to inhibitnuclear import of the vRNP complex [11,39], it wasimportant to pool fractions containing NP but not M1 (asindicated in Fig. 1). These fractions, containing the vRNPTo track the nuclear import of the influenza genome, thevRNA within the purified vRNP complex was biotinylatedin order to attach a streptavidin fluorophore. For this pur-pose, we used the 5' End Tag Nucleic Acid Labeling System(Vector Laboratories), which covalently attached a reac-tive thiophosphate group to the 5' end of the vRNA withinthe vRNP. This allowed for the attachment of biotin-male-imide (a thiol-specific biotin reagent), and the subsequenttagging of fluorescein-streptavidin to yield fluorescein-labeled vRNPs. To verify that the vRNPs were properlybiotinylated, their vRNAs were subjected to Northernblotting using streptavidin alkaline phosphatase. Asshown in Fig. 2A, the vRNA was successfully biotinylatedand ran near the top of the urea gel. Fig. 2B shows thesame sample, but using the SYBR Safe dye to directlydetect the vRNA on the urea gel. The stained vRNA(arrows), similar to the Northern blot, ran near the top ofthe gel. As the vRNA appeared to be larger than 4,000 bpand did not run as a smear or as distinct RNA bands, thisindicates that the vRNA was still associated to the NP oli-gomer as part of the vRNP complex, and not dissociatedfrom the protein components.To confirm that biotin was added at one end of the vRNPs,the biotinylated vRNPs were incubated with streptavidin-gold, and the gold-tagged vRNP complexes were nega-tively stained and visualized by transmission electronmicroscopy. As illustrated in Fig. 2C, the vRNPs had theexpected rod shape, and one end of the rod was labeledwith an electron-dense gold particle.Fluorescein-labeled influenza vRNPs are competent for nuclear importThe nuclear import of fluorescein-labeled influenzavRNPs was studied in the well-established nuclear importassay involving digitonin-permeabilized cells [40]. Digi-tonin permeabilizes the plasma membrane of cells, butretains the integrity of the nuclear envelope; digitonin-permeabilized cells therefore have intact import-compe-tent nuclei, but are depleted of cytosolic nuclear importreceptors. To ensure that the nuclear import assay wasworking properly, a series of controls were performed.First, a fluorescently-labeled dextran with a size thatexceeds the diffusion limit of the NPC was used to controlfor nuclear envelope integrity. As shown in Fig. 3A, the 70kDa dextran labeled with Texas Red entered the plasmamembrane but was almost totally excluded from thenucleus (indicated by the presence of fluorescence signalin the cytoplasm but not in the nucleus). This indicatesthat the plasma membrane was permeabilized, while theintegrity of the nuclear envelope was maintained.To ensure that the digitonin-permeabilized cells sup-Page 3 of 12(page number not for citation purposes)complexes, were pooled, concentrated, and used for sub-sequent fluorescent labeling.ported nuclear import, we used a positive control importsubstrate, the simian virus 40 large T antigen NLS cross-Virology Journal 2007, 4:49 http://www.virologyj.com/content/4/1/49linked to BSA (NLS-BSA) and fluorescently labeled it withCy3 (Cy3-NLS-BSA). As shown in Fig. 3B, Cy3-NLS-BSAefficiently accumulated in the nucleus of digitonin-per-meabilized cells in the presence of exogenous cytosol andan energy-regenerating system. Quantitation of the fluo-rescence intensity in the nucleus and cytoplasm revealedthat Cy3-NLS-BSA accumulated in the nucleus at levels3.5-fold greater than in the cytoplasm (Fig. 3C). In con-trast, in the absence of exogenous cytosol and an energy-regenerating system, Cy3-NLS-BSA was found mainlythroughout the cytoplasm, and unable to accumulateeffectively in the nucleus. Although quantitation of thefluorescence intensity indicated that a small amount ofCy3-NLS-BSA was in the nucleus in the absence of exoge-nous cytosol and an energy-regenerating system (Fig. 3C),this was probably due to residual cytosolic import factorsand energy remaining in the permeabilized cells, whichhas been previously reported [24]. In agreement with thisexplanation, blocking the NPCs with the monoclonalantibody 414 prevented nuclear import (data not shown).Next, we tested whether the fluorescein-labeled influenzavRNPs (fluorescein-vRNP) were competent for nuclearimport. As illustrated in Fig. 3B, similar to Cy3-NLS-BSA,fluorescein-vRNP efficiently accumulated in the cellnucleus under permissive conditions (+cytosol + energy).As revealed from the quantitation of the fluorescenceconditions was comparable to that of Cy3-NLS-BSA. Con-sistent with previous reports, the vRNP complexes wereespecially prone to accumulation at the nucleolus [37,41].Taken together, these results demonstrate that the nuclearimport assay with digitonin-permeabilized cells is func-tional, and that the fluorescein-labeled vRNPs are compe-tent for nuclear import.Peptides mimicking NLS1 or NLS2 on NP inhibited the nuclear import of influenza vRNPsTo understand the contributions of the two putative NLSson NP to the nuclear import of influenza vRNPs, peptidesthat mimic the NP NLS1 or NLS2 were used to competewith the vRNPs for their nuclear import receptors (theimportin αs). Our hypothesis for these peptide competi-tion experiments was that if a peptide served as an NLSthat is responsible for the nuclear import of influenzavRNP, that peptide would bind to its nuclear importreceptor. Thereby, the receptor would be unavailable tobind to and import the vRNPs into the nucleus. Figure 4Ashows a representative peptide competition experimentperformed with fluorescein-vRNPs in digitonin-permea-bilized cells. As shown in Fig. 4A, a control peptide con-sisting of a mutated NLS [42] did not inhibit the nuclearimport of fluorescein-vRNPs. Similarly, quantitation ofthe nuclear-to-cytoplasmic fluorescence in conditionsFractionation profile of the purification of vRNP complexes from influenza A by glycerol gradient centrifugationigure 1Fractionation profile of the purification of vRNP complexes from influenza A by glycerol gradient centrifuga-tion. 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.Page 4 of 12(page number not for citation purposes)intensity (Fig. 3C), the level of nuclear accumulation offluorescein-vRNP under permissive and non-permissivewith and without control peptide revealed that thenuclear accumulation of fluorescein-vRNP was similar inVirology Journal 2007, 4:49 http://www.virologyj.com/content/4/1/49the two samples. In contrast, nuclear accumulation of flu-orescein-vRNP was reduced in the presence of either theNLS1 peptide or the NLS2 peptide. Moreover, nuclearBiotinylation of influenza vRNPsF gure 2Biotinylation of influenza vRNPs. (A) Northern blot of biotinylated influenza vRNA in the vRNP complexes. The biotinylated vRNPs were subjected through urea gel electro-phoresis, 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 dif-ferent 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 asso-ciated with one end of the vRNPs.Nuclear import of fluorescein-labeled influenza vRNPsFigure 3Nuclear 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 inde-pendent 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 fluores-cence for the experimental conditions shown in A and B. Each bar graph shows the mean value and standard error from 100–110 individual cells.Page 5 of 12(page number not for citation purposes)import of fluorescein-vRNP was further reduced whenboth NLS1 and NLS2 peptides were included.Virology Journal 2007, 4:49 http://www.virologyj.com/content/4/1/49The results displayed in Fig. 4 are for conditions in whichthe NLS1 peptide or NLS2 peptide was included in thepresence of the control peptide added at a 1:1 ratio. (Allconditions contained the same total molarity of peptides,so the control peptide was added to maintain constantpeptide concentrations when comparing with the NLS1 +NLS2 double peptide competition condition.) Experi-ments were also performed with just the NLS1 peptide orNLS2 peptide in the absence of a control peptide, and asimilar inhibition profile was obtained (results notshown). In either case, inhibition of either NLS1 or NLS2alone did not completely inhibit the nuclear import of thefluorescein-vRNP. This indicates that NLS1 and NLS2 actindependently of each other to promote nuclear import ofthe vRNPs since inhibition of only one NLS still resultedin a certain degree of vRNP nuclear accumulation. Fur-thermore, this inhibitory effect was additive because whena combination of NLS1 + NLS2 peptides were added, a sig-nificantly larger reduction in the nuclear import of thevRNPs occurred (Fig. 4).Antibodies against NLS1 or NLS2 of NP inhibited the nuclear import of influenza vRNPsTo verify the results of the peptide competition experi-ments, a second approach consisting of antibody inhibi-tion was used. For this purpose, antibodies against NLS1or NLS2 of NP were applied in the nuclear import assaywith digitonin-permeabilized HeLa cells using fluores-cein-vRNP in the presence of exogenous cytosol and anenergy-regenerating system. Our hypothesis was thatthese antibodies would bind to the vRNP complexes andprevent the association of importinα to the vRNPs,thereby inhibiting fluorescein-vRNP nuclear import. As acontrol antibody, we applied anti-BSA. As shown in Fig. 5,the anti-BSA control antibody did not affect the nuclearimport of fluorescein-vRNP. However, in the presence ofeither the anti-NLS1 or anti-NLS2 antibodies, nuclearaccumulation of fluorescein-vRNP was substantiallydecreased. Furthermore, similar to the peptide competi-tion studies, antibody inhibition of both NLS1 and NLS2resulted in an even greater decrease in fluorescein-vRNPnuclear import compared to inhibition with a single anti-body (Fig. 5). Results shown in Fig. 5 are for experimentsin which the control antibody was included with the anti-NLS1 antibody or anti-NLS2 antibody to maintain a con-stant total antibody concentration. However, similarresults were obtained when only the anti-NLS1 antibodyor anti-NLS2 antibody was added without the controlantibody (results not shown).The antibody inhibition results generally agree with thoseof the peptide competition experiments described above(Fig. 4). However, inhibiting the NLSs with antibodiesCompetition of influenza vRNP nuclear import with peptides against NP NLSsFigur  4Competition 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 digi-tonin-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 micros-copy, and representative images of three independent exper-iments 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 stand-ard error from 100–110 individual cells.Page 6 of 12(page number not for citation purposes)seemed to be more effective in inhibiting the nuclearimport of the fluorescein-vRNPs, than competing withVirology Journal 2007, 4:49 http://www.virologyj.com/content/4/1/49these sequences. Furthermore, a difference was observedin the inhibition of NLS1 versus NLS2 with the antibod-ies, but not with the peptides. The discrepancy betweenthese antibody results, which showed a differencebetween the two NLSs versus the peptide competitionresults, occurred probably because the antibodies directlyinhibited nuclear import by blocking the NLSs on thevRNP, while the peptides indirectly inhibited nuclearimport by competing for the vRNP nuclear import recep-tors. If so, the antibody inhibition results would indicatethat while both NLS1 and NLS2 of NP may be involved inmediating the nuclear import of virally-derived vRNPcomplexes, the contribution of NLS1 is slightly greater.DiscussionUsing influenza vRNPs purified under acidic conditions,and thus mimicking physiologically-relevant influenzainfections as closely as possible, we have found that inhi-bition of either NLS1 or NLS2 on influenza NP signifi-cantly inhibited the nuclear import of influenza vRNPcomplexes. Therefore, both NLS1 and NLS2 on NP areinvolved in mediating the nuclear import of incomingvRNP complexes. These two sequences act independentlyof each other, as peptide competition with or antibodyinhibition of only one of these sequences still resulted ina certain, though less pronounced, degree of nuclearimport of vRNPs. Furthermore, when both NLS1 andNLS2 were competed with peptides or blocked by anti-bodies, the nuclear import of the vRNPs was even moredrastically reduced.Some differences, however, existed in the ability of theNLS peptides versus the anti-NLS antibodies to inhibitnuclear import of the vRNPs. These differences were likelydue to the nature of the competition experiments, sincepeptide competition with the vRNPs for the cytosolicnuclear import receptors is less specific than direct inhibi-tion of the vRNP NLSs with antibodies. The antibody inhi-bition experiments may hence provide a more accuratepicture of the relative contributions of NLS1 and NLS2 toinfluenza vRNP nuclear import. From these results, itappears that peptides mimicking or antibodies againstthese conserved NLS regions on NP may be an effectivemeans of interrupting a critical stage in the influenza A lifecycle.Interestingly, performing a sequence alignment (usingClustal W [43]) of NP from different influenza A strains,we can observe that each of NLS1 and NLS2 on NP arehighly conserved among influenza A strains. However,NLS1 and NLS2 do not share much similarity with anyregion on NP of influenza B or C, as analyzed from a Clus-tal W alignment. This is in agreement with previous stud-Inhibition of influenza vRNP nuclear import with antibodies aga nst NP NLSsFigure 5Inhibition 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.Page 7 of 12(page number not for citation purposes)ies that have not been able to pinpoint the NLS in NP forinfluenza B [44], so it appears that influenza B and C mayVirology Journal 2007, 4:49 http://www.virologyj.com/content/4/1/49utilize NLSs that are different from those of influenza A.With respect to NLS1, the only residue conserved amonginfluenza A, B, and C is a conserved arginine at position 8of the influenza A NP, which agrees with previous studiesindicating that that residue is one of the most importantresidues involved in mediating the nuclear import ofrecombinant NP [29,30]. For NLS2, residue 214 on influ-enza A NP is probably one of the most important residuesas an arginine or lysine is found in that position in influ-enza A, B, and C.The exact length of NLS2 also remains to be determined.Previous studies had implicated that NLS2 was a bipartiteNLS of 19 amino acids long, spanning residues 198 to 216[31]. However, recent structural data has questioned thatthis NLS functions as a bipartite classical NLS since thecrystal structure of NP showed that the two clusters ofbasic residues of the bipartite NLS2 on NP were locatedtoo close together in space to be functional as a bipartiteNLS [5]. Even though NLS2 may not be a bipartite NLS,the relevance of residues 213, 214, and 216 on NP inmediating the nuclear import of recombinant NP appearsto be significant [31]. However, which other residues inNLS2 are important in mediating influenza nuclearimport remains to be determined.Our studies here with influenza vRNPs also confirm find-ings with recombinant NP that NLS1 is the stronger of thetwo NLSs [29,31,37]. From the crystal structure of NP [5],it is probably reasonable to assume that NLS1, being anN-terminal sequence near the edge of NP, may be moreaccessible to the binding of cytosolic nuclear import fac-tors than NLS2. However, having both NLS1 and NLS2 asfunctional NLSs on influenza vRNPs could serve a vitalpurpose to the virus. If, in the event that the N-terminalNLS1 is inadvertently cleaved off by any proteases in itshost cell, the vRNP may still have an extra NLS (NLS2) tomediate its nuclear import. Nonetheless, it appears thatwith so many copies of NP, it does seem to be a redundantfunction. However, it is not clear how many of these NLSsare exposed when NP oligomerizes and associates withthe vRNA. Therefore, further studies at understanding thekinetics, cellular targets, conformational states, and role inviral replication of NLS1 and NLS2 will be required tobring further light to their roles in influenza cellular traf-ficking and replication.Previous work by other groups have concentrated mainlyon the nuclear import of recombinant NP [5,27-31,37].These studies have provided a better understanding of therole of the various NLSs on NP in the nuclear import ofnewly-synthesized NP, which occurs after the initialnuclear import of the entire vRNP complex and the subse-(1995) [27], and more recently Cros et al. (2005) [26],formed in vitro-assembled NP-RNA complexes by incubat-ing recombinant NP with in vitro-synthesized influenzavRNA. To study the nuclear import of the influenzagenome, however, vRNPs purified from influenza virionswould likely be the preferred substrates over in vitro-formed vRNA-NP complexes. This is because the actualassembly of NP into an oligomeric structure, and theinteractions of NP with the vRNA in actual influenzainfections, would likely result in structural differencesbetween in vitro-formed RNA-NP complexes versusvirally-produced and assembled vRNPs. For example, cer-tain NLSs may be exposed or hidden according to how NPactually interacts with itself in the oligomer and how NPinteracts with the vRNA. In addition, NP molecules withinactual influenza vRNPs that are produced in mammaliancells may have differences in their post-translational mod-ifications compared to recombinant NP molecules pro-duced in bacteria. Furthermore, any conformationalchanges in the structure of the vRNPs after influenzaexport from the cell, viral entry into a new cell, and duringor after their exit from endosomes are not taken intoaccount by in vitro-formed RNA-NP complexes. The meth-odological differences in the preparation of vRNPs maytherefore explain the differences observed in studies usingin vitro-formed RNP and our results reported here usingnaturally-occurring, influenza-derived vRNP complexes.For example, Cros et al. [27] found that disruption ofNLS2 on NP has no effect on the nuclear accumulation ofin vitro-formed vRNA-NP complexes, while we showedhere that interfering with NLS2 on NP diminished thenuclear import capability of influenza-purified vRNPs(Figs. 4 and 5). Likewise, Ozawa et al. [37] found thatNLS2, but not NLS1, deletion mutants of NP were unableto target effectively to nucleolar regions, while we showedhere that nucleolar localization still occurred whetherNLS1 or NLS2 on NP was inhibited.Much work on understanding the nuclear import of theinfluenza genome still remains. For example, the nuclearaccumulation sequence (NAS) within influenza-assem-bled vRNP complexes is still a mystery. The NAS on NPwas originally found to mediate nuclear accumulation ofNP in Xenopus oocytes [45], but more recently has beenfound to be a cytoplasmic retention signal in mammaliancells [30,31,46]. It would therefore be useful to under-stand in greater detail the role of the NAS and how itsfunction relates to that of NLS1 and NLS2. In addition, therole of M1 in preventing nuclear import of vRNP is stillunclear [11,39]. For example, M1 may be acting indi-rectly, where interaction of M1 with the vRNPs causes thevRNPs to change their structural conformation [47] andthus expose or hide certain NLSs. Alternatively, M1 mayPage 8 of 12(page number not for citation purposes)quent synthesis of new NP in the cytoplasm [12,14]. Tostudy the nuclear import of influenza vRNPs, O'Neil et al.be binding directly to the NLSs of vRNPs to inhibit theirnuclear import. The studies completed to date also raiseVirology Journal 2007, 4:49 http://www.virologyj.com/content/4/1/49the question as to under what conditions and in what con-formational states of NP would NLS1 and NLS2 act tomediate the nuclear import of vRNPs. Further unravelingthe answers to these questions may give us a moredetailed understanding of how the various NLSs on theinfluenza vRNPs work together or independently to medi-ate influenza A nuclear import.ConclusionIn summary, we have showed in this study that inhibitionof either NLS1 or NLS2 on NP from influenza-derivedvRNP complexes significantly decreased the extent ofnuclear localization of the influenza genome. Further-more, inhibiting both NLSs resulted in an additive effect,causing an even greater decrease in vRNP nuclear accumu-lation. This indicates that both NLS1 and NLS2 on NPplay a critical role in nuclear import by acting independ-ently to mediate the nuclear import of incoming influenzaA vRNP complexes. The importance of our findings in thedesign and development of novel influenza antiviral ther-apeutics is critical, as both NLSs will likely require to beinhibited to more completely abolish influenza nuclearimport and thus viral replication.MethodsPurification of influenza vRNP complexesVirally-derived influenza vRNPs were purified accordingto Kemler et al. [38] with minor modifications: 1 ml of theH3N2 X-31 A/AICHI/68 strain of influenza A (CharlesRiver Laboratories, Wilmington, MA) at 2 mg ml-1 waswashed in 30 mM Tris, pH 7.5, 20 mM MES, and 150 mMNaCl, and then centrifuged for 10 minutes, 4°C, at109,000 × g using a TLA-120.2 rotor in an Optima Max-Ecentrifuge (Beckman Coulter, Fullerton, CA). The pelletwas resuspended in 0.5 ml disruption buffer (100 mMMES, pH 5.5, 100 mM KCl, 5 mM MgCl2, 5% glycerol, 50mM octylglucoside (Sigma, St. Louis, MO), 10 mg ml-1lysolecithin (Sigma), and 1.5 mM dithiothreitol (Sigma)).After shaking and vortexing at 31°C, this sample wasloaded onto a glycerol gradient containing 1 ml 70% glyc-erol, 0.75 ml 50% glycerol, 0.375 ml 40% glycerol, and1.8 ml 33% glycerol in buffer containing 50 mM MES, pH5.5, and 150 mM NaCl. Centrifugation was performed for3.75 hours at 4°C in an MLS-50 rotor at 217 000 × g. Frac-tions were analyzed on a reducing SDS gel containing10% polyacrylamide. Gels were stained in 0.025%Coomassie brilliant blue G-250 (Sigma). Peak fractionscontaining the vRNPs were pooled, washed in UltrapureDEPC-treated water (Invitrogen, Carlsbad, CA) by ultra-centrifugation in a TLA-120.2 rotor at 157,000 × g for 4.5hours at 4°C, and concentrated by resuspending the pel-lets in DEPC-treated water. The A280 of the concentratedvRNP was measured, and samples were aliquoted and fro-was calculated using its extinction coefficient of 55,350 M-1 cm-1 (as determined via ProtParam [48]), to calculate theamount of peptides or antibodies required for the compe-tition and inhibition studies.Biotinylation of influenza vRNP complexesThe 5' EndTag Nucleic Acid Labeling kit (Vector Laborato-ries, Burlingame, CA) was used to covalently attach a reac-tive thiophosphate group to the 5' end of the vRNA in thevRNP complex: The 5' phosphate from the vRNA wasremoved with alkaline phosphatase at 37°C for 30 min-utes, followed by transferal of the thiophosphate fromATPγS to the vRNA 5' end with T4 polynucleotide kinaseat 37°C for 30 minutes. Biotin-maleimide (Sigma), whichbinds to the sulfur on the thiophosphate, was then incu-bated with the thiophosphorylated vRNP complex at65°C for 30 minutes.Urea gel electrophoresis and Northern blotting of biotinylated vRNAPurified influenza vRNPs were diluted in loading dye con-taining TBE buffer (45 mM Tris, pH 8, 45 mM boric acid,1 mM EDTA), 0.1% SDS, 0.08 mg ml-1 yeast RNA (RocheApplied Science, Basel, Switzerland) to scavenge anyRNases, glycerol, and the tracking dyes bromophenol blueand xylene cyanol. The sample was heated at 93°C for 3minutes, and then applied to a urea polyacrylamide gel(8.3 M urea and 6% polyacrylamide in TBE buffer). ThevRNA was visualized by staining for 30 minutes at roomtemperature with the SYBR Safe dye (Invitrogen). RNAmolecular weight standards (Sigma) were used to deter-mine the relative mobility of the vRNA.For Northern blotting, the unstained gel was transferredonto nitrocellulose, and blotted with the UltraSNAPDetection Kit (Vector Laboratories). This kit containedalkaline phosphatase-streptavidin to allow for the visuali-zation of biotinylated influenza vRNA.Negative staining and transmission electron microscopyBiotinylated vRNPs were incubated with streptavidin-gold(10-nm diameter) (Ted Pella, Redding, CA) overnight at4°C. The sample was then absorbed onto 2% parlodion/carbon-coated electron microscopy grids for 5 minutes,washed with buffer containing 20 mM Tris, pH 7.4, and120 mM KCl, and negatively stained with 1% uranyl ace-tate. Grids were visualized in a Hitachi H7600 transmis-sion electron microscope (Hitachi High TechnologiesAmerica, Schaumburg, IL).Conjugation of nuclear import substrates with fluorophoresAs a control, bovine serum albumin (BSA) was covalentlyPage 9 of 12(page number not for citation purposes)zen at -80°C. As NP is the major component present inthe pooled fractions, an estimate of the molarity of NPattached to a peptide (CGGGPKKKRKVED) containingthe NLS of the simian virus 40 large T antigen at a ratio ofVirology Journal 2007, 4:49 http://www.virologyj.com/content/4/1/495:1 of NLS:BSA (conjugated by Sigma Genosys). The NLS-BSA was then conjugated to the Cy3 fluorophore (Amer-sham Biosciences, Piscataway, NJ), via incubation with0.1 M sodium bicarbonate, pH 9.3, for 1 hour at roomtemperature. Biotinylated vRNP was conjugated tostreptavidin-fluorescein (Vector laboratories) by incuba-tion with streptavidin-fluorescein for one hour on ice.Nuclear import assay in digitonin-permeabilized HeLa cellsAdherent HeLa cells (American Type Culture Collection)were grown in a 37°C incubator containing 5% CO2 inDulbecco's modified Eagle medium (HyClone, Logan,UT) supplemented with 9% fetal bovine serum (Sigma),penicillin-streptomycin (Sigma), 1 mM sodium pyruvate(Cellgro, Herndon, VA), and 2 mM L-glutamine (Cellgro).The nuclear import assay is based on that of Adam et al.[40]. Briefly, the HeLa cells were seeded onto glass coverslips such that they were 60–70% confluent the next day.The HeLa cells were then rinsed with import buffer (20mM HEPES, pH 7.4, 110 mM potassium acetate, 1 mMEGTA, 5 mM sodium acetate, 2 mM magnesium acetate,and 2 mM dithiothreitol), and permeabilized with 20 μgml-1 digitonin in import buffer at room temperature for 5minutes. After further washes with import buffer, thecover slips containing digitonin-permeabilized cells wereinverted and incubated with the import mixture contain-ing Cy3-labeled cNLS-BSA or fluorescein-labeled vRNPfor 45 minutes at 37°C in the presence or absence of exog-enous 20% cytosol (rabbit reticulocyte lysate, Promega(Madison, WI)) and an energy-regenerating system (0.4mM ATP, 0.45 mM GTP, 4.5 mM phosphocreatine and 18U ml-1 phosphocreatine kinase (all from Sigma)). To pre-vent nonspecific binding, 1.6 mg ml-1 BSA (Sigma) wasadded. Protease inhibitors (chymostatin, leupeptin,antipain, and pepstatin, all from Sigma) at 10 μg ml-1 werealso included. The cells were then washed, fixed with 4%paraformaldehyde in import buffer, and mounted withthe Prolong Gold antifade reagent containing DAPI (Inv-itrogen). As a control, 70 kDa dextran Texas Red (Invitro-gen) was applied to ensure that permeabilization of theplasma membrane, but not the nuclear membrane, hadoccurred [49]. In some experiments 100 μg/ml of themonoclonal antibody MAb414 (Covance), which recog-nizes proteins of the NPC and block nuclear import, wasused.Peptide competition studiesPeptides bearing the sequences of NLS1(1MASQGTKRSYEQM13) and NLS2(198KRGINDRNFWRGENGRKTR216) on influenza A NPwere synthesized by Pacific Immunology (San Diego,CA). As a control peptide, a mutated version of a cNLS-thereby does not support nuclear import [42], was used(synthesized by Sigma Genosys). For the peptide compe-tition experiments, a 500-fold molar excess of the NLS1 orNLS2 peptides (500:1 of peptides: NP) was used in thenuclear import assay. For these experiments, the peptideswere pre-incubated with exogenous cytosol for one hourat 4°C prior to addition of fluorescein-vRNP and initia-tion of the import assay. To maintain constant peptideconcentrations, a 1000-fold molar excess of combinedpeptides was used for each sample. The control peptidewas therefore used where only a single NLS1 or NLS2 pep-tide was added. (For example, a 1000-fold molar excess ofcontrol peptide only was used for the control sample, anda 500-fold excess of control peptide + 500-fold excess ofNLS1 peptide was used for the NLS1 peptide sample).Experiments were also performed with an excess of justthe NLS1 or NLS2 peptides without containing controlpeptide.Antibody inhibition studiesPolyclonal antibodies to the above peptides mimickingNLS1 or NLS2 on NP were produced and affinity purifiedby Pacific Immunology. The specificity of these antibodieswas checked by both dot blots and Western blots. Bothantibodies specifically reacted with their correspondentpeptide and with purified vRNPs.For the nuclear import studies, each antibody was used atan eight-fold molar excess (8:1 of antibodies:NP) in thenuclear import assay. Anti-BSA (Sigma) was used as a con-trol antibody. Antibodies were initially incubated with thefluorescein-vRNP for one hour at 4°C prior to initiationof the import assay. To maintain constant antibody con-centrations, a 16-fold molar excess of combined antibod-ies was used for each sample. The control antibody wastherefore used where only a single antibody was added.Experiments were also performed with just the anti-NLS1or the anti-NLS2 antibodies without containing controlantibody.Fluorescence microscopyConventional fluorescence microscopy was performed ona Zeiss Axioplan 2 (Carl Zeiss, Oberkochen, Germany).Confocal laser scanning microscopy was performed on aZeiss Pascal LSM 5 (Carl Zeiss).Quantification of nuclear importTo quantify the nuclear import of the fluorescently-labeled cargo, the ratio of the nuclear-to-cytoplasmic flu-orescence signal was determined. For this purpose, theintensity of the nuclear and cytoplasmic fluorescence wasmeasured using ImageJ (National Institutes of Health,Bethesda, MD) according to Schedlich et al. [49]. CellsPage 10 of 12(page number not for citation purposes)bearing peptide (CYTPPKTKRKV), which contains a thre-onine (underlined) at position 7 instead of a lysine andimaged under conventional fluorescence microscopy wereused for quantitation because it more accurately reflectedVirology Journal 2007, 4:49 http://www.virologyj.com/content/4/1/49the total amount of fluorescence in the nucleus and thecytoplasm, as opposed to confocal microscopy, whichonly imaged the cells at one plane. To quantify the nuclearimport, the background was first subtracted with ImageJ.Next the mean intensity of a defined area (20 pixels by 20pixels) in the nucleus was measured and divided by themean intensity of the same amount of area in the cyto-plasm from the same cell. The area of the nucleus was cho-sen as close to the centre of the nucleus as possible,usually covering about 80% of the nucleus stained. Thecytoplasmic area was chosen to be representative of thefluorescence intensity of the entire cytoplasm of the cell,usually covering about 40% of the total cytoplasm. Thestaining of the nuclear envelope was not included in thequantification. As photographs were imaged at intensitiesbelow saturation, there should not have been any prob-lems with oversaturation of a certain area of the cell.Between 100–110 cells were quantified for each condi-tion.Competing interestsThe author(s) declare that they have no competing inter-ests.Authors' contributionsWWHW carried out the experiments and drafted the man-uscript. YHBS performed the preliminary studies to workout certain experimental protocols, and commented onthe manuscript. NP conceived the study and experimentaldesign, coordinated the study, and helped to draft themanuscript. All authors read and approved the final man-uscript.AcknowledgementsWe thank Dr. George Mackie and Janet Hankins for helpful advice on RNA handling and analysis. We also thank Dr. David Jans for his generous sug-gestions on the quantification of nuclear import. This work was supported by grants from the Canada Foundation for Innovation (CFI), the Canadian Institute of Health Research (CIHR), and the Natural Sciences and Engi-neering Research Council of Canada (NSERC).References1. Baudin F, Bach C, Cusack S, Ruigrok RW: Structure of influenzavirus RNP. I. Influenza virus nucleoprotein melts secondarystructure in panhandle RNA and exposes the bases to thesolvent.  Embo J 1994, 13:3158-3165.2. Compans RW, Content J, Duesberg PH: Structure of the ribonu-cleoprotein of influenza virus.  J Virol 1972, 10:795-800.3. 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