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The directionality of the nuclear transport of the influenza A genome is driven by selective exposure… Wu, Winco W; Panté, Nelly Jun 2, 2009

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ralssBioMed CentVirology JournalOpen AcceResearchThe directionality of the nuclear transport of the influenza A genome is driven by selective exposure of nuclear localization sequences on nucleoproteinWinco WH Wu and Nelly Panté*Address: Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, British Columbia, V6T 1Z4, CanadaEmail: Winco WH Wu - winco@zoology.ubc.ca; Nelly Panté* - pante@zoology.ubc.ca* Corresponding author    AbstractBackground: Early in infection, the genome of the influenza A virus, consisting of eight complexesof RNA and proteins (termed viral ribonucleoproteins; vRNPs), enters the nucleus of infected cellsfor replication. Incoming vRNPs are imported into the nucleus of infected cells using at least twonuclear localization sequences on nucleoprotein (NP; NLS1 at the N terminus, and NLS2 in themiddle of the protein). Progeny vRNP assembly occurs in the nucleus, and later in infection, theseare exported from the nucleus to the cytoplasm. Nuclear-exported vRNPs are different fromincoming vRNPs in that they are prevented from re-entering the nucleus. Why nuclear-exportedvRNPs do not re-enter the nucleus is unknown.Results: To test our hypothesis that the exposure of NLSs on the vRNP regulates thedirectionality of the nuclear transport of the influenza vRNPs, we immunolabeled the two NLSs ofNP (NLS1 and NLS2) and analyzed their surface accessibility in cells infected with the influenza Avirus. We found that the NLS1 epitope on NP was exposed throughout the infected cells, but theNLS2 epitope on NP was only exposed in the nucleus of the infected cells. Addition of the nuclearexport inhibitor leptomycin B further revealed that NLS1 is no longer exposed in cytoplasmic NPand vRNPs that have already undergone nuclear export. Similar immunolabeling studies in thepresence of leptomycin B and with cells transfected with the cDNA of NP revealed that the NLS1on NP is hidden in nuclear exported-NP.Conclusion: NLS1 mediates the nuclear import of newly-synthesized NP and incoming vRNPs.This NLS becomes hidden on nuclear-exported NP and nuclear-exported vRNPs. Thus theselective exposure of the NLS1 constitutes a critical mechanism to regulate the directionality ofthe nuclear transport of vRNPs during the influenza A viral life cycle.BackgroundThe influenza A virus exploits the cellular nuclear trans-port machinery several times during infection (reviewednucleoproteins; vRNPs) – is released into the cytoplasmand imported into the nucleus for replication. Subse-quently, newly-synthesized viral proteins from the cyto-Published: 2 June 2009Virology Journal 2009, 6:68 doi:10.1186/1743-422X-6-68Received: 9 April 2009Accepted: 2 June 2009This article is available from: http://www.virologyj.com/content/6/1/68© 2009 Wu and Panté; 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)in [1]). Early in infection, the influenza A viral genome –consisting of eight complexes of RNA and proteins (ribo-plasm enter the nucleus to form newly-synthesizedvRNPs. Later in infection, newly-assembled vRNPs areVirology Journal 2009, 6:68 http://www.virologyj.com/content/6/1/68exported from the nucleus to the cytoplasm to allow fortheir packaging into progeny virions. The vRNPs containmultiple copies (up to 97) of viral nucleoprotein (NP; 56kDa) forming a core around which the RNA is helicallywrapped (reviewed in [2]). Each NP monomer has at leasttwo nuclear localization sequences (NLS1, spanning resi-dues 1–13 at the N terminus, and NLS2, spanning resi-dues 198–216 in the middle of the protein) that mediatethe nuclear import of NP and vRNPs [3-7]. We have pre-viously found that both NLS1 and NLS2 on NP areresponsible for mediating the nuclear import of vRNPspurified from influenza A virions in permeabilized cells[7]. We also found that NLS1 of NP is the principal medi-ator of the nuclear import of incoming vRNPs becauseNLS1 has higher surface accessibility than NLS2, bothwithin each vRNP molecule and on a greater number ofvRNP molecules [8].Within the nucleus, the original incoming and newly-syn-thesized negative-sense vRNAs act as templates to tran-scribe the positive mRNA strand, which is selectivelyexported into the cytoplasm and used to translate newviral proteins (reviewed in [9]). Some of the newly-syn-thesized viral proteins (NP; the RNA polymerases PA,PB1, and PB2; the nonstructural protein NS1; the matrixprotein M1) are then imported into the nucleus throughtheir respective NLSs. In the nucleus, the newly-synthe-sized NP, PB1, PB2, PA, and the vRNA assemble into newvRNPs (reviewed in [10]). Subsequently, the newly-assembled vRNPs use the cellular export receptor CRM1to exit the nucleus through the nuclear pore complexes[11-13].Nuclear-exported vRNPs are different from incomingvRNPs in that they are somehow prevented from beingimported back into the nucleus. It has been demonstratedthat association of the vRNPs with the viral protein M1regulates nuclear trafficking of influenza vRNPs [14,15].However details of how M1 prevents newly-assembledvRNPs from re-entering the nucleus is unknown. Ourhypothesis is that the NLSs on NP are the key determi-nants for the nuclear transport directionality of the vRNPsby possessing differential exposure. To test this hypothe-sis, we analyzed the exposure of the NLSs on NP in tissueculture cells infected with influenza A virus. We foundthat an exposed NLS1 on NP allows newly-synthesized NPto enter the nucleus, but NLS1 becomes masked or hiddenonce the progeny vRNPs undergo nuclear export. HiddenNLSs on the nuclear-exported vRNPs prevents the nuclearre-entry of the progeny vRNPs. This selective exposure andmasking of NLS1 on vRNPs thus constitutes a criticalmechanism to regulate the directionality of the nucleartransport of the influenza vRNPs.ResultsSpecificity of NP antibodiesWe have previously generated and characterized two pol-yclonal anti-peptide antibodies that specifically recognizeNLS1 and NLS2 on NP [7,8]. In this study, we used theseanti-NLS antibodies to analyze the exposure of these NLSswithin cells infected with influenza A virus or transfectedwith the cDNA of NP. Total NP was detected by using amonoclonal antibody specific for NP. To ensure that allthree of the NP monoclonal, anti-NLS1, and anti-NLS2antibodies were specific for NP and not for componentsof the cell, we first compared the antibody labeling ininfected cells with that in mock-infected cells. We foundthat each of the respective antibodies gave a strong signalin infected cells compared with mock-infected cells inwhich no virus was added (Fig. 1). A similar specificity ofthe anti-NP monoclonal, anti-NLS1, and anti-NLS2 anti-bodies was observed in cells transfected with the cDNA ofNP compared with mock-transfected cells (results notshown).Besides testing for the specificity of the anti-NP antibod-ies, the results from Fig. 1 also indicated that NLS1 wasgenerally more exposed than NLS2, and exposed in agreater number of influenza A virus-infected cells. This isin agreement with our previous studies examining theimmunogold labeling of purified vRNPs with the anti-NLS1 or anti-NLS2 antibodies [8], and with our conclu-sion that NLS1 is stronger that NLS2 in mediating thenuclear import of the influenza vRNPs [7].Exposure of NLS1 and NLS2 in influenza-infected cellsWe performed double-immunolabeling studies with themonoclonal NP antibody in conjunction with either thepolyclonal NP anti-NLS1 or with the polyclonal NP anti-NLS2 antibody to analyze the exposure of the NLSs incells infected with the influenza A virus. As illustrated inFig. 2, the NP monoclonal antibody detected NP in boththe nucleus and cytoplasm of infected cells (Fig. 2c–d),with 28% of the infected cells showing only nuclear stain-ing (Fig. 3a). Similarly, the NLS1 epitope on NP wasexposed in both the nucleus and cytoplasm (Fig. 2e). Incontrast, the NLS2 epitope was only exposed in thenucleus of the infected cells (Fig. 2f). Quantitative analy-sis showed that 100% of the infected cells labeled with theanti-NLS2 antibody had only nuclear staining of anti-NLS2, while 35% of the infected cells labeled with theanti-NLS1 antibody had only nuclear staining of anti-NLS1 (Fig. 3a).To distinguish between incoming vRNPs and newly syn-thesized NP and progeny vRNPs, we next performed asimilar double-immunolabeling experiment with cellsPage 2 of 12(page number not for citation purposes)infected with influenza A virus in the presence ofcycloheximide (a protein synthesis inhibitor). As illus-Virology Journal 2009, 6:68 http://www.virologyj.com/content/6/1/68trated in Fig. 4, there was no NP fluorescence signal incells treated with cycloheximide. This indicates that theNP being labeled in the infected cells (Fig. 2) representsindeed newly-synthesized NP. Therefore, this limits thetype of cytoplasmic NP detected in infected cells to beeither newly-synthesized NP or newly-assembled vRNPsthat have undergone nuclear export.From the above results, it was unclear why these infectedcells did not contain an exposed NLS2 in the cytoplasmeven though the cells contained NP in the cytoplasm. ThevRNPs. To distinguish whether the cytoplasmic NP isnewly-synthesized NP or nuclear-exported vRNPs, weused leptomycin B (LMB) to inhibit the nuclear export ofvRNPs. These experiments with LMB detect newly synthe-sized vRNPs that is trapped in the nucleus. LMB has beensuccessfully used in the past to inhibit the nuclear exportof vRNPs in infected cells [11,13]. We repeated theseexperiments in the presence of LMB, to block vRNPnuclear export and to determine whether the cytoplasmicNP in the infected cells represented newly-synthesized NPor nuclear-exported vRNPs. As documented in Fig. 2k–l,Specificity of NP antibodiesFigure 1Specificity of NP antibodies. Immunofluorescence microscopy of HeLa cells infected with the influenza A virus and immu-nolabeled with the monoclonal NP antibody, or the polyclonal anti-peptide antibodies that recognize the NLS1 and the NLS2 epitopes of NP. DAPI, a DNA marker, was used to determine the total number of cells present. As a control, a mock infection without influenza A virus was also performed. Cells were fixed and prepared for immunofluorescence microscopy 17 hours after infection.Page 3 of 12(page number not for citation purposes)experiment with cycloheximide helped us to concludethat the cytoplasmic NP does not represent incomingand Fig. 3a, we found that in the presence of LMB 78% ofthe infected cells showed only nuclear, and no cytoplas-Virology Journal 2009, 6:68 http://www.virologyj.com/content/6/1/68mic, NP. Quantitative analysis showed that 22% of theinfected cells, however, also still showed cytoplasmic NPin addition to nuclear NP accumulation (Fig. 3b). Becausewe were inhibiting nuclear export, this cytoplasmic NPrepresents newly-synthesized NP that had not yet under-gone nuclear import.Consistent with the notion that there were two pools ofcytoplasmic NP in infected cells untreated with LMB(newly-synthesized NP and newly-assembled vRNPs thathave undergone nuclear export), the experiment in thepresence of LMB yielded cells in which the fluorescencecontained an exposed NLS1 (Fig. 2m). In fact, quantita-tive analysis showed that 26% of infected cells in the pres-ence of LMB still contained both cytoplasmic and nuclearimmunostaining with the anti-NLS1 antibody (Fig. 3b).This indicates that newly-synthesized cytoplasmic NP thathad not yet undergone nuclear import contains anexposed NLS1 epitope.A longer time point in infected cells (30 hours instead of17 hours) was also performed, and there was even less,but still a small amount of cytoplasmic NP staining fromboth the monoclonal and the anti-NLS1 antibodiesExposure of NLS1 and NLS2 in influenza-infected cellsFigure 2Exposure of NLS1 and NLS2 in influenza-infected cells. HeLa cells infected with influenza A virus for 17 hours, in the absence (a-h) or presence (i-p) of the nuclear export inhibitor LMB, were immunolabeled with DAPI (a-b and i-j; blue), a monoclonal anti-NP antibody (c-d and k-l; red), and either the polyclonal anti-NLS1 antibody (e and m; green) or the polyclo-nal anti-NLS2 antibody (f and n; green). Merged images depict merge of the red and green channels for each respective set of cells.Page 4 of 12(page number not for citation purposes)intensity of the cytoplasmic NP was less intense than fromcells without LMB. Of particular note, this cytoplasmic NP(results not shown), indicating that more NP had under-gone nuclear import. Taken together, these results indi-Virology Journal 2009, 6:68 http://www.virologyj.com/content/6/1/68Page 5 of 12(page number not for citation purposes)Quantification of the exposure of NLS1 and NLS2 in influenza-infected cellsFigure 3Quantification of the exposure of NLS1 and NLS2 in influenza-infected cells. Bar graphs of the percentage of infected cells showing fluorescent staining only in the nucleus (a) or both in the cytoplasm and the nucleus (b) for the experi-mental conditions described in Fig. 2. Data shows the mean values and standard error scored from 152 and 82 infected cells in the absence and presence of LMB, respectively.Virology Journal 2009, 6:68 http://www.virologyj.com/content/6/1/68cate that NLS1 (but not NLS2) exposure is a prerequisitefor successful nuclear import of newly-synthesized NP.Exposure of NLS1 and NLS2 in NP-transfected cellsTo distinguish any differences in the localization betweenNP only and NP as part of the vRNP complex, we repeatedthe immunolocalization experiments in cells transfectedwith NP cDNA. Similar to infected cells, 71% of the trans-fected cells showed NP in both the cytoplasm andnucleus, as represented by immmunolabeling with themonoclonal anti-NP antibody (Fig. 5c–d, and Fig. 6b).However, NP NLS1 and NLS2 were only exposed in the65% of the cells with NP NLS1 exposed in the cytoplasm(Fig. 2e and Fig. 3b). According to our results above, thiswould indicate that the cytoplasmic NP in these trans-fected cells represented NP that had been nuclearexported, and not newly-synthesized NP, since NLS1 wasnot exposed in the cytoplasm of transfected cells (Fig. 5eand Fig. 6b) even though 71% of the transfected cellsshowed NP existing in the cytoplasm (Fig 5c–d, and Fig.6b). To confirm this and distinguish between the twopopulations of cytoplasmic NP (nuclear exported ornewly-synthesized), we blocked NP nuclear export withLMB. As expected, LMB completely inhibited NP nuclearLocalization of newly-synthesized NP in influenza-infected cellsFigure 4Localization of newly-synthesized NP in influenza-infected cells. Immunofluorescence microscopy of HeLa cells infected with the influenza A virus in the absence or presence of the protein synthesis inhibitor, cycloheximide. Cells were fixed and immunolabeled with DAPI and the monoclonal anti-NP antibody 17 hours after infection.Page 6 of 12(page number not for citation purposes)nucleus, and not cytoplasm, of transfected cells (Fig. 5e–f,and Fig. 6). This contrasted to infected cells, which yieldedexport, with all the NP being retained in the nucleus of thetransfected cells (Fig. 5k–l, and Fig. 6a). This indicates thatVirology Journal 2009, 6:68 http://www.virologyj.com/content/6/1/68all the cytoplasmic NP in transfected cells in the absenceof LMB (Fig. 5c–d) indeed represented nuclear-exportedNP. Since these cytoplasmic NP molecules did not showimmunolabeling of NLS1 or NLS2 (Fig. 5e–f), nuclearexported-NP has its NLSs hidden or masked.Exposure of NLS1 and NLS2 within the nucleolusWe also observed that in infected cells NP localized to dis-tinct nuclear spots, which were reminiscent of nucleoli. Toverify this we performed double immunolabeling withthe anti-NLS antibodies and a monoclonal antibodyagainst the nucleolar protein fibrillarin. As illustrated inFig. 7a, we found that in influenza-infected cells, NLS1is in contrast to NP-transfected cells, which have NLS1and NLS2 exposed in the nucleoplasm, without any expo-sure in the nucleolus (Fig. 7b). This indicates that one ormore components from the influenza virus play a role inallowing NLS2 to become exposed in the nucleolus ofinfluenza A virus-infected cells.DiscussionWe have previously shown that the NLS1, compared tothe NLS2, epitope on NP is more highly exposed through-out each vRNP molecule [8]. This has the consequencethat NLS1 is a stronger mediator than NLS2 for nuclearimport of vRNPs in vitro [7]. In this study, we analyzed theExposure of NLS1 and NLS2 in NP-transfected cellsFigure 5Exposure of NLS1 and NLS2 in NP-transfected cells. HeLa cells transfected with the cDNA of NP, in the absence (a-h) or presence (i-p) of the nuclear export inhibitor LMB, were immunolabeled with DAPI (a-b and i-j; blue), a monoclonal anti-NP antibody (c-d and k-l; red), and either the polyclonal anti-NLS1 antibody (e and m; green) or the polyclonal anti-NLS2 anti-body (f and n; green). Merged images depict merge of the red and green channels for each respective set of cells.Page 7 of 12(page number not for citation purposes)was not exposed in the nucleolus. NLS2 was, however,exposed both in the nucleoplasm and the nucleolus. Thisdegree of exposure of NLS1 and NLS2 in influenza-infected cells, and found that these NLSs are also differen-Virology Journal 2009, 6:68 http://www.virologyj.com/content/6/1/68Page 8 of 12(page number not for citation purposes)Quantification of the exposure of NLS1 and NLS2 in NP-transfected cellsFigure 6Quantification of the exposure of NLS1 and NLS2 in NP-transfected cells. Bar graphs of the percentage of trans-fected cells showing fluorescent staining only in the nucleus (a) or both in the cytoplasm and the nucleus (b) for the experi-mental conditions described in Fig. 5. Data shows the mean values and standard error scored from 288 and 87 transfected cells in the absence and presence of LMB, respectively.Virology Journal 2009, 6:68 http://www.virologyj.com/content/6/1/68Page 9 of 12(page number not for citation purposes)Exposure of NLS1 and NLS2 within the nucleolusFigure 7Exposure of NLS1 and NLS2 within the nucleolus. Immunofluorescence microscopy of cells infected with the influenza A virus (a) or transfected with the cDNA of NP (b) and immunolabeled with DAPI, the monoclonal anti-fibrillarin antibody (red), and either the polyclonal anti-NLS1 antibody (green) or the polyclonal anti-NLS2 antibody (green). Merged images of anti-fibrillarin (red) with the corresponding anti-NLS antibody (green) are shown.Virology Journal 2009, 6:68 http://www.virologyj.com/content/6/1/68tially exposed in the different cell compartments duringthe course of an infection. Interestingly, NLS2 was onlyexposed in the nucleus, while NLS1 was exposed in thecytoplasm and nucleus. By designing experiments thatallowed us to detect specific forms of cytoplasmic NP andvRNPs, we found that NLS1 is exposed in newly-synthe-sized cytoplasmic NP, confirming once more that NLS1,but not NLS2, is especially critical for the nuclear importof influenza NP [6]. The exposure and role of NLS2 innuclear trafficking of NP and vRNP is less clear. However,our findings that NLS2 is exposed in the nucleolus ofinfected, but not NP-transfected, cells is in agreement witha role of this sequence for viral replication, as it has beenpreviously demonstrated [16].We have also found in this study that nuclear-exported NPcontains a masked NLS1, thereby preventing this mole-cule from re-entering the nucleus. Based on this result, weconclude that the selective exposure and masking of NLS1constitutes a critical mechanism to regulate the direction-ality of nuclear trafficking of vRNPs during the influenzaA viral life cycle. Our results is consistent with a model(Fig. 8) in which NLS1 is exposed in newly-synthesizedNP and also in incoming vRNPs to allow these moleculesto bind to cellular importins and enter the nucleus; uponassembly of NP into newly-synthesized vRNPs in thenucleus, NLS1 becomes masked, so after the vRNPs arenuclear exported, they cannot return to the nucleus. Thehidden NLS1 epitope thereby critically regulates the direc-tionality of the nuclear transport of newly-assembledvRNPs, driving their uni-directional nuclear export andallowing subsequent cytoplasmic assembly and buddingof the complete influenza A virion.Several putative pathways to encrypt NLS1 on nuclear-exported vRNPs and NP may occur. Since the masking ofNLS1 was also observed in transfected, and not onlyinfected, cells, a masked NLS1 epitope is independent ofthe viral M1 matrix protein, viral RNA, or other influenzaA components. NLS1 masking on newly-synthesizedvRNP and NP is also unlikely due to NP oligomerizationbecause we have previously demonstrated that NP oli-gomerized as vRNPs contains an exposed NLS1 [8].Instead, this NLS masking is likely due to an NP post-translational modification, its binding to a cellular pro-tein, or a conformational change in NP. Which of thesemechanisms act to prevent nuclear re-entry awaits furtherstudies.ConclusionOur results indicate that NLS1 is exposed in cells afterinfluenza infection to mediate the nuclear import ofincoming vRNPs and newly-synthesized NP. This NLSing of the NLS1 epitope prevents nuclear re-entry ofnewly-synthesized vRNPs. The molecular mechanism ofthis masking awaits further studies, but we believe thatthis study provides the basic underlying mechanism thatregulates the directionality of the nuclear trafficking ofinfluenza vRNPs. We conclude that selective exposure andmasking of the NLS1 on the vRNP constitutes a criticalmechanism to regulate the directionality of the nucleartransport of vRNPs during the influenza A viral life cycle(Fig. 8).MethodsCells, viruses, antibodiesHeLa cells (American Type Culture Collection) were cul-tured in DMEM (HyClone) supplemented with 9% fetalbovine serum (FBS; Sigma) and maintained at 37°C in ahumidified atmosphere with 5% CO2. Influenza A (A/WSN/1933) NP cDNA in the pCAGGS vector was kindlyprovided by Dr. G. Whittaker (Cornell University). Theaffinity-purified rabbit polyclonal antibodies against theNLSs of NP (NLS1, 1MASQGTKRSYEQM13 and NLS2,198KRGINDRNFWRGENGRKTR216) were produced byPacific Immunology, and have been characterized previ-ously [7,8]. The mouse monoclonal NP and fibrillarinantibodies were purchased from Acris and Abcam, respec-tively. Influenza A virus (A/Aichi/1968) was obtainedfrom Charles River Laboratories.Influenza infectionHeLa cells were plated at 30% confluency the day beforeinfection in growth media containing 9% FBS onto 12-mm glass cover slips in 12-well plates. The next day, thecells were washed with phosphate buffered saline (PBS),and then 1 ml of growth media containing 0.2% FBS wasapplied to each well. 30 μl of the influenza A virus at 2mg/ml (MOI of 1) were applied to the cells. The virus wasallowed to adsorb to the surface of the cells for 40 minutesat room temperature, with gentle rocking every 10–15minutes. The media containing the virus was thenremoved, and replaced with 1 ml of media containing 2%FBS. The cells were incubated for 17 or 30 hours in a 37°Cincubator containing 5% CO2. After these incubationtimes, the cells were prepared for immunofluorescencemicroscopy as described below.For some experiments, the protein synthesis inhibitorcycloheximide (Sigma, St. Louis) at a final concentrationof 1 mM was added to the 2% FBS medium. To inhibitnuclear export, leptomycin B (LMB; Sigma) was added tothe cells 6 hours after replacing the media containing 2%FBS, and cells were incubated for a total of 17 or 30 hoursat 37°C. LMB was used at a concentration of 11 nM,which is effective for the inhibition of the nuclear exportPage 10 of 12(page number not for citation purposes)becomes hidden once progeny vRNPs have been exportedfrom the nucleus. Our data support the model that mask-of NP and vRNPs, as previously reported [6,11].Virology Journal 2009, 6:68 http://www.virologyj.com/content/6/1/68TransfectionsTransfection of HeLa cells with NP-pCAGGS was carriedout on glass coverslips with Lipofectamine 2000 (Invitro-gen) according to the manufacturer's protocol. For someexperiments, cycloheximide (final concentration 1 mM)was added after media was changed, and the cells wereincubated for 24 hours at 37°C. To inhibit nuclear export,LMB (final concentration 11 nM) was added 6 hours afterthe change in media, and the cells were incubated for atotal of 30 hours at 37°C. After these incubation times,the cells were prepared for immunofluorescence micros-copy as described below.Immunofluorescence microscopydehyde, permeabilized with 0.2% Triton X-100 (Sigma)in PBS containing 10% goat serum (Sigma), and labeledwith the NP or the fibrillarin monoclonal antibody andeither the NP polyclonal anti-NLS1 or the NP polyclonalanti-NLS2 antibody for 1 hour, followed by incubationwith secondary antibodies (goat anti-mouse rhodamineand goat anti-rabbit fluorescein, both from Invitrogen).After washing, coverslips were mounted with ProlongGold antifade containing DAPI (Invitrogen). Fluorescencemicroscopy was performed on a Zeiss Axioplan 2.Competing interestsThe authors declare that they have no competing interests.Model of the exposure of the NLS1 on influenza A NP and its role in cellular nuclear transportFigure 8Model of the exposure of the NLS1 on influenza A NP and its role in cellular nuclear transport. The exposure and masking of the NP NLS1 mediates the directionality of the nuclear trafficking of influenza NP and vRNP. NP and vRNPs with an exposed NLS1 are represented by green, and NP and vRNPs with a hidden NLS1 are represented by red.Page 11 of 12(page number not for citation purposes)After infection or transfection, cells were washed threetimes with PBS, fixed for 10 minutes with 4% paraformal-Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central Virology Journal 2009, 6:68 http://www.virologyj.com/content/6/1/68Authors' contributionsWWHW carried out the experiments and drafted the man-uscript. NP conceived the study and experimental design,coordinated the study, and helped to draft the manu-script. All authors read and approved the final manu-script.AcknowledgementsWe thank Dr. Gary Whittaker for kindly providing the NP cDNA. This work was supported by grants from the Canada Foundation for Innovation (CFI), the Canadian Institutes of Health Research (CIHR), and the Natural Sciences and Engineering Research Council of Canada (NSERC). NP is a Michael Smith Foundation for Health Research (MSFHR) Senior Scholar.References1. Whittaker G, Bui M, Helenius A: The role of nuclear import andexport in influenza virus infection.  Trends Cell Biol 1996, 6:67-71.2. Wright PF, Neumann G, Yoshihiro K: Orthomyxoviruses.  In FieldsVirology Edited by: Knipe DM, Howley PM. Philadelphia: LippincottWilliams & Wilkins; 2007:1691-1740. 3. Neumann G, Castrucci MR, Kawaoka Y: Nuclear import andexport of influenza virus nucleoprotein.  J Virol 1997,71:9690-9700.4. Wang P, Palese P, O'Neill RE: The NPI-1/NPI-3 (karyopherinalpha) binding site on the influenza a virus nucleoprotein NPis a nonconventional nuclear localization signal.  J Virol 1997,71:1850-1856.5. Weber F, Kochs G, Gruber S, Haller O: A classical bipartitenuclear localization signal on Thogoto and influenza A virusnucleoproteins.  Virology 1998, 250:9-18.6. Cros JF, Garcia-Sastre A, Palese P: An unconventional NLS is crit-ical for the nuclear import of the influenza A virus nucleo-protein and ribonucleoprotein.  Traffic 2005, 6:205-213.7. Wu WWH, Sun YHB, Panté N: Nuclear import of influenza Aviral ribonucleoprotein complexes is mediated by twonuclear localization sequences on viral nucleoprotein.  Virol J2007, 4:49.8. Wu WWH, Weaver LL, Panté N: Ultrastructural analysis of thenuclear localization sequences on influenza a ribonucleopro-tein complexes.  J Mol Biol 2007, 374:910-916.9. Sidorenko Y, Reichl U: Structured model of influenza virus rep-lication in MDCK cells.  Biotechnol Bioeng 2004, 88:1-14.10. Boulo S, Akarsu H, Ruigrok RW, Baudin F: Nuclear traffic of influ-enza virus proteins and ribonucleoprotein complexes.  VirusRes 2007, 124:12-21.11. Elton D, Simpson-Holley M, Archer K, Medcalf L, Hallam R, McCauleyJ, Digard P: Interaction of the influenza virus nucleoproteinwith the cellular CRM1-mediated nuclear export pathway.  JVirol 2001, 75:408-419.12. Ma K, Roy AM, Whittaker GR: Nuclear export of influenza virusribonucleoproteins: identification of an export intermediateat the nuclear periphery.  Virology 2001, 282:215-220.13. Watanabe K, Takizawa N, Katoh M, Hoshida K, Kobayashi N, NagataK: Inhibition of nuclear export of ribonucleoprotein com-plexes of influenza virus by leptomycin B.  Virus Res 2001,77:31-42.14. Whittaker G, Bui M, Helenius A: Nuclear trafficking of influenzavirus ribonuleoproteins in heterokaryons.  J Virol 1996,70:2743-2756.15. Martin K, Helenius A: Nuclear transport of influenza virus ribo-nucleoproteins: the viral matrix protein (M1) promotesexport and inhibits import.  Cell 1991, 67:117-130.16. Ozawa M, Fujii K, Muramoto Y, Yamada S, Yamayoshi S, Takada A,Goto H, Horimoto T, Kawaoka Y: Contributions of two nuclearlocalization signals of influenza A virus nucleoprotein to viralreplication.  J Virol 2007, 81:30-41.yours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralPage 12 of 12(page number not for citation purposes)


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