Nuclear and Nucleolar Targeting of Inﬂuenza A Virus NS1 ...jvi.asm.org/content/81/11/5995.full.pdf · ... American Society for Microbiology. ... Striking Differences between Different

JOURNAL OF VIROLOGY, June 2007, p. 5995–6006 Vol. 81, No. 110022-538X/07/$08.00�0 doi:10.1128/JVI.01714-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Nuclear and Nucleolar Targeting of Influenza A Virus NS1 Protein:Striking Differences between Different Virus Subtypes�

Krister Melen,1* Leena Kinnunen,2 Riku Fagerlund,1 Niina Ikonen,1 Karen Y. Twu,3Robert M. Krug,3 and Ilkka Julkunen1

Departments of Viral Diseases and Immunology1 and Epidemiology and Health Promotion,2 National Public Health Institute,FIN-00300, Helsinki, Finland, and Section of Molecular Genetics and Microbiology, Institute for Cellular and

Molecular Biology, University of Texas at Austin, Austin, Texas 787123

Received 8 August 2006/Accepted 9 March 2007

Influenza A virus nonstructural protein 1 (NS1A protein) is a virulence factor which is targeted into thenucleus. It is a multifunctional protein that inhibits host cell pre-mRNA processing and counteracts host cellantiviral responses. We show that the NS1A protein can interact with all six human importin � isoforms,indicating that the nuclear translocation of NS1A protein is mediated by the classical importin �/� pathway.The NS1A protein of the H1N1 (WSN/33) virus has only one N-terminal arginine- or lysine-rich nuclearlocalization signal (NLS1), whereas the NS1A protein of the H3N2 subtype (Udorn/72) virus also has a secondC-terminal NLS (NLS2). NLS1 is mapped to residues 35 to 41, which also function in the double-strandedRNA-binding activity of the NS1A protein. NLS2 was created by a 7-amino-acid C-terminal extension (residues231 to 237) that became prevalent among human influenza A virus types isolated between the years 1950 to1987. NLS2 includes basic amino acids at positions 219, 220, 224, 229, 231, and 232. Surprisingly, NLS2 alsoforms a functional nucleolar localization signal NoLS, a function that was retained in H3N2 type virus NS1Aproteins even without the C-terminal extension. It is likely that the evolutionarily well-conserved nucleolartargeting function of NS1A protein plays a role in the pathogenesis of influenza A virus.

The influenza A virus genome consisting of eight separateRNA segments encodes 11 viral structural and nonstructuralproteins. In addition to the viral hemagglutinin, nonstructuralprotein 1 (NS1A) is one of the major viral virulence factors.The evolution of NS1A genes appears to be species specific,and the evolution of the present human NS1A genes began in1918 when H1N1 type viruses emerged and became pandemic(20).

The NS1A protein is a multifunctional protein that partici-pates in both protein-RNA (7, 16, 28, 57) and protein-protein(23, 25, 38) interactions. The NS1A protein contains an N-terminal double-stranded RNA (dsRNA)-binding domain anda C-terminal effector domain (45). The three-dimensionalstructures of the dsRNA-binding and effector domains ofNS1A have been determined (3, 6, 27). The NS1A proteinexists as a dimer, and the structure of its RNA-binding domaindiffers markedly from all other known RNA-binding proteins.The effector domain binds two cellular proteins that are essen-tial for the 3� end processing of cellular pre-mRNAs (5, 26, 38).As a result, the processing of cellular pre-mRNAs, includingbeta interferon (IFN-�) pre-mRNA and the pre-mRNAs ofother antiviral proteins, is inhibited, thereby suppressing theamount of mature IFN-� mRNA that is produced in infectedcells (38, 39, 49, 55). The role of the dsRNA-binding activity iscontroversial and may be virus strain specific. The role of thedsRNA-binding activity of the NS1A protein of the humanH3N2 influenza A/Udorn/72 virus was determined using a re-

combinant virus expressing a NS1 protein lacking dsRNA-binding activity. Analysis of the defect in virus replication dem-onstrated that the primary role of the NS1 dsRNA binding is toinhibit the activation of the IFN-induced 2� to 5� oligo(A)synthetase/RNase L pathway and showed that this dsRNA-binding activity has no role in inhibiting the production ofIFN-� mRNA (34). In contrast, experiments with the mouse-adapted H1N1 influenza A/PR8/34 virus indicated that theRNA-binding domain participates in an NS1A protein-medi-ated inhibition of the activation of retinoic acid-inducible geneI, which is required for cytokine gene expression (19, 31, 50),leading to impaired synthesis of IFN during influenza A virusinfection (33, 44).

Unlike most other RNA viruses, influenza viruses replicatein the nucleus of the host cells. The NS1A protein is efficientlytargeted into the nucleus, and two nuclear localization signals(NLSs) have been identified in the H3N2 subtype influenzaA/Alaska/6/77 virus NS1A protein (15). However, so far themolecular mechanisms mediating the nuclear import of NS1Aproteins have not been determined.

Active nuclear import of proteins targeted to the nucleus ismediated by specific sequence elements, NLSs. A classicalmonopartite NLS is composed of a stretch of four to six argi-nines or lysines (18, 24), while in a bipartite NLS two stretchesof basic amino acids are separated by a spacer 10 to 12 aminoacids long (11). In the cytoplasm NLS-containing proteins arerecognized by importin �, followed or preceded by binding ofimportin � to importin �. Cargo/importin �/importin � proteincomplexes are then translocated into the nucleus through thenuclear pore complex (NPC). Six human importin � isoformshave been identified: importin �1, importin �3, importin �4,importin �5, importin �6, and importin �7 (9, 10, 21, 22, 36,

* Corresponding author. Mailing address: Department of Viral Dis-eases and Immunology, National Public Health Institute, Manner-heimintie 166, FIN-00300, Helsinki, Finland. Phone: 358 9 47448879.Fax: 358 9 47448355. E-mail: [email protected].

� Published ahead of print on 21 March 2007.

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48). Importin � isoforms show significant differences in theirsubstrate specificity and binding mechanisms (12, 22, 32). Thethree-dimensional structure of the importin � NLS-bindingdomain has been determined (8, 14).

Eukaryotes have a specialized nuclear compartment, thenucleolus, which is a relatively large, dynamic, highly organizednonmembranous subcompartment of the nucleus. The nucle-olus is the site for rRNA synthesis, processing, and maturation.Recently, it has become apparent that the nucleolus also has arole in regulating the cell cycle, tumor suppression and onco-genic activities, assembly of signal recognition particle, controlof aging, and modulation of telomerase functions (41–43).Some of these functions are mediated through sequestration oftranscription factors that control the cell cycle (4, 47). Nuclearproteins pass through the nucleolus randomly, and those withaffinity to constitutive nucleolar components are retained. Ithas been suggested that nucleolar localization signals (NoLSs)act as retention signals rather than as classical targeting ortransport signals (2, 4). Many NoLSs overlap with NLSs andcontain basic amino acids (46, 54).

Here we show that influenza A virus NS1A proteins have awell-conserved N-terminal monopartite NLS (NLS1), wherebasic amino acids 35, 38, and 41 are critical for importin �binding and nuclear translocation. Remarkably, the NLS1 iscoincident with the dsRNA-binding epitope of the NS1A pro-tein. Viruses isolated between the years 1950 and 1987 alsohave a 7-amino-acid C-terminal extension (residues 231 to 237)in their NS1A protein that functions, together with other C-terminal basic residues, as an NoLS and as a second NLS(NLS2). Evolutionary analysis and fine mapping of the NoLSand NLS2 signals by site-directed mutagenesis indicated thatthe C-terminal basic amino acids constituted both of thesesignals. Even when the C-terminal extension (residues 231 to237) of H3N2 virus NS1A proteins was deleted, the truncatedNS1A protein (NS1A�231-237) was still localized in the nu-cleolus and contained a functional NLS2.

MATERIALS AND METHODS

Cells. Human A549 lung carcinoma cell line (ATCC CCL 185) was maintainedin continuous culture in minimum Eagle’s medium-� (Invitrogen Corp., Carls-bad, CA) supplemented with 0.6 �g/ml penicillin, 60 �g/ml streptomycin, and10% fetal calf serum (Integro, Zaandam, The Netherlands). Human hepatocel-lular carcinoma HuH7 (37) cells were maintained in minimum Eagle’s medium-�with supplements as above. Spodoptera frugiperda (Sf9) cells were used forbaculovirus expression and maintained in Grace’s insect medium as describedpreviously (52).

Viruses and infections. A549 cells, grown on glass coverslips on 24-well plates,were infected with the following viruses: H3N2 viruses A/Udorn/72, A/Fin/001/73, A/Fin/008/85, A/Beijing/353/89, A/Fin/229/92, A/Fin/455/97, and A/Moscow/10/99; H1N1 viruses A/WSN/33, A/Fin/001/79, A/Fin/001/82, A/Fin/40/86, A/Fin/432/96, and A/New Caledonia/20/99. Virus stocks were cultivated in 8-day-oldembryonated chicken eggs and stored at �70°C. The hemagglutination titers ofthe stock viruses ranged from 64 to 256, and the infectivity of the virus stocks inA549 cells was 1 � 107 to 4 � 107 PFU/ml. The multiplicity of infection used inthe experiments varied from 0.5 to 5 PFU/cell.

Recombinant influenza A/Udorn/72 viruses were created as previously de-scribed (53). A mutant A/Udorn/72 virus with a deletion of amino acids 221 to237 of NS1A (NS1A�221–237) was created by introducing a stop codon atposition 221 of the NS1A gene sequence by using two rounds of PCR and specificoligonucleotide primers. The resulting DNA was sequenced and cloned intopHH21 vector. The virus encoding the mutant NS1A protein was generated bycotransfecting 293T cells with eight plasmids encoding the viral RNA segmentsand four plasmids expressing the PB1, PB2, PA, and NP proteins (53). Cellculture supernatants were collected, the virus was titered by plaque assay on

MDCK cells, and individual plaques were amplified in 10-day-old embryonicchicken eggs.

Antibodies. Rabbit anti-importin �1, �3, and �7 antibodies used in Westernblot analysis were as previously described (22). Secondary horseradish peroxi-dase-conjugated goat anti-rabbit antibodies (1:2,000; Daco, Glostrup, Denmark)were used as suggested by the manufacturer. For confocal laser microscopy,anti-influenza A NS1A protein antibodies were prepared in guinea pigs byimmunizing the animals four times at 4-week intervals with an Escherichia coli-expressed, glutathione-Sepharose-purified (Amersham Biosciences, Bucking-hamshire, United Kingdom) glutathione S-transferase (GST)-NS1A fusion pro-tein (50 �g of protein/immunization/animal). Rabbit anti-human immunodeficiencyvirus type 1 (HIV-1) Rev antibodies have been described previously (30). Sec-ondary antibodies used were rhodamine Red-X- or fluorescein isothiocyanate-labeled goat anti-guinea pig or anti-rabbit immunoglobulins, respectively (1:100;Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).

Plasmids and DNA manipulations. GST-importin �1, �3, �5, �7, and � E. coliexpression constructs have been described previously (32). The coding region ofthe cDNA for human importin �6 (O15131) was modified by PCR to create N-and C-terminal NcoI and XhoI sites, respectively, for further cloning into an E.coli GST fusion vector (pETM-30; kindly provided by G. Stier, EMBL Heidel-berg, Germany). The importin �1, �3, �4, and �7 gene constructs in baculovirusGST expression vectors have been described previously (13). The importin �5gene (NM_002264) was PCR modified and cloned into the BamHI site of aGST-pAc/YMI baculovirus expression vector, and GST-importin �5 was pro-duced as described previously (13).

The wild-type (wt) A/Udorn/72 (H3N2 virus) NS1 gene (V01102) was ex-pressed in E. coli GST (pGEX-3X; Amersham Biosciences) and eukaryoticpcDNA3.1(�) (Invitrogen) expression vectors. The wt A/WSN/33 (H1N1 virus)NS1 gene (M12597) was modified by PCR to create N- and C-terminal BglII sitesfor further cloning into the BamHI site of a pcDNA3.1(�) expression vector(Invitrogen). To create point mutations to A/Udorn/72 and A/WSN/33 NS1cDNAs, a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA)was used.

To create green fluorescent protein (GFP)-NS1A fusion constructs, the C-terminal cDNAs for wt A/Udorn/72 (amino acids 203 to 237) and A/WSN/33(amino acids 203 to 230) NS1 genes were modified by PCR to create N- andC-terminal SalI and NheI sites, respectively, for further cloning into the SalI andNheI cloning site of the pCMX-SAH/Y145F expression vector (40). Mutations toGFP-NS1A chimeric gene constructs were done using the QuikChange Site-Directed Mutagenesis Kit. All oligonucleotides used to modify the genes in thestudy will be provided upon request.

The HIV-1 Rev cDNA in the pBC12/CMV expression vector has been de-scribed previously (30). All DNA manipulations were performed according tostandard protocols, and the newly created gene constructs were partially se-quenced.

Importin binding assay, sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE), and Western blotting. Human GST-importin �1, �3, �4,�5, �6, �7, and � or influenza A virus GST-NS1A fusion proteins were expressedin E. coli BL21 cells or by baculovirus in Sf9 cells, and GST-fusion proteins werepurified as described previously (12, 32).

In vitro translated NS1A wt or NLS mutant proteins (TnT Coupled Reticu-locyte Lysate Systems; Promega, Madison, WI) were 35S labeled (PRO-MIX;Amersham Biosciences) and allowed to bind to Sepharose-immobilized GST orGST-importin fusion proteins on ice for 60 min followed by washing. GST-importin-bound 35S-labeled proteins were separated by 12% SDS-PAGE. Thegels were fixed and treated with Amplify reagent (Amersham Biosciences) asspecified by the manufacturer and autoradiographed. GST pull-down experi-ments from A549 cell extracts were carried out as described previously (13).

Transfections, indirect immunofluorescence, and confocal laser microscopy.For indirect immunofluorescence and confocal laser microscopy, HuH7 cellsgrown on glass coverslips for 24 h were transfected with wt and mutant NS1A,GST-NS1A fusion, and HIV-1 rev gene constructs using FuGENE6 transfectionreagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’sinstructions. Forty-eight hours after transfection, the cells were fixed with 3%paraformaldehyde at room temperature for 20 min, permeabilized with 0.1%Triton X-100 for 5 min, and processed for immunofluorescence microscopy. Thecells positive for NS1A or HIV-1 Rev proteins were visualized and photographedon a Leica TCS NT confocal microscope.

RNA-binding assay. The GST portion of GST fusion proteins in RNA-bindingassays was cleaved using either the protease factor Xa (GST-NS1A A/Udorn/72;E. coli expression construct) or thrombin (GST-importin �1; E. coli expressionconstruct). The RNA-binding experiment was performed as described previously(57).

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RESULTS

All human importin � isoforms bind the influenza virusNS1A protein. Although the influenza A virus NS1A proteinhas been shown to contain NLSs that target it into the host cellnucleus (15), the mechanism of its nuclear import has not bedetermined. To determine whether the NS1A protein can in-teract with different importin � isoforms, GST-importin � fu-sion proteins were expressed in E. coli and in Sf9 insect cells,and pull-down experiments with 35S-labeled the influenzaA/Udorn/72 NS1A protein generated by in vitro translationwere carried out. E. coli-expressed GST-importin �1 and �6isoforms, but not those of importin �3, �5, or �7, were able tobind the influenza A/Udorn/72 NS1A protein (Fig. 1A). Incontrast, all the Sf9-expressed GST-importin � isoforms (�1,�3, �4, �5, and �7) bound the NS1A protein (Fig. 1B). Thesame results were obtained using the influenza virus A/WSN/33NS1A protein (Fig. 1C and D). These results show that theinfluenza NS1A protein is capable of binding to all humanimportin � isoforms. The differences in the binding patterns ofE. coli- and Sf9-expressed importin � isoforms suggest thatposttranslational modifications may be needed for the bindingof the NS1A protein to certain importin � isoforms.

To determine whether the NS1A protein specifically binds toimportin � molecules in human cells, extracts from A549 hu-man lung carcinoma cells were allowed to bind to the GST-NS1A protein, followed by immunoblotting of the bound pro-teins with antibodies specific for anti-importin �1, �3, or �6(�7 cross-reactive). Importins �1 and �6/7 and, to a lesserextent, importin �3 were found to bind to immobilized GST-NS1A (Fig. 1E). Since the recombinant and natural importins�1 and �6 were found to interact with the NS1A protein, E.coli-expressed GST-importins �1 and �6 were used in subse-quent binding experiments.

The NLS1 of the NS1A protein is highly conserved amonginfluenza A virus strains. It has been shown previously that theNS1A protein of the H3N2 type influenza virus A/Alaska/6/77has two NLSs. NLS1 was suggested to comprise amino acids 34to 38 (DRLRR) (15). This sequence is located in helix 2/2� ofthe dimeric RNA-binding domain (6, 27). We aligned 172human, 650 avian, 103 swine, and 24 equine influenza A virusNS1A protein sequences found in the GenBank (29) to deter-mine whether the amino acid sequence of NLS1 is conservedamong influenza A viruses isolated from different species andat different times (Fig. 2A). Only 1 out of the 172 humanstrains and 12 out of the 650 avian strains differed from theconsensus sequence. It should be noted that almost all of thesedifferences are characterized by the substitution of a similaramino acid (I substituted for L or K substituted for R). In theavian virus A/pintai/Alberta/119/79, there is an alanine in placeof an arginine at position 38. No differences from the consen-sus sequence were detected in swine or equine virus strains.Several amino acids C-terminal to the DRLRR sequence werealso well conserved. Amino acid 41 was K or R in all 990 strainsanalyzed (Fig. 2A). Amino acid 44 was almost as well con-served, since only 5 out of the 990 strains analyzed had anotheramino acid apart from K or R at this position. Amino acid K20was also highly conserved (data not shown). For site-directedmutagenesis of the NLS1, we selected conserved basic aminoacids K20, R35, R37, R38, K41, and R44 as targets.

FIG. 1. Influenza A virus NS1 binds to all importin � isoforms.(A) 35S-labeled and in vitro translated NS1A protein from A/Udorn/72virus strain (H3N2) was allowed to bind to E. coli-expressed andSepharose-immobilized GST-importin �, �1, �3, �5, �6, or �7 at �4°Cfor 1 h. After being washed twice with binding buffer, importin-boundNS1A was dissolved in Laemmli sample buffer, separated by 12%SDS-PAGE, and autoradiographed. A similar gel was also stained withCoomassie blue to visualize the amount of Sepharose-immobilizedGST-importin � and � isoforms. C, control; M, Sepharose matrix-bound in vitro translated NS1A. (B) 35S-labeled and in vitro translatedNS1A protein (A/Udorn/72) was allowed to bind to Sf9-expressed andSepharose-immobilized GST and GST-importin �1, �3, �4, �5, or �7as described above. 35S-labeled and in vitro translated NS1A proteinfrom A/WSN/33 virus strain (H1N1) was allowed to bind to E. coli-expressed (C) or Sf9-expressed (D) and Sepharose-immobilized GST-importins as described above. C, the control of in vitro translated NS1.(E) Cell extracts of cultured A549 cells were prepared, and the pro-teins in cell extracts were allowed to bind to E. coli-expressed andSepharose-immobilized GST and GST-NS1A (A/Udorn/72) at �4°Cfor 1 h. Sepharose-bound proteins were dissolved in Laemmli samplebuffer followed by 8% SDS-PAGE and Western blotting with anti-importin �1, �3, and �7 antibodies. As a control, cell extracts of A549cells containing 20 �g of protein were stained in lane C. A similar gelwas also stained with Coomassie blue to visualize the amount ofSepharose-immobilized GST and GST-NS1A A/Udorn/72.

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A putative NLS2 is found in the NS1A proteins of a subsetof influenza A virus strains. The NLS2 in the NS1A protein ofinfluenza virus A/Alaska/6/77 was mapped to amino acids 203to 237 at the C terminus (15), but the specific amino acids

comprising the NLS2 were not identified. The sequence in thisNS1A protein from amino acids 219 to 237, KRKMARTARSKVRRDKMAD (Fig. 2B), resembles a classical bipartite NLS,in which two short basic amino acid sequences (underlined)

FIG. 2. Variation of the amino acid sequence around NLS1 and NLS2 of influenza A virus NS1A protein. (A) Variation of NLS1 (amino acids34 to 44). The number of human, avian, swine, and equine NS1A protein sequences from different virus strains is shown at right. The previouslyidentified putative NLS1 (amino acids 34 to 38) (14) is underlined, and critical arginines (R) at positions 35 and 38 and lysine (K) at position 41,which regulate importin � binding, are shaded. (B) Variation of putative C-terminal NLS2 in selected influenza A virus NS1A proteins. Criticalamino acids 219, 220, 224, 227, 229, 231, and 232 that regulate NS1A protein interaction with importin � (this study) are shaded.



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FIG. 3. Influenza virus A/Udorn/72 NS1A protein has two importin � binding sites, and A/WSN/33 NS1A protein has one importin � bindingsite. (A) Influenza virus A/Udorn/72 NS1A protein binds to importin � protein via two separate NLSs. 35S-labeled and in vitro translatedA/Udorn/72 NS1A wt and point mutant proteins were allowed to bind to E. coli-expressed and Sepharose-immobilized GST, GST-importin �, andGST-importin �1 proteins as indicated in the figure. Importin-bound NS1A was dissolved in Laemmli sample buffer, separated by 12%SDS-PAGE, and autoradiographed. A similar gel was also stained with Coomassie blue to visualize the amount of Sepharose-immobilized GSTand GST-importin � and �1 proteins. For the translation control 1 �l of 35S-labeled and in vitro translated NS1 wt and six point mutants wereseparated by 12% SDS-PAGE and autoradiographed. (B) 35S-labeled and in vitro translated A/Udorn/72 NS1A wt and seven point mutant proteinscovering putative NLS1 and NLS2 as indicated in the figure. The binding experiment was carried out as described for panel A. (C) 35S-labeled andin vitro translated A/WSN/33 NS1A wt and four point mutant proteins were allowed to bind to E. coli-expressed and Sepharose-immobilized GSTand GST-importin �1 proteins as indicated in the figure. The binding experiment was as described for panel A.



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are separated by a nonspecific 9-amino-acid spacer. To deter-mine which amino acids in the putative NLS2 of theA/Udorn/72 NS1A protein are required for NLS function, wecarried out site-directed mutagenesis of basic amino acids 219,220, 224, 227, 229, 231, and 232. A similar, or identical, puta-tive NLS2 is found in all human influenza A viruses isolatedbetween 1950 and 1989, including influenza A/Udorn/72, thestrain used in the present study. These virus strains, which arecomprised of all human HN types during this period (H1N1,H2N2, and H3N2), encode an NS1A protein that contains 237amino acids (Fig. 2B). In contrast, the NS1A proteins of allother human influenza A strains are shorter. The vast majorityhave a stop codon at position 230, thereby disrupting the pu-tative NLS2. The NS1A protein of influenza A/WSN/33 is inthis category. In avian virus strains, the length of the NS1Aprotein is between 217 and 230 amino acids (Fig. 2B).

Importin � binds both NLS1 and NLS2 of the NS1A proteinof influenza virus A/Udorn/72. To determine whether both theNLS1 and the putative NLS2 of A/Udorn/72 NS1A proteinmediate the binding to importin �, we carried out pull-downexperiments with E. coli-expressed GST-importin �1 and[35S]methionine-labeled A/Udorn/72 NS1A proteins contain-ing specific mutations in the NLS1 and/or the putative NLS2sequences (Fig. 3A). Mutations in NLS1 of R37A and R38Ayielding NLS1(R37A R38A) or in the putative NLS2 yieldingthe individual mutants NLS2(K219A R220A) or NLS2(R231AR232A) or a two-step mutation of first NLS2(K219A R220A)and then NLS2(R231A R232A) did not eliminate the bindingof the A/Udorn/72 NS1A protein to importin �1. In contrast,when both NLS1 and NLS2 sequences were simultaneouslymutated [NLS1(R37A R38A) plus NLS2(K219A R220A) orNLS1(R37A R38A) plus NLS2(R231A R232A)], A/Udorn/72NS1A protein binding to the importin �1 was eliminated. Thesame result was obtained with E. coli-expressed GST-importin�6 (data not shown). These results show that both NLS1 andNLS2 mediate importin � binding, and that NLS2 likely func-tions as a classical bipartite NLS consisting of a proximal part,K219 and R220, and a distal part, R231 and R232.

To identify the amino acids in the NLS1 of the A/Udorn/72 NS1A protein that are required for importin � binding,single mutations were made in this sequence on top of theNLS2 mutant construct [NLS2(K219A R220A) followed byNLS2(R231A R232A)]. In this context, single point mutations,either R35A, R38A, or K41A, completely eliminated importin�1 binding, whereas K20A, R37A, or R44A single mutationsdid not significantly reduce binding (Fig. 3B). These resultswere fully confirmed with binding to E. coli-expressed GST-importin �6 or Sf9-expressed GST-importins �5 and �7 (datanot shown). It should be noted that R38, which is required forimportin � binding, is also the amino acid that is absolutelyrequired for dsRNA binding (57).

The binding of the influenza virus A/WSN/33 NS1A proteinto importin � is mediated solely by the NLS1 in the dsRNA-binding domain. Since the NS1A protein of influenzaA/WSN/33 has a stop codon at position 230, it lacks the puta-tive NLS2. Consequently, it would be expected that mutationsin the NLS1 of this NS1A protein would eliminate importin �binding. To determine whether this is the case, we carried outpull-down experiments with E. coli-expressed GST-importin�1 and [35S]methionine-labeled A/WSN/33 NS1A protein with

point mutations in the NLS1 sequence (Fig. 3C). Mutations ofR35A, R38A, or K41A completely eliminated the binding ofthe WSN NS1A protein to importin �1. These results wereconfirmed with binding to importin �6 (data not shown). Theseare the same amino acids that are required for the binding ofNLS1 of A/Udorn/72 to importin �. These results also showthat the A/WSN/33 NS1A protein lacks a C-terminal NLS2that binds importin �. The two conserved basic amino acids,K219 and R220, that are part of NLS2 in the A/Udorn/72 NS1protein are not able to mediate importin � binding withoutamino acids R231 and R232. Alignment of human influenza Avirus NS1A protein C termini revealed that many virus strainslack the C-terminal NLS2 (Fig. 2B).

Importin �1 does not compete with dsRNA for binding theNS1A protein. Based on previous site-directed mutagenesisexperiments, R38 and K41 in the NS1A protein are the criticalamino acids for dsRNA-binding (57). As R38 and K41 are alsoessential in the binding of NS1A to importin �1 (Fig. 3), westudied by direct binding experiments using highly purifiedproteins whether dsRNA would compete with importin �1 forbinding to the NS1A protein. Our results clearly show thatimportin �1 does not compete with dsRNA for binding to theNS1A protein (Fig. 4). This was the case regardless of theorder of addition of dsRNA and importin �. Since bothdsRNA and importin � can bind to NS1A simultaneously, wecan infer that the binding sites of these molecules are actuallynot totally overlapping.

FIG. 4. Importin �1 does not compete with dsRNA for binding tothe NS1A protein. (A) Gel shift assay of complexes formed betweenradiolabeled 55-bp dsRNA and C-terminally deleted A/Udorn/72NS1A(1–215) protein in the absence and presence of importin �1. Theindicated polypeptides [40 or 400 nM of NS1A(1–215) and 4 to 1,600nM of importin �1] were incubated with the 55-bp dsRNA (10,000cpm; 1 nM), and the polypeptide-RNA complexes were separatedfrom free RNA by nondenaturing gel electrophoresis (6% acrylamide;bis:acrylamide 1:100). (B) Gel shift assay of complexes formed be-tween radiolabeled 55-bp dsRNA and importin �1. The last lane showsthe complex formed between this dsRNA and the NS1A(1–215) pro-tein.



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NS1A proteins containing mutated NLS sequence(s) fail toaccumulate in the nucleus. To determine the intracellular lo-cation of wt and NLS mutant A/Udorn/72 NS1A proteins,HuH7 hepatoma cells were transiently transfected with plas-mids encoding these NS1A proteins, followed by analysis withlaser scanning confocal microscopy (Fig. 5A). wt A/Udorn/72NS1A proteins, as well as the NLS1 or NLS2 mutant proteins,were found in the cell nucleus (Fig. 5A). However, when boththe NLS1 protein carried the mutation NLS1(R35A R38A)and/or NLS1(K41A) and the NLS2 protein carried the muta-

tions NLS2(K219A R220A) plus NLS2(R231A R232A), theA/Udorn/72 NS1A protein was distributed throughout the cy-toplasm, and only weak nuclear accumulation was seen. Incontrast, the A/Udorn/72 NS1A protein was localized in thenucleus when the NLS2 mutations were combined with thesingle point mutations K20A, R37A, or R44A. In the case ofthe NS1A protein of A/WSN/33, mutations within the NLS1,specifically, the single point mutations R35A, R38A, and K41Aand the double mutation R38A K41A, were sufficient to renderNS1A protein predominantly cytoplasmic with only weak nu-clear staining (Fig. 5B). The intracellular distribution of allNS1A mutant proteins fully correlated with their ability to bindto importin � (compare Fig. 3 and 5). However, it was unex-pected that some NLS mutant NS1A proteins that lacked im-portin � binding still weakly entered the nucleus. Since NS1Aprotein is a relatively small protein, it is possible that some ofit can passively diffuse into the nucleus.

Surprisingly, the NLS2 of the NS1A protein of influenzaA/Udorn/72 is retained after deletion of amino acids 231 to237. Based on the above results, it may be predicted thatdeletion of the amino acid extension (residues 231 to 237) ofthe NS1A protein of the influenza A/Udorn/72 virus woulddisrupt the bipartite NLS2 and thereby eliminate the C-termi-nal sequence of the NS1A protein from binding to importin �.To test this prediction, we carried out GST-importin pull-downexperiments with the NS1A�231–237 protein that also carriedthe mutant NLS1(R37A R38A) (Fig. 6A). Unexpectedly, thismutant NS1A protein still efficiently bound to GST-importin�6. In addition, this mutant NS1A protein was localized in thenucleus (Fig. 6B). This result indicated that the truncatedC-terminal region retained NLS2 function, presumably as amonopartite NLS. To identify specific basic amino acids thatmediate importin �6 binding, we changed both K219 and R220to A residues in the NS1A�231-237(K37A K38A) mutant pro-tein. These additional mutations eliminated both importin �binding and nuclear localization. Consequently, the retainedNLS2 in the Udorn NS1A protein required K219 and R220.However, additional basic amino acids are needed because theWSN NS1A protein, which lacks the C-terminal extension,contains K219 and R220 but lacks NLS2 function. Two otherbasic amino acids (R224 and K229) are also required for NLS2function (data not shown). WSN NS1A protein has a G ratherthan an R at position 224. In addition, other H1N1 type viruseshave a negatively charged E rather than a positively charged Kat position 229 (see Fig. 2B). In particular, a negatively chargedamino acid within an NLS/NoLS seems to eliminate all bindingcapacity.

Localization of the NS1A protein in influenza A virus-in-fected cells: identification of a C-terminal NoLS. Analysis ofthe intranuclear localization of the NS1A protein in virus-infected A549 cells revealed that in A/Udorn/72-infected cells,the NS1A protein was found in not only the nucleus but alsothe nucleolus at 6 to 12 h after infection (Fig. 7A). In contrast,in A/WSN/33-infected cells the NS1A protein localized in thenucleus but failed to accumulate significantly in the nucleolus.These results suggested that the C-terminal end of the UdornNS1A protein containing the NLS2 was responsible for thenucleolar localization of its encoded NS1A protein. To test thispossibility, we generated a recombinant A/Udorn/72 virus thatencoded an NS1A protein with a deletion in its C-terminal end

FIG. 5. Point mutations to NLSs regulate nuclear/cytoplasmic dis-tribution of the influenza A virus NS1A protein. (A) HuH7 cells weretransiently transfected with wt or NLS mutant (mt) influenzaA/Udorn/72 NS1A gene constructs as indicated in the figure. NLS1,NLS1(R37A R38A); NLS2, NLS2(K219A R220A) plus NLS2(R231AR232A). (B) HuH7 cells were transiently transfected with wt or NLSmutant (mt) influenza A/WSN/33 NS1A gene constructs as indicatedin the figure. The cells were stained with rabbit anti-NS1A protein andsecondary rhodamine-labeled anti-rabbit antibodies. Bar, 5 �m.



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(NS1A�221–237). This virus was not attenuated, and it repli-cated with the same kinetics as wt Udorn virus. However, themutant NS1A�221–237 protein completely failed to accumu-late in the nucleolus (Fig. 7A), demonstrating that the C-terminal 17 amino acids of the A/Udorn/72 virus NS1A proteincontains a functional NoLS.

Because the C-terminal extension (residues 231 to 237) isnot required for the NLS2 function of the Udorn NS1A pro-tein, it was possible that this extension is also not required forNoLS function. To test this possibility, we utilized three H3N2-type viruses (A/Beijing/353/89, A/Finland/229/92, and A/Fin-land/455/97), each of which encodes an NS1A protein thatlacks the C-terminal extension but has an amino acid sequencefrom position 219 to 230 that is identical to that of A/Udorn/72(Fig. 2B). In A549 cells infected with these H3N2 viruses,nucleolar localization of the NS1A protein was observed in 78to 91% of the cells (Fig. 7B). This is similar to the results withA/Udorn/72, whose NS1A protein contains the C-terminal ex-

tension. These results indicate that the shared sequence from219 to 230 in the NS1A proteins of H3N2 viruses is sufficientfor NoLS function, as is the case for NLS2 function (seeabove). This was not the case for influenza A/WSN/33 (Fig.6A), as well as for two other H1N1 viruses that lack the C-terminal extension (A/Finland/432/96 and A/New Caledonia/20/99) (Fig. 2B and 7B). In A549 cells infected with these

FIG. 6. C-terminal deletion mutant influenza A/Udorn/72 NS1A�231–237 protein binds to importin �6. (A) 35S-labeled and in vitro trans-lated A/Udorn/72 NS1A wt and three mutant proteins were allowed tobind to E. coli-expressed and Sepharose-immobilized GST and GST-importin �6 proteins as indicated in the figure. Importin-bound NS1Awas dissolved in Laemmli sample buffer, separated by 12% SDS-PAGE, and autoradiographed. A similar gel was also stained withCoomassie blue to visualize the amount of Sepharose-immobilizedGST and GST-importin (GST-imp) �6 proteins. For the translationcontrol, 1 �l of 35S-labeled and in vitro translated NS1 wt and threemutants were separated by 12% SDS-PAGE and autoradiographed.(B) HuH7 cells were transiently transfected with wt or C-terminaldeletion mutant influenza A/Udorn/72 NS1A gene constructs as indi-cated in the figure. NLS1, NLS1(R37A R38A); NLS2, NLS2(K219AR220A). Bar, 5 �m. FIG. 7. Intracellular localization of NS1A protein during influenza

A virus infection. (A) A549 cells grown directly on coverslips wereinfected with wt influenza A/Udorn/72, recombinant A/Udorn/72NS1A�221–237 with a stop codon at position 221 of the NS1A proteingene, or wt A/WSN/33 viruses for 4 to 24 h as indicated in the figure.After fixation, the cells were stained with rabbit anti-NS1A and fluo-rescein isothiocyanate-labeled anti-rabbit antibodies, followed by anal-ysis with confocal laser microscopy. Bar, 5 �m. (B) A549 cells wereinfected with different wt H3N2 or H1N1 influenza A virus strains asindicated in the figure. Nuclear and nucleolar localization of NS1Aprotein was detected at 6 and 12 h after infection by indirect immu-nofluorescence microscopy. For each virus the percentage of cellsexpressing NS1A protein in the nucleolus was calculated from 300NS1A protein-expressing cells.



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H1N1 viruses, only weak nucleolar localization of the NS1Aprotein was observed in approximately 7 to 15% of the cells.Consequently, the NS1A sequence from residue 219 to 230 ofthese H1N1 viruses, which lack basic amino acids at positions224 and 229 (Fig. 2B), does not exhibit an NoLS function. Infact, even with the H1N1 viruses whose NS1A proteins containthe C-terminal extension (residues 231 to 237), for example,A/Finland/001/79 and A/Finland/40/86, nucleolar localizationis weaker than that observed with H3N2 viruses: nucleolarlocalization was observed in only approximately 25 to 41% ofthe infected cells. These results indicate that the basic residuesat positions 224 and 229 in the NS1A protein of H3N2 virusesfunction in the C-terminal NoLS of the NS1A protein.

C-terminal arginines and lysines form the functional NoLSof the NS1A protein. To verify the identity of the amino acidsthat constitute the NoLS of the NS1A protein of A/Udorn/72,we constructed plasmids expressing GFP fusion proteins con-taining the C-terminal amino acids 203 to 237 of this virus[GFP-NS1A(203–237)]. This plasmid was transiently trans-fected into HuH7 cells, and the subcellular localization of theGFP-containing proteins was analyzed by fluorescence micros-copy (Fig. 8A). GFP expressed alone was distributed through-out the cells. In contrast, GFP-NS1A(203–237) accumulatedpredominantly in the nucleoli. Coexpression of this proteinand the HIV-1 Rev protein, which has previously been local-ized into the nucleolus (29), showed an excellent colocaliza-tion, establishing that GFP-NS1A(203–237) is indeed localizedin the nucleoli (Fig. 8A, lower panels). Double mutations,K219A R220A, R224A K229A, or R231A R232A, in theNS1A(203–237) sequence of the GFP fusion protein destroyedthe NoLS, since these mutant proteins failed to accumulateinto the nucleoli (Fig. 8A). Interestingly, the GFP-NS1A(203–230) protein, which lacks residues 231 to 237 (RRDKMAD) ofthe C-terminal extension, maintained its nucleolar localization,although a bit weakened, indicating that the presence of theC-terminal extension is not absolutely required for nucleolarlocalization.

As indicated by the results shown in Fig. 7, the NS1A proteinof the H1N1 A/WSN/33 virus, which lacks a C-terminal exten-sion, possess only a weak functional NoLS. Consistent withthese results, the GFP-NS1A(203–230) A/WSN/33 fusion pro-

FIG. 8. C-terminal end of NS1A protein in H3N2 type influenza Aviruses encode a functional NoLS. C-terminal fragments of NS1Agenes encoding amino acids 203 to 237 in A/Udorn/72 and amino acids

203 to 230 in A/WSN/33 were inserted into GFP expression vectorpCMX-SAH/Y145F to express GFP-NS1A fusion proteins [GFP-NS1A(9203-230/237)]. (A) HuH7 cells were transiently transfectedwith GFP-wt, mutant, or deletion A/Udorn/72 NS1A gene constructsfor 48 h as indicated in the figure. The intensity of nucleolar localiza-tion was scored by immunofluorescence microscopy as no nucleolarstaining (�) or weak (�), moderate (��), or strong (��) nucleolarstaining. Critical basic amino acids involved in nuclear/nucleolar tar-geting are marked in boldface and underlined. Mutated amino acidsare marked in red. To verify nucleolar localization, HuH7 cells weretransiently transfected with GFP-wt NS1A(203–237) A/Udorn/72 andHIV-1 Rev gene constructs for 48 h as indicated in the figure. Afterfixation the cells were stained with anti-HIV-1 Rev antibodies, andcolocalization with GFP-NS1A(203–237) protein was detected withconfocal microscopy. (B) HuH7 cells were transiently transfected withGFP-wt and mutant A/WSN/33 NS1A gene constructs for 48 h asindicated in the figure. Critical and mutated amino acids are marked asabove. Bars, 5 �m.



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tein completely failed to accumulate in the nucleoli (Fig. 8B).Next, we determined whether NoLS function could be acti-vated by the introduction of basic residues at 224 and/or atboth 224 and 229. Mutation of G224 to R led to partial nucle-olar localization, whereas mutation of both G224 and E229 tobasic residues (R and K, respectively) resulted in full nucleolarlocalization. Consequently, basic residues at both of these po-sitions were sufficient for a functional NoLS.

Next we concentrated on analyzing the role of the C-termi-nal extension in nucleolar targeting. The C-terminal extension(residues 231 to 237) by itself did not render the GFP-NS1A(203–230) A/WSN/33 fusion protein nucleolar (Fig. 8B).However, if G224 was mutated to R, this C-terminal extensionrendered the protein faintly nucleolar. These results, cou-pled with those described above, demonstrate that the NoLSof the NS1A protein is comprised of basic residues at posi-tions 219, 220, 224, and 229, and when the NS1A protein hasa C-terminal extension, basic residues at positions 231 and232 are also part of the NoLS and will strengthen its func-tion (Fig. 8B and 9A).

DISCUSSION

In the present study we show that influenza A virus NS1Aprotein has a strong arginine- or lysine-rich NLS (NLS1) in theN-terminal part of the molecule, which is very well conservedamong all known avian, human, and other mammalian NS1Aproteins. NLS1 was found to mediate an interaction of theNS1A protein with all six human importin � isoforms, indicat-ing that the nuclear import of the NS1A protein takes place viathe classical importin �/� nuclear import pathway. This is likelyto happen in all types of cells, since different importin � iso-forms, although not always all of them, are expressed in all cells(21). The critical amino acids regulating the functionality ofNLS1 were identified to be R35, R38, and K41. It is remark-able that the amino acids which form the NLS1 of the NS1Aprotein are also the same ones that participate in dsRNA-binding activity of the protein (16, 28, 57, 59). Previous site-directed mutagenesis experiments have shown that R38 andK41 are the most essential ones for dsRNA-binding (57).

Because of these mutagenesis results, it was surprising thatthe NS1A protein was able to bind to dsRNA and importin �simultaneously. This finding suggests that although the mostimportant amino acids comprising the NLS1 (R35, R38, andK41) and dsRNA binding (R38 and K41) sites of the NS1Aprotein are practically the same, the binding sites of dsRNAand importin � are not totally overlapping, enabling both mol-ecules to bind to the NS1A protein at the same time. Hope-fully, future structural analyses will reveal the exact bindingsites of dsRNA and importin � on the NS1A protein. It isnoteworthy that relatively high concentrations of these mole-cules are required before an interaction between these mole-cules can be demonstrated, suggesting a relatively low bindingaffinity for both dsRNA and importin � binding to the NS1Aprotein. In fact, a low affinity of the NS1A RNA-binding do-main for dsRNA has previously been demonstrated (7). None-theless, it is likely that the RNA-binding domain of newlysynthesized NS1A protein molecules is efficiently bound toimportin � molecules that mediate nuclear import, because atmost times of infection the vast majority of the NS1A protein

is found in the nucleus (Fig. 8). Based on our competitionexperiments, these NS1A proteins would be capable of import-ing dsRNA molecules into the nucleus.

In addition to NLS1 which overlaps with the dsRNA-bindingdomain, the C terminus of the NS1A proteins of H3N2 andH2N2 viruses contains an NLS2, which also functions as anNoLS, thereby localizing these NS1A proteins in the nucleolus(data summarized in Fig. 9). To further verify that the C-

FIG. 9. (A) Schematic representation of NS1A intracellular target-ing signals. Both WSN and Udorn virus NS1A proteins have an NLSconstituting basic residues 35, 38, and 41 (NLS1). The Udorn NS1Aprotein also has a 7-amino-acid C-terminal extension (residues 231 to237) and a second NLS (NLS2) at the end of the molecule, constitutingarginines or lysines at positions 219, 229, 224, 229, 231, and 323. Thesame residues form a functional NoLS. NLS and NoLS signals areshown in bold and underlined. (B) Mechanisms of nuclear import ofUdorn virus NS1A protein. Newly synthesized dimeric Udorn NS1Aprotein interacts via its NLS1 or NLS2 with different cytoplasmicimportin (Imp) � (all 6 isotypes)/importin � complex, followed bynuclear translocation of the complex via the NPC. Most cytoplasmicNS1A is likely bound to importin � interfering with dsRNA binding toNS1A in the cytoplasm. In the nucleus NS1A protein is released fromthe transport complex, and importin � and importin � are transportedback into the cytoplasm through the NPC. After the release of impor-tin �, the C-terminal NoLS of the NS1A protein is exposed, andthe NS1A protein is targeted into the nucleolus. Simultaneously, thedsRNA-binding domain of the NS1A protein is also exposed, and theprotein becomes competent to bind dsRNA. This leads to sequestra-tion of dsRNA and lack of activation of the oligoadenylate synthetase/RNase L antiviral pathway. RBD, dsRNA-binding domain.



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terminal NLS2 also functioned as an NoLS during virus infec-tion, we generated a recombinant H3N2 Udorn virus encodingan NS1A protein that lacks the last 17 C-terminal amino acids.The NS1A protein expressed by the wt Udorn virus localized inthe nucleolus, whereas the NS1A protein expressed by themutant virus failed to do so. From an evolutionary point ofview, it was of interest that the C-terminal extension of 7 aminoacids (amino acids 231 to 237) was found in viruses isolatedbetween the years 1950 and 1989. This extension existed in notonly the H3N2 NS1A proteins but also H1N1 and H2N2 vi-ruses as well (Fig. 2). A functional NLS2/NoLS of the UdornNS1A protein required the basic arginine or lysine residues atpositions 219, 220, 231, and 232. Evolutionary and mutationalanalyses (Fig. 2 and 8) revealed that R at position 224 and K atposition 229 also played a role in the formation of a functionalNLS2/NoLS. When the residues 224 and 229 were R and K,respectively, the NLS2/NoLS function was not lost, even if theC-terminal extension was eliminated by a stop codon at posi-tion 231 in the NS1A reading frame of the H3N2 viruses, aswas the case in viruses that were isolated after the year 1989.An additional interesting observation was that, when the argi-nines at positions 231 and 232 were mutated to alanines, theNLS2/NoLS function was lost even if residues 224 and 229were intact. This may indicate that the mutated C-terminalextension (residues 231 to 237) of NS1A protein may fold insuch a way that it interferes with the interaction of basic resi-dues 224 and 229 with importin �. Consequently, our dataindicate that the C-terminal extension with its basic residues atpositions 231 and 232 was able to create a functional secondNLS and NoLS for the NS1A protein. However, this extensionwas no longer needed for NLS2/NoLS function, if the C ter-minus contained an arginine and a lysine at positions 224 and229, respectively, as was the case in the evolutionary lineage ofH3N2 type viruses (Fig. 2).

In contrast, the H1N1 NS1A proteins, which contain theC-terminal extension, show only weak nucleolar localization,which is lost when the C-terminal extension was eliminated inH1N1 viruses isolated after the year 1989. The absence of anNoLS was thus traced to the absence of basic residues atpositions 224 and 229. In addition, the C-terminal end of theH5N1 (avian virus) NS1A protein resembles the sequences ofthe present H1N1 type viruses, and is thus likely to lack theC-terminal NLS2/NoLS.

The most likely interpretation of our results is that the im-portant function of the C-terminal region of the NS1A proteinsof the H3N2 and H2N2 viruses is to target these NS1A pro-teins to the nucleolus. Nucleolar localization of the NS1Aprotein is not unique, since many other viruses apart frominfluenza A virus encode proteins that are targeted into thenucleolus (17). The nucleolar localization of dengue, Kunjin,and Japanese encephalitis virus core proteins apparently playsa critical role in virus replication and pathogenesis in mamma-lian cells (35, 56, 58). It is possible that viral proteins localizedin the nucleolus interfere with normal cell cycle regulationand/or associate with or reorganize nucleolar proteins likenucleolin, B23, and fibrillarin (17). Nucleolar sequestration ofregulatory proteins may be another mechanism by which viralproteins regulate cellular gene expression, cell proliferation,and apoptosis (1, 4, 51). It has been shown previously thatlocalization of the influenza virus NS1A protein in the nucle-

olus inhibits rRNA synthesis (23). Our working hypothesis isthat the primary roles of the nucleolar localization of H3N2and H2N2 NS1A proteins are on host functions and thus onpathogenesis, particularly because the H3N2 Udorn virus thatexpresses an NS1A protein lacking its 17 C-terminal aminoacids does not exhibit any apparent defect during replication intissue culture experiments (unpublished data). Consequently,an important future question will be to determine whethernucleolar localization of the NS1A protein plays a role in thepathogenesis of H3N2 and H2N2 influenza A virus infections.

ACKNOWLEDGMENTS

We thank Reijo Pyhala for the viruses and valuable discussions. Wealso thank Sinikka Sopanen and Raija Tyni for providing us with thecells, Anja Villberg and Riitta Santanen for growing different influenzaviruses, and Hanna Valtonen and Johanna Lahtinen for their excellenttechnical assistance.

This study was supported by the Medical Research Council of theAcademy of Finland and the Sigrid Juselius Foundation (I.J.) and bygrant AI11772 from the National Institutes of Health of the U.S.Department of Health and Human Services (R.M.K.).

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