Influenza Virus NS1 Protein Induces Apoptosis in Cultured Cells

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Influenza Virus NS1 Protein Induces Apoptosis in Cultured Cells

STACEY SCHULTZ-CHERRY,1_{* NAOMI DYBDAHL-SISSOKO,}2_{GABRIELE NEUMANN,}3 YOSHIHIRO KAWAOKA,3_{ANDVIRGINIA S. HINSHAW}3

Southeast Poultry Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Athens, Georgia 306051_;

Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention,

Atlanta, Georgia 303332_{; and Department of Pathobiological Sciences, School of Veterinary Medicine,}

University of Wisconsin—Madison, Madison, Wisconsin 537063

Received 2 April 2001/Accepted 24 May 2001

The importance of influenza viruses as worldwide pathogens in humans, domestic animals, and poultry is well recognized. Discerning how influenza viruses interact with the host at a cellular level is crucial for a better understanding of viral pathogenesis. Influenza viruses induce apoptosis through mechanisms involving the interplay of cellular and viral factors that may depend on the cell type. However, it is unclear which viral genes induce apoptosis. In these studies, we show that the expression of the nonstructural (NS) gene of influenza A virus is sufficient to induce apoptosis in MDCK and HeLa cells. Further studies showed that the multimer-ization domain of the NS1 protein but not the effector domain is required for apoptosis. However, this mutation is not sufficient to inhibit apoptosis using whole virus.

Apoptosis is essential in many physiological processes, in-cluding tissue atrophy, development of the immune system, and tumor biology (19, 21, 28, 73). Apoptosis also plays an important role in the pathogenesis of many infectious diseases, including those caused by viruses (4, 27, 46, 48). Many virus infections result in apoptosis of host cells, and several viruses have evolved mechanisms to inhibit apoptosis (52, 62). Al-though there is no obvious advantage for the induction of apoptosis by a cytopathogenic virus, influenza viruses induce apoptosis in numerous cell types both in vivo (29) and in vitro (6, 18, 24, 30, 39, 49, 50, 57).

Influenza viruses induce apoptosis in cells that are permis-sive for virus replication like macrophages, Madin-Darby Ca-nine Kidney (MDCK) and mink lung epithelial (Mv1Lu) cells (18, 24, 30, 37), and cells which do not support viral replication, such as HeLa cells or lymphocytes. The mechanism of influ-enza virus-induced apoptosis is not known in detail. However, it appears to involve both cellular and viral factors and may depend on the cell type. Influenza virus-induced apoptosis is inhibited bybcl-2(37), v-FLIP, and crmA(59) and involves caspase activation (59). There is also evidence for indirect activation of apoptosis during infection. In HeLa cells, Fas antigen, a transmembrane protein belonging to the tumor ne-crosis factor receptor superfamily (36), and the Fas ligand are upregulated during influenza virus infection and are partially responsible for apoptosis in infected cells (10, 54–56, 67). Through these studies, we have a better understanding of which cellular pathways may be involved in influenza virus-induced apoptosis. However, it is still unclear which viral genes induce apoptosis in cells that support productive viral replica-tion.

It is possible that the expression of any of the individual influenza virus genes may induce apoptosis in the infected cell.

The neuraminidase protein (NA) appears to induce apoptosis through indirect and direct mechanisms. Indirectly, NA acti-vates transforming growth factor ␤ (TGF-␤) in vivo and in vitro (49). TGF-␤is a multifunctional growth-regulatory pro-tein that induces apoptosis in many cell types, including lym-phocytes (23) and MDCK cells (45, 49). Neutralizing antibod-ies against TGF-␤only partially inhibit influenza virus-induced apoptosis, suggesting that NA can also induce apoptosis di-rectly. These findings were further supported by Morris et al. (30), who showed that NA induces apoptosis in different cell lines by a TGF-␤-independent, virus-dependent mechanism.

In MDCK cells, apoptosis occurs early in the course of viral replication (18). Therefore, it is likely that viral genes ex-pressed early in replication and that interfere with normal cellular processes or associate with cell proteins involved in apoptosis may induce apoptosis directly. In these studies, we focused on the role of the nonstructural (NS) gene.

The NS gene is the smallest segment of the influenza A virus genome and is transcribed into a colinear mRNA encoding two proteins, NS1 and NS2 (also called NEP) (22, 38). Unlike NEP, NS1 is found only in infected cells. NS1 regulates nu-merous cellular functions during influenza virus infection by binding to polyadenylated mRNAs, inhibiting nuclear export (3, 16, 26, 40, 43); binding to small nuclear RNAs (snRNA), specifically to key components of the spliceosome, blocking pre-mRNA splicing (2, 8, 25, 44, 69) and inhibiting the poly-adenylation of host cell mRNA (31); and interacting with sev-eral host cell proteins (26, 31, 71, 72). The RNA-binding ac-tivities of NS1 are based on the interaction of two functional domains: an RNA-binding domain at the amino end of the protein (amino acids 19 to 38) that binds to poly(A) sequences in mRNAs (43), and an effector domain (amino acids 134 to 161) that interacts with cellular proteins to inhibit mRNA nuclear export (40). These domains are highly conserved within the NS1 gene (20, 68), suggesting that NS1 is evolution-arily conserved.

Arguably, one of NS1’s most important functions is inhibit-ing the activation of the double-stranded RNA (dsRNA) ki-* Corresponding author. Mailing address: Southeast Poultry

Re-search Laboratory, USDA-ARS, 934 College Station Rd., Athens, GA 30605. Phone: (706) 546-3464. Fax: (706) 546-3161. E-mail: sschultz @seprl.usda.gov.

7875

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nase (PKR), thus preventing the interferon (IFN)-mediated antiviral response (11, 12, 17, 26). Takizawa et al. showed that a mutation in the catalytic domain of PKR partially suppresses influenza virus-induced cell death (58). Based on the ability of NS1 to interfere with host cell RNA functions and block the activation of PKR, we propose that NS1 is involved in influ-enza virus-induced apoptosis. These studies show that expres-sion of NS1 in different cell types is sufficient to induce cell death. Further, using NS1 mutants, we show that the RNA-binding/dimerization domain but not the effector domain is required for NS1-induced apoptosis in cell culture.

MATERIALS AND METHODS

Virus growth and cell culture. A/Turkey/Ontario/7732/66 (A/Ty/Ont/66) (H5N9; University of Wisconsin influenza virus repository) was propagated in the allantoic cavities of 10- or 11-day old embryonated chicken eggs for 48 h at 35°C. The allantoic fluid was harvested, centrifuged for clarification, and stored at⫺70°C.

MDCK and HeLa cells were grown in modified Eagle’s medium (MEM; Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal bovine serum (FBS; Life Technologies) and 2 mM glutamine. All cells were maintained at 37°C in 5% CO2.

Construction of plasmids.Full-length NS gene from A/Ty/Ont/66 was PCR amplified, ligated into the TA vector (Invitrogen, Carlsbad, Calif.), and then subcloned into the expression plasmid pUHD 10-3 (kindly provided by Hermann Bujard) at theEcoRI site. Expression vector pUHD 10-3 contains the heptamer-ized tetracycline repressor gene operators upstream of the cytomegalovirus pro-moter in two different orientation sites (15, 47, 51). Clones were screened for proper orientation by restriction enzyme digestion and sequence analysis.

All of the A/Udorn/72 NS1 genes described in this paper were expressed using an NS1 gene containing a 3⬘splice site mutation (NS13⬘SS) ligated into the pBC12 vector via theBamHI site as described previously (2, 40, 68, 70). All mutations were confirmed by dideoxynucleotide sequencing. The cDNAs of NS13⬘SS (NS1), NS13⬘SS DM (no NS1 produced), NS1 M2 mutant (R19 and K20 changed to A [RK 19/20 AA]) and NS1⌬117-161 (Table 1) were cloned into theBamHI site of pUHD 10-3. Plasmids were transformed intoEscherichia coli

DH5␣ competent cells (Life Technologies), amplified, and purified using a Qiagen (Valencia, Calif.) purification kit.

Reassortant virus.Two viruses containing a mutation in the NS1 RNA-bind-ing domain (RK 19/20 AA or RK 19/20 AD) were constructed in an A/WSN/33 backbone by reverse genetics as described elsewhere (33, 34). Parental WSN and mutant viruses were propagated in MDCK cells grown in MEM containing 5% bovine serum albumin (BSA) and 1␮g ofL-1-p-tosylamino-2-phenylethyl chlo-romethyl ketone–trypsin (Sigma Immunochemicals, St. Louis, Mo.) per ml. Viral titers were determined by 50% tissue culture infective dose.

Stable expression system. The pUHD 10-3 expression plasmid containing full-length NS, NS1, and NS1 mutants and the transcriptional transactivator (pUHD 15-1) expression plasmid containing the tetracycline repressor gene fused with the viral VP16 coding sequence containing the neomycin cassette (kindly provided by Wen-Hwa Lee) (51) were electroporated into MDCK or HeLa cells. Briefly, cells were electroporated with 10␮g of DNA of both plas-mids, and G418-resistant cell lines expressing the protein of interest were se-lected. The cell lines were maintained in MEM containing 10% FBS in the presence of 2.5␮g of anhydrotetracycline per ml (14) and 400␮g of G418 per ml. Incubating the cells in tetracycline-free medium induced expression of the full-length or mutant NS gene.

DNA fragmentation assay.Fragmentation of cellular DNA into the charac-teristic apoptotic ladder was assessed as previously described (49), with minor modifications. Briefly, confluent monolayers of cells expressing pUHD 10-3 expression vector, NS gene, or NS1 mutants were washed with phosphate-buff-ered saline (PBS) and incubated for various times in tetracycline-free MEM containing 1% FBS. Prior to processing of the DNA, cell monolayers were trypsinized and cell numbers were determined. DNA was harvested by centri-fuging the cells, resuspending the cell pellet in 300␮l of cold cell lysis buffer (10 mM Tris, 0.5% Triton X-100 [pH 7.5]), and incubating it on ice for 30 min. The lysates were centrifuged at 12,500 rpm for 10 min at 4°C, and the supernatants were extracted once with buffered phenol and once with chloroform. The DNA was precipitated with 300 mM NaCl and ethanol. DNA samples were resus-pended in 15␮l of Tris-EDTA buffer (10 mM Tris, 1 mM EDTA [pH 7.5]) treated with RNase A (Life Technologies). Equal cell numbers were loaded and electrophoresed through a 2% SeaKem GTG agarose gel FMC Bioproducts, Rockland, Maine). The gel was stained with ethidium bromide to visualize DNA fragmentation. Cell viability was also assessed by trypan blue exclusion. Unin-fected cells and cells inUnin-fected with A/Ty/Ont/66 collected at identical times served as controls.

Western blot analysis.Infected or DNA-transfected cell monolayers were lysed, and total protein concentration was determined by the bicinchoninic acid assay (Pierce, Rockford, Ill.). Equal protein concentrations were heated for 3 min at 100°C in sample buffer containing␤-mercaptoethanol and resolved by sodium dodecyl sulfate–5 to 20% gradient polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to nitrocellulose, the proteins were blocked with 1% powdered milk in Tris-buffered saline with 0.1% Tween 20 (TTBS; Sigma Immunochemicals) for 30 min at room temperature. The nitrocellulose was probed for NS protein expression using mouse monoclonal or rabbit polyclonal antibodies diluted 1:1,000 in TTBS and incubated for 1 h at room temperature. Proteins were detected by incubation with a secondary antibody conjugated to horseradish peroxidase followed by enhanced chemiluminescence (Amersham, Arlington Heights, Ill.) according to the manufacturer’s protocols.

RESULTS

Expression of NS proteins induces apoptosis.Previous

stud-ies showed that HeLa cells undergo apoptosis during influenza virus infection. To determine if the expression of NS1 and NS2 was sufficient to induce apoptosis, HeLa cells were transfected with the full-length NS gene from A/Ty/Ont/66 in a tetracy-cline-regulated system, and cells expressing NS proteins were selected and screened for protein expression by Western blot analysis. NS protein expression was evident 24 h after tetracy-cline withdrawal and increased slightly at 48 h. NS protein levels in the NS-expressing cells were much lower than levels in infected cells (Fig. 1A). In spite of low protein levels, DNA fragmentation was observed in NS-expressing cells within 8 h after the removal of tetracycline (Fig. 1B). DNA fragmentation was not observed in HeLa cells in the presence of tetracycline. Similar studies were performed with MDCK cells. MDCK cells were transfected with the full-length NS gene from A/Ty/ Ont/66 in a tetracycline-regulated system, and two clones (NS D1 and NS D5) expressing NS proteins were selected. Similar to the case for HeLa cells, the levels of NS protein were much lower in all clones tested than in infected cells (Fig. 2A). In spite of low protein levels, NS D1 and NS D5 were tested for DNA fragmentation after the removal of tetracycline. In-creased fragmentation was observed in both NS D1 and NS D5 in a time-dependent manner (Fig. 2B). DNA fragmentation was observed as early as 5 h after the removal of tetracycline in clone D1; however, clone D5 had slower kinetics, and ladder-ing was not evident until 24 h after tetracycline removal (Fig. 2B). No DNA fragmentation was observed in MDCK cells expressing only vector after the removal of tetracycline. Similar results were observed in mink lung epithelial and chicken em-bryo fibroblast (CEF) cells (data not shown). These results TABLE 1. Description of NS mutants

Mutant Position of_{mutation (phenotype)}aaachange _localizationCellular

NS13⬘SS Only NS1 protein Nuclear

NS13⬘SS DM No NS1 protein

M2 aa 19 and 20 RK3AA (⫺) Nuclear

NS⌬117-161 aa 117–161 Deletion Nuclear

a_{aa, amino acid.}

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show that NS induces apoptosis in different cell types regard-less of their ability to support productive viral replication.

NS1 induces apoptosis.The NS gene of influenza virus

en-codes two proteins, NS1 and NEP (NS2). Attempts to express NEP in the tetracycline system were unsuccessful. Therefore, we examined the induction of DNA laddering in two different MDCK clones expressing NS1. Similar to expression of the NS proteins, NS1 expression induced DNA laddering in a time-dependent manner (Fig. 3A). Both clones (NS1 C4 and NS1 C9) showed clear apoptosis 24 h after the removal of tetracy-cline, with more intense laddering observed at 48 h. The ex-pression of NS1 protein is required for apoptosis in this system. Two cellular clones expressing an NS1 splice site mutation containing two stop sites (E8 and G4), which makes no NS1 protein, failed to induce DNA laddering after tetracycline withdrawal (Fig. 3B). No fragmentation was observed in the MDCK cells expressing vector alone. Similar to the NS protein analysis, the clones expressing NS1 had significantly less NS1 protein than did infected cells.

The effector domain of NS1 is not required for apoptosis.

NS1 has a number of described functional domains, including

an RNA-binding/dimerization domain and an effector domain that interacts with cellular proteins (3, 25, 26, 32, 40–44, 53, 68). To determine if either of these domains is required for the induction of apoptosis, MDCK cells expressing NS1 proteins with a mutation in the functional RNA-binding domain/dimer-ization (amino acids 19 and 20 mutated from RK to AA [M2]) or a deletion of the effector domain (amino acids 117 to 161) were tested for DNA fragmentation. The deletion of the ef-fector domain had no effect on DNA laddering (Fig. 3B). Fragmentation was observed at 5 h and then increased through 48 h, similar to clones expressing full-length NS1. In contrast, a mutation in the RNA-binding/dimerization functional do-main resulted in no DNA fragmentation even at 48 h after tetracycline removal (Fig. 3B). This result could be explained by the loss of NS1 protein expression in cells expressing the RNA-binding/dimerization domain. However, Western blot analysis shows that this clone was expressing NS1 protein within 5 h after removal of tetracycline and that protein levels were still detectable at 48 h (Fig. 3C). These studies show that expression of the NS1 protein is sufficient to induce apoptosis in numerous cell types by a mechanism that is independent of the effector domain.

FIG. 1. Expression of NS induces apoptosis in HeLa cells. (A) Confluent cultures of HeLa cells expressing pUHD 10-3 containing the NS gene from A/Ty/Ont/66 were washed two times with PBS and then incubated for 5, 24, or 48 h in medium with (⫹) or without (⫺) anhydrotetracycline. Cell monolayers were lysed, and 5 mg of total protein was loaded per lane under reducing conditions and resolved by SDS-PAGE. After being transferred to nitrocellulose, proteins were probed for NS with a mouse monoclonal antibody against NS. Bands were detected by enhanced chemiluminescence as instructed by the manufacturer. The arrow indicates the location of NS1. Molecular size markers are indicated on the left. MDCK cells infected with A/Ty/ Ont/66 at an MOI of 0.5 for 24 h served as a positive control (virus lane). (B) Confluent cultures of HeLa cells in 25-cm2_{flasks expressing} pUHD 10-3 containing the NS gene from A/Ty/Ont/66 were washed two times with PBS and then incubated for 8 or 24 h in medium containing anhydrotetracycline (⫹) or anhydrotetracycline-free me-dium (⫺) containing 1% FBS at 37°C in 5% CO2. DNA was collected and analyzed for DNA fragmentation by agarose gel analysis.

FIG. 2. Expression of NS induces apoptosis. (A) Confluent cultures of MDCK cells expressing pUHD 10-3 containing the NS gene from A/Ty/Ont/66 were washed two times with PBS and then incubated for 5, 24, or 48 h in medium without anhydrotetracycline. Cell monolayers were lysed, and 5 mg of total protein was loaded per lane under reducing conditions and resolved by SDS-PAGE. After being trans-ferred to nitrocellulose, proteins were probed for NS with a mouse monoclonal antibody against NS. Bands were detected by enhanced chemiluminescence as instructed by the manufacturer. The arrow in-dicates the location of NS1. Molecular size markers are indicated on the left. MDCK cells infected with A/Ty/Ont/66 at an MOI of 0.5 for 24 h served as a positive control (virus lane), and cells expressing empty vector (pUHD) served as a negative control. Results for two different clones (D1 and D5) are shown. (B) Confluent cultures of MDCK cells in 25-cm2_{flasks expressing pUHD 10-3 containing the NS} gene from A/Ty/Ont/66 or empty vector (pUHD) were washed two times with PBS and then incubated for 5, 24, or 48 h in anhydrotetra-cycline-free MEM containing 1% FBS at 37°C in 5% CO2. DNA was collected and analyzed for DNA fragmentation by agarose gel analysis. Two different clones, D1 and D5, expressing NS protein were analyzed.

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Reassortant WSN virus containing a mutation in the NS1

RNA-binding domain induces apoptosis. The generation of

the plasmid-based reverse genetics system provides a powerful opportunity to understand the importance of single gene mu-tations within the context of whole virus (33, 34). Based on the results described above, the RNA-binding/dimerization region of the NS1 protein is required for NS-induced apoptosis in MDCK cells. To determine if this mutation is sufficient to delete influenza virus-induced apoptosis, we attempted to gen-erate viruses whose NS1 proteins have mutations that abolish RNA-binding/dimerization activity. Two different mutations were generated, RK 19/20 AA and RK 19/20 AD. Both of the mutant viruses were infectious, although they were highly at-tenuated in MDCK cells compared to parental WSN virus, averaging a 3-log-lower virus titer. To determine if the mutant viruses could induce apoptosis in MDCK cells, cells were in-fected with a multiplicity of infection (MOI) of 2 and examined for DNA laddering at 24 and 48 h postinfection (p.i.). A high MOI was used to ensure maximum levels of apoptosis inde-pendent of replication differences between the parental strain and the mutant viruses. At 24 h p.i., the RK 19/20 AA mutant virus induced laddering similar to that observed with WSN virus (Fig. 4). In contrast, no laddering was observed at 24 hr p.i. with the RK 19/20 AD virus; however, both viruses induced apoptosis by 48 h, similar to the WSN parental virus. Similar results were seen in mink lung epithelial and CEF cells. These results suggest that while the RNA-binding/dimerization do-main is required for NS1-induced apoptosis, a mutation in this region is not sufficient to inhibit influenza virus-induced apo-ptosis.

DISCUSSION

The above results show that the expression of the NS1 pro-tein of influenza virus in MDCK and HeLa cells is sufficient to induce apoptosis. Western blot analysis showed that very little NS1 protein was expressed in the NS clones compared to influenza virus-infected cells. MDCK cells are difficult to trans-fect, and even after electroporation, only 1 to 10% of the surviving cells expressed NS. Additionally, the NS1 protein FIG. 3. The RNA-binding domain of NS1 is required for apoptosis.

(A) Confluent cultures of MDCK cells expressing pUHD 10-3 con-taining the NS1 gene from A/Udom/72 or empty vector (pUHD) were washed two times with PBS and then incubated for 5, 24, or 48 h in anhydrotetracycline-free MEM containing 1% FBS at 37°C in 5% CO2. DNA was collected and analyzed for DNA fragmentation by agarose gel analysis. Two different clones, C4 and C9, expressing NS1 protein were analyzed. (B) Confluent cultures of MDCK cells express-ing pUHD 10-3 containexpress-ing an NS mutant that fails to produce NS1 protein (NS13⬘SS DM clones E8 and G4), a mutation in the RNA-binding domain (M2), full-length NS (clone D1), or a deletion of the effector domain (NS1⌬117-161) were washed two times with PBS and then incubated for 5, 24, or 48 h in anhydrotetracycline-free MEM containing 1% FBS at 37°C in 5% CO2. DNA was collected and analyzed for DNA fragmentation by agarose gel analysis. (C) Conflu-ent cultures of MDCK cells expressing pUHD 10-3 containing the NS M2 (RNA-binding domain mutation), full-length NS gene from A/Ty/ Ont/66, or NS1⌬117-161 were washed two times with PBS and then incubated for 5, 24, or 48 h in medium without anhydrotetracycline. Cell monolayers were lysed, and 5 mg of total protein was loaded per lane under reducing conditions and resolved by SDS-PAGE. After being transferred to nitrocellulose, proteins were probed for NS with a rabbit polyclonal antibody against NS. Bands were detected by en-hanced chemiluminescence as instructed by the manufacturer. The arrow indicates the location of NS. Molecular size markers are indi-cated on the left. MDCK cells infected with A/Ty/Ont/66 at an MOI of 0.5 for 24 h served as a positive control (virus lane).

FIG. 4. Influenza viruses containing mutated NS1 RNA-binding sites induce apoptosis. Confluent cultures of MDCK cells were washed two times with PBS, incubated with MEM alone (lane 1) or MEM with A/WSN parental virus (lane 2), A/WSN NS1 RK 19/20 AA (lane 3), or NS1 RK 19/20 AD (lane 4) at an MOI of 2 and incubated for 1 h at 37°C in 5% CO2. After the 1-h binding period, the cells were washed with PBS to remove residual virus and incubated for 24 or 48 h in MEM containing 5% BSA. DNA was collected and analyzed for DNA fragmentation by agarose gel analysis.

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proved to be very toxic to MDCK cells. We were unable to generate stable cell lines expressing NS1 protein even under the control of the tetracycline-regulated system. The system was slightly leaky even in the presence of high concentrations of anhydrotetracycline, a stable tetracycline derivative. During selection of clones, cells expressing high concentrations of NS died; therefore, the G418 selection time was shortened from 2 weeks to 1 week to allow analysis of NS-expressing clones. Attempts were also made to express NS1 in MDCK cells by using the ecdysone system, with similar results (data not shown). Similar problems were observed in mink lung epithe-lial and CEF cells but not HeLa cells. It is unclear why the HeLa cells were better able to support expression of the NS protein.

Further studies using well-described NS mutants showed that the RNA-binding domain, but not the effector domain, is essential for the induction of apoptosis. Previous studies showed that mutating amino acids R19 and K20 to alanine resulted in a loss of dsRNA binding and U6 snRNA binding (32). More recently, Wang et al. showed that R19 is essential for dimerization of the NS1 protein (70). These results suggest that RNA binding and/or dimerization of NS1 protein may be required for the induction of apoptosis by NS. The mutation of the effector domain would have no effect on dimerization or RNA binding. In support of these findings, a laboratory variant of A/Turkey/Oregon/71 virus, which encodes an NS1 protein that is only 125 amino acids long and lacks an effector domain (35), also induces apoptosis (data not shown).

Using a WSN backbone, influenza viruses containing muta-tions in the RNA-binding domain of the NS1 protein were generated and examined for apoptosis. The growth of the mutated viruses was highly attenuated compared to the WSN parental strain in MDCK cells (data not shown). However, the mutated and parental viruses grew to equal titers in Vero cells and CEF cells prepared from 6-day-old embryos (unpublished data). Similar results were shown with viruses containing long deletions in the NS1 protein or lacking the NS1 gene entirely (5, 12, 17). These results suggest that mutating the RNA-binding/dimerization region of NS1 protein may inhibit NS1’s ability to inhibit PKR activation and IFN response. Despite attenuation in MDCK cells, the mutated viruses induced apo-ptosis similarly to parental WSN virus when used at an MOI of 2. In addition, apoptosis was inhibited with caspase inhibitors (59), suggesting that the viruses used similar cellular pathways leading to cell death.

The induction of apoptosis in the NS1 mutant viruses may also be due to the activation of the IFN-induced PKR and IFN (10, 54–56, 58, 67). During virus infection, PKR is activated by its interaction with dsRNA, resulting in a cascade of effects including activation of transcriptional factors leading to apo-ptosis (13). NS1 inhibits the activation of PKR through up-regulation of a cellular PKR inhibitor (61), by binding directly to PKR (17), and by inhibiting IFN regulatory factor 3 (60). Takizawa et al. showed that PKR is involved in influenza virus-induced apoptosis (55, 58). The NS1 mutant viruses may be unable to inhibit PKR activation by dsRNA, leading to PKR autophosphorylation and increased IFN levels, resulting in cell death. Studies examining this hypothesis are under way. In the vector system, no dsRNA should be generated and PKR should remain inactive. However, it is possible that dsRNA is

generated by the plasmid, and IFN levels may be elevated, resulting in apoptosis. Studies are under way to determine how expressed NS1 is inducing apoptosis and what cellular factors are involved.

We and others showed that NA induces apoptosis directly (30) and through the activation of TGF-␤(49). It is probable that a portion of the apoptosis observed with the NS mutant viruses is due to NA, whether directly or through TGF-␤ ac-tivation. Finally, the expression of other viral proteins may induce apoptosis. During the NS studies, we also expressed the matrix (M) and nucleoprotein (NP) genes of A/Ty/Ont/66. Expression of the M gene in the tetracycline-regulated system had no effect on cell viability, and it was possible to make a stable cell line expressing M proteins. In contrast, NP also induced apoptosis in MDCK cells, although with different ki-netics than NS1 (data not shown).

The question still remains as to whether virus-induced apo-ptosis is advantageous for viral spread or is advantageous to the host by possibly reducing viral titers. Depletion of lympho-cytes in chickens infected with the highly virulent avian influ-enza Ty/Ont virus (18, 65, 66) and mice infected with the highly virulent human A/Hong Kong/483/97 virus (64) is associated with apoptosis. It has been suggested that influenza virus-induced apoptosis of lymphocytes may be important in viral pathogenesis in highly pathogenic influenza virus. In addition, cells undergoing influenza virus-induced apoptosis are taken up by dendritic cells, inducing a cytotoxic T-cell response (1). Similarly, macrophages phagocytose infected cells through an apoptosis-dependent mechanism (9). These results suggest that understanding the role of apoptosis in influenza virus pathogenesis will be complex and may be dependent on the cell type and environment. However, these studies will increase our understanding of viral pathogenesis and may lead to new ther-apies for influenza virus infection.

ACKNOWLEDGMENTS

We are very appreciative to Robert Krug (University of Texas at Austin) for providing the NS1 and NS2 constructs used in these stud-ies, for providing technical assistance in establishing the cell lines, and for critically reading and revising the manuscript. We are also grateful to Martha McGregor and Laura Kelley for expert technical assistance, to Hermann Bujard (University of Heidelberg) for the tetracycline-responsive system, to Robert Webster (St. Jude’s Children’s Hospital) for the monoclonal antibodies against NS proteins, and to Chris Olsen, Diane Larsen, Matthew Koci, Holly Sellers, and Terry Tumpey for critically reading and revising the manuscript.

This work was supported by Public Health Service research grants from the National Institute of Allergy and Infectious Diseases to V.S.H. and Y.K. S.S-C. was supported initially by a postdoctoral train-ing fellowship in tumor virology through the McArdle Cancer Center at the University of Wisconsin followed by USDA CRIS project 661232000020.

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