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Journal of Virology, September 2001, p. 7875-7881, Vol. 75, No. 17
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
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 multimerization 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, including 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). Although 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 permissive for
virus replication like macrophages, Madin-Darby Canine 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 influenza 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 by bcl-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 necrosis 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 replication.
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 activates transforming growth
factor In MDCK cells, apoptosis occurs early in the course of viral
replication (18). Therefore, it is likely that viral genes expressed 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 numerous 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 polyadenylation of host cell mRNA (31);
and interacting with several host cell proteins (26, 31, 71,
72). The RNA-binding activities 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
evolutionarily conserved.
Arguably, one of NS1's most important functions is inhibiting the
activation of the double-stranded RNA (dsRNA) kinase (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 influenza virus-induced apoptosis.
These studies show that expression 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.
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
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.17.7875-7881.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Influenza Virus NS1 Protein Induces Apoptosis in Cultured
Cells
Madison, Madison, Wisconsin 537063
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
(TGF-) in vivo and in vitro (49). TGF- is a multifunctional growth-regulatory protein that induces apoptosis in
many cell types, including lymphocytes (23) and MDCK cells (45, 49). Neutralizing antibodies against TGF- only
partially inhibit influenza virus-induced apoptosis, suggesting that NA can also induce apoptosis directly. 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.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C.
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 the EcoRI site. Expression vector pUHD 10-3 contains the heptamerized tetracycline repressor gene operators upstream of the cytomegalovirus promoter 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 the BamHI 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 the BamHI
site of pUHD 10-3. Plasmids were transformed into Escherichia
coli DH5
competent cells (Life Technologies), amplified, and
purified using a Qiagen (Valencia, Calif.) purification kit.
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Reassortant virus. Two viruses containing a mutation in the NS1 RNA-binding 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 µg of L-1-p-tosylamino-2-phenylethyl chloromethyl 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 µg of DNA of both plasmids, and G418-resistant cell lines expressing the protein of interest were selected. The cell lines were maintained in MEM containing 10% FBS in the presence of 2.5 µg of anhydrotetracycline per ml (14) and 400 µg 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 characteristic 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-buffered 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 centrifuging the cells, resuspending the cell pellet in 300 µl 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 resuspended in 15 µl 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. Uninfected cells and cells infected 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.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Expression of NS proteins induces apoptosis.
Previous studies
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 tetracycline-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 tetracycline 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.
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NS1 induces apoptosis.
The NS gene of influenza virus encodes
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 tetracycline, with more intense laddering observed at 48 h. The expression 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.
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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/dimerization (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 effector 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 domain 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.
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 mutations 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
generate 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 attenuated 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 infected 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 independent 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 domain is
required for NS1-induced apoptosis, a mutation in this region is not
sufficient to inhibit influenza virus-induced apoptosis.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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The above results show that the expression of the NS1 protein 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 transfect, and even after electroporation, only 1 to 10% of the surviving cells expressed NS. Additionally, the NS1 protein 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 epithelial 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 mutations 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 apoptosis 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 apoptosis (13). NS1 inhibits the activation of PKR through upregulation 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- activation. 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 kinetics than NS1 (data not shown).
The question still remains as to whether virus-induced apoptosis is advantageous for viral spread or is advantageous to the host by possibly reducing viral titers. Depletion of lymphocytes in chickens infected with the highly virulent avian influenza 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 therapies for influenza virus infection.
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ACKNOWLEDGMENTS |
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We are very appreciative to Robert Krug (University of Texas at Austin) for providing the NS1 and NS2 constructs used in these studies, 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 training fellowship in tumor virology through the McArdle Cancer Center at the University of Wisconsin followed by USDA CRIS project 661232000020.
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FOOTNOTES |
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* Corresponding author. Mailing address: Southeast Poultry Research Laboratory, USDA-ARS, 934 College Station Rd., Athens, GA 30605. Phone: (706) 546-3464. Fax: (706) 546-3161. E-mail: sschultz{at}seprl.usda.gov.
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Materials and Methods
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Discussion
References
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Journal of Virology, September 2001, p. 7875-7881, Vol. 75, No. 17
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.17.7875-7881.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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