Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Muthgasse 18B, A-1190 Vienna, Austria
Correspondence
Andrej Egorov
a.egorov{at}iam.boku.ac.at
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ABSTRACT |
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These authors contributed equally to this work.
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INTRODUCTION |
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The major target for influenza virus is the ciliated epithelial cells; later on in the course of infection, mononuclear cells also become involved (Kaufmann et al., 2001). It has been shown that virus-infected epithelial cells produce only a limited number and amount of pro-inflammatory cytokines and chemokines in response to influenza A virus infection (Adachi et al., 1997
; Matsukura et al., 1996
). In contrast, influenza A virus-infected monocytes/macrophages are capable of producing higher quantities and a broader spectrum of different pro-inflammatory cytokines and chemokines, e.g. IFN-
/
, interleukin (IL) 1
, IL6, tumour necrosis factor alpha (TNF-
), IL18, CCL3 (MIP-1
), CCL4 (MIP-1
), CCL5 (RANTES), CCL2 (MCP-1), CCL7 (MCP-3), CCL20 (MIP-3
) and CXCL10 (IP-10) (Julkunen et al., 2001
; Kaufmann et al., 2001
; Matikainen et al., 2000
).
Influenza A virus is a negative-strand RNA virus belonging to the family Orthomyxoviridae. The genome of influenza A virus consists of eight RNA segments that encode 1011 proteins, depending on the viral strain. Among them, the NS1 and PB1-F2 proteins have been shown to interfere with the antiviral measures of the host's innate immunity, thus enabling effective replication of the virus in the host. The PB1-F2 protein does this by way of promoting the apoptotic death of immunocompetent cells (Chen et al., 2001). The NS1 protein accomplishes this interference by way of multiple mechanisms, employing several functional domains. First, the dimerized NS1 protein, via its N-terminal RNA-binding domain (RBD), can bind to dsRNA, thus preventing the activation of PKR (Bergmann et al., 2000
; Hatada et al., 1999
; Lu et al., 1995
) and several transcription factors, such as IRF-3 (Talon et al., 2000
) and NF-
B (Wang et al., 2000
). As a consequence, virus-induced apoptosis is delayed (Takizawa et al., 1996
) and the induction of type I IFNs is inhibited in primary infected cells (Talon et al., 2000
; Wang et al., 2000
). Secondly, the NS1 protein, through the function of its effector domain (ED), is able to downregulate the formation and nuclear export of cellular mRNAs that interfere with the cellular 3'-end processing and splicing machineries, thus inhibiting the maturation of cellular pre-mRNAs (Chen et al., 1999
; de la Luna et al., 1995
; Krug et al., 2003
; Noah et al., 2003
). Thirdly, the NS1 protein is able to selectively enhance the translation of viral mRNAs, but not cellular mRNAs (de la Luna et al., 1995
). Thus, the NS1 protein can antagonize the production of cellular proteins at several levels transcriptional, post-transcriptional and translational (Chen et al., 1999
; de la Luna et al., 1995
; Krug et al., 2003
; Noah et al., 2003
; Talon et al., 2000
; Wang et al., 2000
).
As the NS1 protein is considered to be an IFN antagonist (Egorov et al., 1998; García-Sastre et al., 1998
; Noah et al., 2003
), we hypothesized that the NS1 protein might also be involved in the control of the production of other pro-inflammatory cytokines. For this reason, we investigated cytokine release in primary human macrophages infected with various influenza A virus NS1 mutants. Here, we have demonstrated that mutant viruses lacking the entire NS1 gene or expressing the non-dimerized (non-functional) NS1 protein were strong inducers of IL1
/IL18 production in virus-infected macrophages. At the same time, an NS1 mutant virus encoding a functional N-terminal RNA binding/dimerization domain and lacking the C-terminal ED was able to antagonize IL1
and IL18 production, but failed to inhibit the production of IFN-
, TNF-
, IL6 and CCL3 (MIP-1
). Our data indicate that influenza A virus NS1 protein controls the production of pro-inflammatory cytokines in infected macrophages through the function of its N- and C-terminal domains. Moreover, we demonstrated that the N-terminal domains of the NS1 protein are essential for preventing caspase-1 activation, thus inhibiting the caspase-1-dependent post-translational processing of pro-IL1
/pro-IL18 in primary human macrophages.
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METHODS |
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Viruses, cells and cell infection.
Vero cell-adapted influenza A/PR/8/34 (H1N1) virus (PR8 wt), together with NS1 mutant viruses [PR8/NS1125, PR8/delNS1 and PR8/NS1del4080; obtained as described previously (Egorov et al., 1998)], were used in this study (Fig. 1
). All of the tested viruses were propagated in Vero cells (WHO certified, obtained from ATCC), which were infected at a low m.o.i. of 0·001 and incubated at 37 °C for 48 h in Dulbecco's modified Eagle's medium/Ham's F15 (Biochrom F4815) supplemented with a protein-free additive (proprietary formulation; Polymun Scientific) containing 1 µg trypsin ml1 (Sigma). Infectious titres of generated viral stocks were assessed by plaque assays utilizing Vero cells. Harvested macrophages were washed twice with RPMI 1640 medium and seeded into six-well plates (1x106 cells per well) and infected with wild-type or NS1 mutant viruses at an m.o.i. of 2. After 1 h virus adsorption at 37 °C, the inoculum was removed and cells were washed twice with RPMI 1640 medium and incubated in RPMI 1640 medium supplemented with 20 ng GM-CSF ml1 at 37 °C. Cells and cell-culture supernatants were harvested at various time points post-infection (p.i.) and further processed for testing by ELISA, Western blotting, Quantikine, apoptosis and caspase-1 activity assays.
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Cytokine ELISAs.
The amounts of IFN-, IL6, TNF-
, CCL3 (MIP-1
), IL1
and IL18 in the culture supernatants were determined at 18 h p.i. by using quantitative cytokine-specific ELISA kits purchased from PBL (IFN-
) or R&D [IL6, TNF-
, CCL3 (MIP-1
), IL1
, IL18], following the manufacturer's instructions.
Isolation of RNA and assessment of IL1-specific mRNA.
Total RNA was extracted from virus-infected macrophages at 5 h p.i. by using TRIzol reagent (Invitrogen) following the manufacturer's instructions. The obtained RNA was quantified with an Agilent 2100 Bioanalyser using an RNA6000 Nano Assay kit (Agilent Technologies) plus RNA 6000 ladder marker (Ambion). Equal amounts of RNA (2 µg per sample) were assayed for the presence of IL1- or
-actin-specific mRNA by using Quantikine kits (R&D). Samples and a manufacturer-supplied calibrator (standard) were hybridized with the IL1
- or
-actin-specific biotin-labelled oligonucleotide probe and the digoxigenin-labelled detection probe in microplates. The hybridization solution was then transferred to streptavidin-coated microplates, the specific mRNAprobe hybrid was captured and unbound material was washed away. Extensive washing was followed by incubation with anti-digoxigenin alkaline phosphatase conjugate. Excess conjugate was washed away and substrate was added. The development of the colour reaction, proportional to the amount of IL1
- or
-actin-specific mRNA, was enhanced by the addition of an amplifier solution. The intensity of the colour reaction was determined by using a microplate reader and the concentration of IL1
- or
-actin-specific mRNA in each sample was calculated from calibration curves.
Western blot analysis.
Virus-infected macrophages were lysed at 5 h p.i. and 20 µg protein per lane was analysed by 420 % gradient SDS-PAGE (Novex) using the Tris/glycine buffer system. After separation, proteins were transferred from the gel to PVDF membranes (Millipore) by using a Novex electrotransfer apparatus at 400 mA for 2 h. The membranes were blocked with PBS containing 0·1 % Tween 20 (TPBS) and 5 % non-fat milk. Blots were stained for 2 h at room temperature with rabbit anti-human IL1 mAb (R&D) or polyclonal rabbit anti-human caspase-1 antibody (Santa Cruz). Intensive washing of the membranes with TPBS was followed by staining (1 h at room temperature) with anti-rabbit peroxidase conjugate (Sigma). Visualization of the IL1
- and caspase-1-specific protein bands was carried out by using an ECL PLUS kit (Amersham Biosciences).
Fluorometric caspase-1 activity assay.
Day 7 macrophages (1x106) were infected with wild-type or NS1 mutant viruses (at an m.o.i. of 2). At 5 h p.i., cells were lysed and tested for caspase-1 activity by using a caspase-1 fluorometric kit (R&D). All reagents used in the assay were supplied by the manufacturer. The enzymic reaction for caspase-1 was performed in 96-well flat-bottomed microplates. Cell lysates (100 µl) and supernatants were mixed with 100 µl 2x reaction buffer supplemented with 1 µl 1 M dithiothreitol and 10 µl of the caspase-1 fluorogenic peptidic substrate WEHD-AFC. Subsequently, the microplate was incubated at 37 °C and 2 h later analysed with a fluorescence-detecting microplate reader. Cleavage of the peptidic substrate by caspase-1 led to the release of the fluorochrome, which, when excited at a wavelength of 400 nm, emitted fluorescence at 505 nm. The detected fluorescence signal was proportional to the level of caspase-1 enzymic activity in the cell lysates.
Measurement of the extent of virus-induced apoptosis.
The extent of apoptosis induced in virus-infected macrophages at 2, 4 and 6 h p.i. was assessed by using a Cell Death Detection ELISAPLUS kit (Roche) according to the manufacturer's instructions. This assay is based on a quantitative sandwich-enzyme immunoassay principle, utilizing mouse mAbs directed against DNA and histones, respectively, and allows the specific determination of mono- and oligonucleosomes of degraded DNA in the cytoplasmic fraction of cell lysates from apoptotic cells.
Inhibition of virus-induced apoptosis by using a PKR inhibitor, universal caspase inhibitor and caspase-1 inhibitor.
Macrophages were left untreated or were pre-treated for 30 min with 2 mM PKR inhibitor (2-aminopurine), 25 µM universal caspase inhibitor (Z-VAD-FMK) or 25 µM caspase-1-specific inhibitor (Z-WEHD-FMK) and then infected with the PR8/NS1del4080 virus (at an m.o.i. of 2). The ratio of infected to uninfected control cells (apoptosis death index) was determined at 6 h p.i. by using a Cell Death Detection ELISAPLUS kit as above.
Inhibition of PKR.
Macrophages were left untreated or were pre-treated for 30 min with 4, 3, 2 or 1 mM PKR inhibitor (2-aminopurine) and subsequently infected with wild-type or NS1 mutant viruses at an m.o.i. of 2) in the presence or absence of 2-aminopurine. Cell-culture supernatants were collected at 18 h p.i. and assessed for the presence of IL1 by ELISA.
Inhibition of caspase-1, -8, -9 and -3.
Macrophages were left untreated or were pre-treated for 30 min with 25 µM caspase-1 inhibitor (Z-WEHD-FMK), caspase-8 inhibitor (Z-IETD-FMK), caspase-9 inhibitor (Z-LEHD-FMK) or caspase-3 peptidic inhibitor (Z-DEVD-FMK) (all from R&D). Subsequently, cells were infected with wild-type or NS1 mutant viruses (m.o.i. of 2) in the presence or absence of each caspase inhibitor. Cell-culture supernatants were collected at 18 h p.i. and assessed for the presence of IL1 and IL18 by ELISA.
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RESULTS |
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NS1 protein is required for virus replication in primary human macrophages
We found that the PR8 wt virus could replicate in human macrophages infected at an m.o.i. of 0·01, giving a yield in the range of 104105 p.f.u. ml1. In contrast, none of the NS1 mutant viruses tested was able to replicate in macrophages. Infection of macrophages at an m.o.i. of 2 resulted in a striking difference in the onset of virus-induced apoptosis. Macrophages infected with PR8/delNS1 or PR8/NS1del4080 virus developed apoptosis at 6 h p.i. (Fig. 2), whereas macrophages infected with wild-type or PR8/NS1125 mutant virus showed signs of apoptosis at 12 h p.i. (data not shown).
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NS1 protein restricts the production of IL1 in influenza A virus-infected macrophages at the post-translational level
We investigated whether the enhanced production of IL1 by macrophages infected with the PR8/delNS1 or PR8/NS1del4080 virus could be attributed to enhanced expression of the IL1
gene. High background levels of IL1
-specific mRNA were detected in uninfected macrophages, presumably due to the presence of GM-CSF in the culture medium. All of the tested viruses enhanced the accumulation of IL1
-specific mRNA to a similar extent (Fig. 4
a). These data suggested that, under our experimental conditions, the NS1 protein did not interfere with IL1
production at either the transcriptional or post-transcriptional level. We next assessed the accumulation and processing of pro-IL1
protein in macrophages by Western blotting using anti-IL1
mAbs. As shown in Fig. 4(b)
, infection of macrophages with any of the tested viruses did not result in enhanced accumulation of pro-IL1
protein. However, the biologically active 17 kDa form of IL1
was detected only in macrophages infected with the PR8/delNS1 or PR8/NS1del4080 mutant virus (Fig. 4b
).
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DISCUSSION |
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The PR8/NS1125 virus, encoding NS1 protein with an intact N terminus (125 aa), but possessing a large C-terminal deletion, was not able to prevent the production of IFN-, TNF-
, IL6 or CCL3 (MIP-1
). Nevertheless, the PR8/NS1125 mutant was as efficient as the PR8 wt virus at antagonizing the production of IL1
and IL18. It is known that the C-terminal domains of the NS1 protein interfere with cellular mRNA 3'-end processing machinery, thus inhibiting the maturation and nuclear export of cellular mRNAs (Krug et al., 2003
). Our data indicated that the NS1 protein might also employ a similar mechanism in order to control the production of IFN-
, TNF-
, IL6 and CCL3 (MIP-1
), although further experiments are necessary to elucidate the precise mechanisms of their regulation. It should be mentioned that the inability of the PR8/NS1125 virus to antagonize IFN-
, TNF-
, IL6 and CCL3 (MIP-1
) could not be attributed to the insufficient dimerization of its 125 aa NS1 protein, as it was able to bind dsRNA in vitro as efficiently as the full-sized NS1 protein (data not shown). Moreover, the PR8/NS1125 mutant virus was capable of controlling virus-induced apoptosis of infected macrophages to the same extent as the PR8 wt virus.
On the other hand, viruses lacking the entire NS1 gene or expressing NS1 protein with an impaired RBD and dimerization domain induced 1050 times more biologically active IL1 and three to five times more biologically active IL18 than the wild-type or PR8/NS1125 viruses. The PR8/NS1del4080 mutant virus, encoding an almost-intact C-terminal ED, displayed a phenotype very similar to that of the PR8/delNS1 mutant virus. Therefore, we believe that the non-dimerized NS1 protein encoded by the PR8/NS1del4080 virus was also deficient in functions mediated by its C-terminal domains. It was noteworthy that the production of IFN-
, TNF-
, IL6 and CCL3 (MIP-1
) by this group of mutant viruses was diminished when compared with the PR8/NS1125 virus, which was mainly due to the fast onset of apoptosis of the PR8/NS1del4080 or PR8/delNS1 virus-infected macrophages.
Thus, our data imply that the NS1 protein can downregulate cytokine production by utilizing both of its functional domains. In our experimental set-up, the N-terminal part of the NS1 protein appeared to be crucial for the inhibition of IL1 and IL18 production, whereas the C-terminal part was important for the regulation of IFN-
, TNF-
, IL6 and CCL3 (MIP-1
) production in influenza A virus-infected human macrophages.
Previous reports have shown that the C-terminal domain of the NS1 protein mediates inhibition of IFN- pre-mRNA maturation in infected cells. In our experimental set-up, we found that the C-terminal part of the NS1 protein of PR8 wt virus could inhibit the accumulation of IFN-
-specific mRNA (data not shown), but did not have any effect on the accumulation of IL1
-specific mRNA. A similar enhancement of pre-existing levels of IL1
-specific mRNA was detected in macrophages infected with any of the investigated viruses, including the PR8 wt virus.
Pro-IL1 and pro-IL18 are synthesized as biologically inactive precursors that must be cleaved by caspase-1 in order to become active (Ghayur et al., 1997
; Gu et al., 1997
; Li et al., 1995
). Therefore, these two cytokines might be also regulated at the post-translational level. We could detect the mature form of IL1
protein only in macrophages infected with NS1 mutant viruses encoding non-functional NS1 proteins (PR8/delNS1 and PR8/NS1del4080). The treatment of infected cells with caspase-1 inhibitor downregulated the release of the mature form of IL1
by 90 %, confirming the key role of this enzyme in the post-translational processing of pro-IL1
. Surprisingly, the release of IL1
was also significantly repressed by other caspase inhibitors (caspase-9, -8 and -3). The caspase-9-specific inhibitor, in particular, reduced the production of IL1
as efficiently as the caspase-1 inhibitor. We assume that caspase-9, -8 and -3 might also be involved in the regulation of IL1
and IL18 production by having a direct or indirect impact on the activation of caspase-1 (Cohen, 1997
; Srinivasula et al., 1996
).
It was previously shown that pro-caspase-1 is expressed constitutively in primary human macrophages and that its expression is not enhanced upon viral infection (Pirhonen et al., 1999, 2001
). We also observed equal amounts of pro-caspase-1 in virus-infected and uninfected macrophages. Nevertheless, the enhanced activity of caspase-1 was detected only in macrophages infected with the PR8/delNS1 and PR8/NS1del4080 viruses. Our findings imply that the NS1 protein restricts the production of the mature forms of IL1
and IL18 by preventing the activation of caspase-1. However, it is difficult to elucidate the precise mechanism of this interference at the molecular level, as the exact mechanism by which caspase-1 is activated is not yet known in detail.
In our experiments, we also observed that enhanced activity of caspase-1 in macrophages infected with the PR8/delNS1 or PR8/NS1del4080 viruses correlated with an early onset of apoptosis in these cells. It was previously reported that the PR8/delNS1 virus rapidly induced apoptosis in IFN-competent cells, but not in IFN-deficient cells (Zhirnov et al., 2002). Thus, the NS1 protein, by inhibiting some of the IFN-induced factors, can prevent virus-induced apoptosis. In this context, PKR is considered to be one of the major factors responsible for influenza A virus-induced apoptosis (Takizawa et al., 1996
). It was demonstrated that the NS1 protein could prevent the activation of PKR in PR8/delNS1 virus-infected cells (Bergmann et al., 2000
). Influenza A virus-induced apoptosis in HeLa cells has been shown to be PKR-dependent and accompanied by the activation of caspase-8 and -3, but not caspase-1 (Takizawa et al., 1995
, 1996
, 1999
). The involvement of caspase-1 in PR8/delNS14080 virus-induced apoptosis was shown in our experiments by the repression of macrophage death following the addition of caspase-1-specific inhibitor to infected macrophages. Moreover, treatment of PR8/delNS14080-infected cells with a PKR inhibitor (2-aminopurine) resulted in complete inhibition of apoptosis and IL1
release. Based on these findings, we concluded that the mechanism by which NS1 protein inhibits caspase-1 activation in macrophages might include prevention of PKR activation. However, the link between PKR or other Ser/Thr kinases and caspase-1 activation remains to be elucidated.
The cytokines IL1 and IL18 belong to the class of major regulators of the antiviral immune response (Nakanishi et al., 2001
). It was shown that influenza A virus-infected IL1
/IL18 knockout mice displayed a higher mortality rate than control mice, thus indicating the importance of IL1
and IL18 for host antiviral defence (Kozak et al., 1995
; Liu et al., 2004
). Enhanced induction of IL1
and IL18 by the PR8/delNS1 and PR8/delNS14080 viruses might stimulate a robust immune response, due to the immuno-potentiating properties of IL1
and IL18 (Bradney et al., 2002
; Staats et al., 2001
). Recently, we found that these two viruses could elicit a significant Th1-type immune response that was sufficient to protect mice against wild-type virus challenge. Interestingly, mice immunized with the PR8/NS1del4080 virus developed the strongest release of type I IFNs and IL1
in the mouse respiratory tract (unpublished data). We believe that the results of this study may contribute towards the development of a new generation of safe and immunogenic live influenza A virus vaccines based on NS1 mutant viruses encoding targeted modifications within the NS1 protein.
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ACKNOWLEDGEMENTS |
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Received 6 July 2004;
accepted 30 September 2004.