Báo cáo khoa học: Regulation of arginase II by interferon regulatory factor 3 and the involvement of polyamines in the antiviral response

Regulation of arginase II by interferon regulatory factor 3 and the involvement of polyamines in the antiviral response Nathalie Grandvaux1,2, Franc¸ ois Gaboriau3, Jennifer Harris1,4, Benjamin R. tenOever1,2, Rongtuan Lin4 and John Hiscott1,2,4

1 Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, Montreal, Canada 2 Department of Medicine and Oncology, McGill University, Montreal, Canada 3 INSERM U522, Regulations des Equilibres Fonctionnels du Foie Normal and Pathologique, CHRU Pontchaillou, Rennes, France 4 Department of Microbiology and Immunology, McGill University, Montreal, Canada

Keywords antiviral response; arginase II; interferon regulatory factor 3 (IRF-3); polyamine; spermine

Correspondence J. Hiscott, Molecular Oncology Group, Lady Davis Institute for Medical Research, 3755 chemin de la Cote Sainte Catherine, Montreal, Quebec, Canada H3T1E2 Fax: +514 340 7576 Tel: +514 340 8222 Ext. 5265 E-mail: john.hiscott@mcgill.ca

(Received 11 December 2004, revised 6 April 2005, accepted 20 April 2005)

doi:10.1111/j.1742-4658.2005.04726.x

The innate antiviral response requires the induction of genes and proteins with activities that limit virus replication. Among these, the well-character- ized interferon b (IFNB) gene is regulated through the cooperation of AP-1, NF-jB and interferon regulatory factor 3 (IRF-3) transcription fac- tors. Using a constitutively active form of IRF-3, IRF-3 5D, we showed previously that IRF-3 also regulates an IFN-independent antiviral response through the direct induction of IFN-stimulated genes. In this study, we report that the arginase II gene (ArgII) as well as ArgII protein concentra- tions and enzymatic activity are induced in IRF-3 5D-expressing and Sendai virus-infected Jurkat cells in an IFN-independent manner. ArgII is a critical enzyme in the polyamine-biosynthetic pathway. Of the natural polyamines, spermine possesses antiviral activity and mediates apoptosis at physiological concentrations. Measurement of intracellular polyamine con- tent revealed that expression of IRF-3 5D induces polyamine production, but that Sendai virus and vesicular stomatitis virus infections do not. These results show for the first time that the ArgII gene is an early IRF-3-regula- ted gene, which participates in the IFN-independent antiviral response through polyamine production and induction of apoptosis.

recently described noncanonical IKK-related kinases, IKKe and tank-binding kinase (TBK)-1, which regu- late IRF-3 phosphorylation and activation [6,7].

IFNs are well-characterized components of

the IFN-stimulated gene (ISG)

The establishment of an antiviral defense requires the co-ordinate activation of a multitude of signaling cas- cades in response to virus infection, ultimately leading to the expression of genes encoding cytokines, inclu- ding type I interferons (IFNs), chemokines and pro- that both impede pathogen replication and teins, stimulate innate and adaptive immune responses [1–3]. Among the kinases activated are mitogen-activated protein kinase, Jun-N-terminal kinase (JNK) and p38, which phosphorylate AP-1 [4,5], IjB kinase (IKK), which regulates the activation of NF-jB [4], and the

the innate host defense, which act through engagement of specific cell surface receptors and trigger the acti- vation of the janus kinase (JAK) ⁄ signal transducer and activator of transcription (STAT) signaling pathway. factor Induction of transcrip- (ISGF)-3 [ISGF3c(IRF-9) ⁄ STAT1 ⁄ STAT2] tion factor mediates the induction of a network of

Abbreviations FITC, fluorescein isothicyanate; HSV, herpes simplex virus; IFN, interferon; IRF-3, interferon regulatory factor 3; ISG, IFN-stimulated gene; ISPF, 1-phenylpropane-1,2-dione-2-oxime; ISRE, IFN-stimulated responsive element; JAK, janus kinase; JNK, Jun-N-terminal kinase; LPS, lipopolysaccharide; ODC, ornithine decarboxylase; PI, propidium iodide; SeV, Sendai virus; STAT, signal transducer and activator of transcription; VSV, vesicular stomatitis virus.

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ISGs

through IFN-stimulated responsive antiviral element (ISRE) consensus sequences ([2,8]). Among the ISGs, IRF-7 contributes to the amplification of the IFN response [9–11].

are

genes

ISRE-containing

5D-expressing Jurkat cells. Furthermore, we show that Sendai virus (SeV) infection induced ArgII expression in a type I-IFN-independent manner in Jurkat T cells and macrophages. IRF3 5D expression also resulted in the induction of spermine, which inhibits virus repli- cation and mediates apoptosis. Together, these results illustrate a new mechanism by which IRF-3 may con- tribute to the development of the IFN-independent antiviral state.

Results

Induction of ArgII expression and activity by IRF-3 5D in Jurkat T cells

In addition to the IFN-dependent pathway, many antiviral induced in response to virus infection without the need for prior de novo IFN synthesis [12–14]. IRF-3 is ubiquitously present in a latent form in the cytoplasm of uninfected cells and upon stimulation mediates gene transcription through recognition of ISRE sequences. Thus, IRF-3 was considered as a potential candidate to regulate ISGs in the early events of innate response to virus infection. In a previous study, we used a constitutively active form of IRF-3 (IRF-3 5D) to stimulate tran- scription of genes in the absence of virus infection [15] and to profile by microarray analysis genes that are directly responsive to IRF-3 [14]. This study showed that IRF-3 participates in the development of the anti- viral state, not only through induction of IFNb gene expression, but also through a specific IFN-independ- ent activation of a subset of the antiviral ISGs such as ISG 54, 56 and 60. Moreover, other genes were found to be IRF-3 responsive, including the gene encoding arginase II (ArgII).

Using DNA microarray analysis, we previously repor- ted that the ArgII gene was up-regulated in the Jurkat T cell line following inducible expression of the consti- tutively active form of IRF-3, IRF-3 5D [14]. Up-regu- lation of ArgII gene expression was observed after treatment of the tetracycline inducible cell line, rtTA- in the IRF-3 5D-Jurkat, with doxycycline for 36 h, presence of neutralizing antibodies against IFNs [14]. ArgII mRNA was strongly induced in IRF-3 5D- expressing Jurkat cells, compared with control cells (Fig. 1A). Furthermore, a dramatic induction of ArgII was detected by immunoblot in IRF-3 5D-expressing Jurkat cells at 24 h, and was sustained throughout doxycycline treatment (Fig. 1B). Arginase activity was likewise greatly increased after IRF-3 5D expression by doxycycline, with a profile that mirrored protein expression (Fig. 1C).

ArgII expression and enzymatic activity are induced in Jurkat and Raw 264.7 cells infected with paramyxovirus

the host

regulate numerous processes,

ArgII is the extrahepatic isoform of the arginase type enzymes, and ArgI is the hepatic-specific counter- part [16]. The two isoforms possess the same enzymatic activity for converting l-arginine into l-ornithine and urea, a critical step in the polyamine biosynthesis path- way. Subcellular localization of the two isoforms dif- fers, with ArgI located in the cytoplasm and ArgII in the mitochondria [16]. Whereas ArgI is well character- ized as an essential enzyme of the urea cycle, the func- tion of Arg II in extrahepatic tissues, which do not possess urea cycle activity, is not well understood. Inducible expression of active ArgII has been reported in macrophages upon stimulation with bacterial lipo- polysaccharide (LPS), cAMP, and the ThII cytokine interleukin 4 [17–19]. Most importantly, induction of ArgII has been demonstrated in response to Helico- bacter pylori infection, suggesting that it may be part response to pathogen infection [20]. of Natural polyamines (spermine, spermidine and putres- cine) including cell growth and differentiation, immune response regula- tion, and apoptosis [21]. However, their role in the apoptotic process remains somewhat paradoxical, as polyamines have been reported to both induce and block apoptosis [21,22].

The up-regulation of ArgII was next studied in the con- text of SeV infection, a negative single-strand RNA paramyxovirus known to be a strong activator of IRF- 3 phosphorylation [23]. ArgII protein expression and arginase activity were detected at 24 h and increased the 5–10-fold between 48 and 60 h (Fig. 2A). At mRNA concentration, ArgII was induced 7 h after SeV infection (Fig. 4A), suggesting a delay between mRNA induction and protein detection. Inducible ArgII expression has been previously described in macro- phages [17–20], therefore we examined it in RAW 264.7 macrophages after SeV infection. As shown in Fig. 2B, ArgII protein concentration and enzymatic activity were also increased 5–10-fold 24–48 h after infection. This shows for the first time that the ArgII gene is inducible after SeV infection.

In this study, we confirmed biochemically the DNA microarray results by demonstrating up-regulation of ArgII mRNA, protein and enzymatic activity in IRF3

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Fig. 2. Virus-inducible expression of ArgII in T lymphocytes and macrophages. Jurkat cells (A) and Raw 264.7 cells (B) were infec- ted with SeV (40 HAU per 106 cells) for the indicated times. Cell lysates were analyzed for arginase activity. A540 was measured, and arginase activity was determined as mUÆ(mg protein))1. This experiment is representative of three experiments and is expressed as mean ± SEM from triplicate determinations. In the lower panels, whole-cell extracts (50 lg) were subjected to SDS ⁄ PAGE and ana- lyzed by immunoblotting with antibodies against ArgII. Membranes were stripped and reprobed with antibodies against actin.

tions were increased by virus infection but not by IFN treatment, whereas the IFN-responsive ISG56 gene was induced by both virus and IFN, indicating that virus-induced ArgII expression was IFN-independent (Fig. 3).

ArgI and ornithine decarboxylase (ODC) are not induced in response to virus infection

As the two isoforms of arginase, I (hepatic isoform) and II (extrahepatic isoform), may contribute to the arginase activity measured in the previous experiment,

Fig. 1. IRF-3 5D-inducible expression of ArgII. RtTA-Neo-IRF-3 5D and rt-TA-IRF-3 5D Jurkat cells were induced with doxycycline for the indicated time in the presence of IFN-neutralizing antibodies. (A) Total RNA was extracted and subjected to RT-PCR analysis for ArgII and GAPDH expression. (B) Whole-cell extracts (50 lg) were subjected to SDS ⁄ PAGE and analyzed by immunoblotting with anti- bodies against ArgII. Membranes were stripped and reprobed with antibodies against IRF-3 and actin. (C) Cells were lyzed and ana- lyzed for arginase activity by colorimetric assay, as described in Experimental procedures, through measurement of the production of urea. A540 was measured and arginase activity was determined as mUÆ(mg protein))1. This experiment is representative of three experiments and is expressed as mean ± SEM from triplicate de- terminations.

ArgII induction in response to virus infection is IFN-independent

Fig. 3. IFN-independent expression of ArgII. Jurkat cells were trea- ted with either SeV for 48 h or with type I IFN (1000 UÆmL)1) for 0–48 h. Whole-cell extracts (50 lg) were resolved by SDS ⁄ PAGE and transferred to nitrocellulose membrane. The membrane was probed with antibodies against ArgII. After being stripped, mem- branes was reprobed with antibodies against ISG56 and actin.

IRF-3-regulated genes may be activated as part of the early or delayed phase of the antiviral response [8]. Indeed, these genes are modulated through ISRE consensus sites, which can be targeted by ISGF3, in response to IFN stimulation or by IRFs. As IRF-3 5D alone is not sufficient to induce IFN production [24], the result described above suggested that IFN was not involved in ArgII expression. To directly assess whe- ther ArgII up-regulation could be amplified by IFN production, Jurkat cells were treated with type 1 IFN (1000 UÆmL)1) for 0–48 h. ArgII protein concentra-

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(Fig. 4C)

Jurkat cells was studied. Kinetic analysis of ODC mRNA by RT-PCR (Fig. 4A) and ODC protein con- centration by immunoblot revealed that ODC expression was not regulated at the mRNA or in IRF- protein level after virus infection. Similarly, 3 5D-expressing Jurkat cells, ODC was not up-regula- ted at the protein level (data not shown).

Spermine inhibits vesicular stomatitis virus (VSV) replication in Jurkat T cells

B

C

To assess whether natural polyamines have a direct effect on viral replication, VSV, a negative single- strand RNA rhabdovirus which strongly stimulates the IFN pathway and also induces ArgII expression (data not shown), was used in the next experiment. Jurkat cells were infected with VSV for 14 h in the presence or absence of increasing concentrations of putrescine, spermidine and spermine and assayed for virus repli- cation using a sensitive, quantitative plaque assay (Fig. 5A,B). In the absence of polyamine, the VSV titer (pfu)ÆmL)1, reached 2.3 · 106 plaque-forming units whereas in the presence of physiological concentrations of spermine [20,25,26], the virus titer decreased in a dose-dependent manner. At a concentration of 25 lm, the VSV titer was reduced to 5.4 · 104 pfuÆmL)1, and at concentration of 100 lm, the virus titer was reduced more than 3 logs, to 6.3 · 102 pfuÆmL)1. In the pres- ence of spermidine, the titer of VSV was slightly decreased to 5 · 105 pfuÆmL)1 at a concentration of 100 lm, whereas putrescine did not affect virus yield. Immunoblot analysis of cells treated in the presence of 25 lm and 100 lm polyamine confirmed that spermine treatment dramatically inhibited the expression of VSV glycoprotein, nucleocapsid, polymerase and mat- rix proteins (G, N, P and M) during the lytic cycle (Fig. 5C).

Fig. 4. Induction of ArgII by SeV. (A) Total RNA was extracted from Jurkat cells infected with SeV (40 HAUÆmL)1) for the indicated times or from mouse liver tissue. Time-course expression of mRNA from ArgI, ArgII and ODC was analyzed by RT-PCR. (B, C) Whole- cell extracts from Jurkat cells infected with SeV for the indicated times and from mouse liver and kidney tissues were resolved by SDS ⁄ PAGE and transferred to nitrocellulose membrane. Mem- branes were probed with antibodies against ArgI (B) or human ODC (C). After being stripped, membranes were reprobed with antibodies against actin. Mouse liver and kidney tissues, respect- ively, were used as positive and negative control for ArgI expres- sion [22].

Spermine antiviral effect is dependent on apoptosis

regulation of ArgI in the context of virus infection was also analyzed. No increase in ArgI mRNA (Fig. 4A) or protein levels (Fig. 4B) was observed in Jurkat cells in response to SeV infection.

ArgII is involved in the biosynthesis of natural poly- amines (putrescine, spermidine and spermine) through conversion of l-arginine into l-ornithine [16]. The lat- ter is in turn used by ODC to produce putrescine, the precursor of spermidine and spermine. To further ana- lyze the regulation of the polyamine-synthetic pathway in virus infection, ODC expression in SeV-infected

IRF-3 5D has been shown to mediate apoptosis [24,27], and several reports have also described a role for ArgII and ⁄ or polyamine in the regulation of apop- tosis [21,22]. Thus, the possibility that the antiviral effect of spermine is mediated by induction of apop- tosis was analyzed. For this purpose, the effect of spermine (50 lm) on viral replication was analyzed in the presence of Z-VAD-FMK, a general inhibitor of caspase activity, or Me2SO (control). In the presence of Me2SO, virus titer was significantly decreased by spermine compared with untreated cells (Fig. 6, lanes 2 and 3). However, when cells were pretreated with

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Fig. 6. The spermine antiviral effect requires caspase activation. Jurkat cells were pretreated with Z-VAD-FMK (100 lM) or an equal volume of Me2SO for 1 h before infection with VSV (m.o.i. 0.001) for 14 h in serum-free medium in the absence or presence of sper- mine (50 lM). Supernatants were analyzed for VSV titer using a standard plaque assay. Plaques were counted and titers calculated as pfuÆmL)1. Values are representative of two experiments and are expressed as mean ± SEM from triplicate determinations. Note that the difference in the quantitative effect of spermine (compare with Fig. 5) on virus titer is due to the presence of Me2SO (data not shown).

Fig. 5. Spermine treatment inhibits VSV replication. Jurkat cells were infected with VSV (m.o.i. 0.001) for 14 h in serum-free med- ium in the absence or presence of the indicated concentration of putrescine (triangles), spermidine (squares) or spermine (circles). Supernatants were analyzed for VSV titer using a standard plaque assay. Plaques were counted and titers calculated as pfuÆmL)1(A). (B) Representative plaque assays from cells treated with 100 lM putrescine, spermidine or spermine. (C) Whole-cell extracts (20 lg) from cells treated with 25 lM and 100 lM polyamine in (A) were analyzed by immunoblotting using antibodies against VSV.

tial component of the antiviral effect triggered by sper- mine. To directly demonstrate that spermine enhanced virus-induced apoptosis, annexin V ⁄ propidium iodide (PI) staining of apoptotic cells was quantified in VSV- infected Jurkat T cells in the absence or presence of spermine. As shown in Fig. 7, the presence of spermine during VSV infection strongly potentiated virus- induced apoptosis. At 8 h postinfection, VSV-induced apoptosis was low (2.6% annexin V+ ⁄ PI– and 3.1% annexin V+ ⁄ PI+), whereas in the presence of spermine significant levels of apoptotis were detected (7.9% annexin V+ ⁄ PI– and 30.4% annexin V+ ⁄ PI+). Intere- stingly, spermine alone induced significant apoptosis (3.5% annexin V+ ⁄ PI– and 15.9% annexin V+ ⁄ PI+). No effect of spermidine or putrescine was observed (data not shown). Thus, spermine was the only natural polyamine with the capacity to induce apoptosis and to augment apoptosis during virus infection.

titer was comparable in the Z-VAD-FMK, virus absence and presence of spermine (Fig. 6, lanes 4 and 5). This shows that activation of caspases is an essen-

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Fig. 7. Spermine potentiates VSV-induced apoptosis. Jurkat T cells were infected with VSV (m.o.i. 0.01) in the absence or presence of 100 lM spermine. At the indicated times, cells were harvested and double-stained with FITC–annexin V ⁄ PI as indicated in Experimental procedures. The upper panel represents the percentage of cells that were annexin V positive (annexin V+ ⁄ PI– and annexin V+ ⁄ PI+) by flow cytometry. Plots in the lower panel illustrate the 8 h time point. Data are representative of two independent experiments.

Fig. 8. IRF-3 5D expression, but not SeV infection, triggers polyam- ine production in Jurkat cells. (A) rt-TA-IRF-3 5D Jurkat cells were left uninduced (light-shaded bars) or induced with doxycycline (1 lgÆmL)1) for 30 h (dark-shaded bars). (B) Jurkat cells were left untreated (light-shaded bars) or infected with SeV (80 HAU per 106 cells) for 52 h (dark-shaded bars). Cells were harvested, and per- chloric acid extracts were used to quantify the intracellular concen- tration of spermine, spermidine and putrescine as described in Experimental procedures. These results are representative of two independent experiments, each with duplicate measurements. The SE was estimated by the percentage of variation observed over the two independent experiments.

Spermine and spermidine are induced in IRF-3 5D-expressing, but not virus-infected, Jurkat cells

produced in response to IRF-3 activation, but not during SeV or VSV infection.

Discussion

Finally, to evaluate whether polyamines, and partic- ularly spermine, were produced in response to IRF-3 activation, rtTA-IRF-3 5D-Jurkat cells were treated with doxycycline for 30 h, and the pool of intracellular polyamines was measured by dansylation and LC ⁄ MS analysis as described in Experimental Procedures. As shown in Fig. 8A, production of spermine and spermi- dine was significantly induced in IRF-3 5D-expressing Jurkat cells compared with control cells. Intracellular polyamine content was also measured after virus infec- tion, and polyamine production was not induced after SeV infection (Fig. 8B) or VSV infection (data not shown). Thus, the final products of the polyamine- biosynthetic pathways, spermine and spermidine, are

In previous studies, we showed that IRF-3 mediates an antiviral response in an IFN-independent manner, in part due to the IRF-3-dependent expression of ISGs, such as ISG-54, 56 and 60. We now report that activa- tion of IRF-3 stimulates the ArgII gene in an IFN- independent manner. ArgII is a mitochondrial enzyme involved in the polyamine synthesis pathway through the catalysis of l-ornithine production from l-arginine. Of the natural polyamines, spermine and to a lesser extent spermidine, possess antiviral activities resulting from their potential to induce apoptosis, and both

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polyamines were induced in response to the expression of a constitutively active form of IRF-3.

ited by polyamine [37]. Polyamine depletion inhibited TNF-a-induced JNK activation and subsequently pre- vented caspase-3 activation in intestinal epithelial IEC- 6 cells, thereby delaying TNF-a-induced apoptosis [38]. As both NF-jB and JNK pathways are activated by virus infection, these pathways may be targets of the pro-apoptotic activity of spermine.

This study shows for the first time that ArgII expres- sion is up-regulated in the context of virus infection. Previous studies reported the induction of ArgII in response to LPS, cAMP, or H. pylori [20,28–30], with ArgII expression up-regulated at mRNA, protein and activity levels after H. pylori infection. Furthermore, ArgI and ODC expression were not up-regulated at the transcriptional level after H. pylori infection [20], a result that correlates with the present experiments in virus-infected cells. In Jurkat T cells, basal level ODC mRNA and protein expression was observed, and this was not modulated after virus infection.

the possibility that

Spermine and to a lesser extent spermidine inhibited VSV multiplication, but inhibition was abolished when cells were treated with the caspase inhibitor, Z-VAD- FMK, suggesting that spermine-mediated apoptosis may be part of the host antiviral response. Further- more, enhanced virus-induced apoptosis occurred in the presence of spermine (Fig. 7). However, we cannot rule out spermine production in vivo in response to virus infection induces sufficient apoptosis to limit the levels of virus multiplication, thus mimicking an antiviral effect. An alternative mechanism, that spermine acts by inhibition of virus entry, was examined using recombinant VSV-GFP virus, and virus entry was not inhibited by spermine (data not shown).

simplex virus

The pathways involved in ArgII gene regulation are not well characterized, but a role for NF-jB has been suggested based on the use of chemical inhibitors; pyr- rolidine dithiocarbamate was shown to inhibit ArgII induction in rat alveolar macrophages stimulated with LPS, whereas ArgII expression in LPS-stimulated Raw264.7 cells was not inhibited by pyrrolidine dithio- carbamate [28]. In Raw 264.7 cells cocultured with H. pylori, ArgII expression was inhibited by MG-132 [20], suggesting indirectly an involvement of NF-jB in ArgII regulation. Our study is thus the first direct demonstration of the involvement of IRF-3 in ArgII regulation in response to virus infection. IRF-3 is also activated in response to LPS in a TLR-4-dependent mechanism [31,32]; thus IRF-3 may also participate in the LPS-mediated or H. pylori-mediated induction of ArgII via a TLR-4-dependent pathway.

A limited number of studies have examined the rela- tionship between polyamine production and herpes virus replication. Polyamine depletion was shown to replication [39,40], block human cytomegalovirus whereas inhibition of polyamine biosynthesis produced different effects on herpes (HSV)-1, HSV-2 or pseudorabies virus replication [41–43]. HSV inhibited polyamine biosynthesis by inhibiting protein synthesis, whereas human cytomegalovirus infection induced spermine and spermidine expression in fibro- blasts [41,44]. Another study reported induction of ArgI and ArgII mRNA in the cornea during HSV infection, but protein concentrations and arginase activity were not analyzed [45]. Conversely, proteose– peptone-activated and IFNc-activated macrophages exhibited increased arginase activity and were resistant to HSV infection by a mechanism that was prevented by the addition of arginine, suggesting an essential role for arginase in antiviral activity [46,47]. In retrospect, however, these results may simply reflect the consump- tion of arginine by inducible nitric oxide synthase, which competes with arginase for the arginine sub- strate, to produce nitric oxide, an antiviral compound produced by macrophages [48,49].

The role of polyamines in apoptosis is controversial; both induction of and protection against apoptosis by polyamines have been demonstrated [21,22]. In agree- ment with the present study, an apoptosis process dependent on ArgII and ODC was reported in response to H. pylori infection of macrophages [20]. The present study describes a role for ArgII up- regulation and the polyamine-synthesis pathway in IRF-3 5D-induced apoptosis. Although IRF-3 can sti- mulate apoptosis in Jurkat cells [24], the molecular mechanisms responsible for triggering it in response to IRF-3 have not been defined. ISG56 was induced in response to IRF-3, and because ISG56 is involved in the inhibition of protein translation and cell prolifer- ation [33,34], it may participate in IRF-3-mediated apoptosis. Another potential mechanism involves sper- mine, which induced apoptosis in Jurkat cells and enhanced virus-induced apoptosis at physiological con- centrations [20,25,26]. Polyamines are known to modu- late DNA–protein interactions; specifically, spermine has been shown to induce NF-jB activation in breast cancer cells [35,36], whereas Oct-1 binding was inhib-

Spermine, spermidine and putrescine are induced in response to IRF-3 5D expression, but not in response to SeV or VSV infection, although these two viruses trigger IRF-3 phosphorylation ⁄ activation. Based on this surpri- sing result, it is possible that SeV and VSV may have evolved strategies to antagonize polyamine synthesis the polyamine-mediated apoptotic and to evade

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response. The molecular mechanisms used by viruses to block polyamine synthesis are under investigation.

138 bp

[50], 362 bp; human and TCACACGTGCTTGATT-3¢ murine ArgI, 5¢-ATTGGCTTGAGAGACGTGGACCCT-3¢ and 5¢-TTGCAACTGCTGTGTTCACTGTTC-3¢, 369 bp; human ODC, 5¢-TGTTGCTGCTGCCTCTACGTT-3¢ and [51]; 5¢-GCTGGCATCCTGTTCCTCTACTT-3¢, human b-actin, 5¢-ACAATGAGCTGCTGGTGGCT-3¢ and 5¢-GATGGGCACAGTGTGGGTGA-3¢; murine b-actin, 5¢-TGGAATCCTGTGGCATCCATGAAAC-3¢ and 5¢-TA AAACGCAGCTCAGTAACCGTCCG-3¢. Human GAPDH primers were included in the Advantage RT-PCR kit.

In conclusion, this study shows for the first time the induction of ArgII mRNA, protein and enzymatic activity in the context of virus infection in an IRF-3- dependent and IFN-independent manner. Moreover, expression of a constitutively active form of IRF-3 leads to induction of spermine, which possesses pro- apoptotic and antiviral activities. These results thus illustrate a potential new mechanism by which IRF-3 contributes to the development of the antiviral state.

Immunoblot analysis

Experimental procedures

Reagents

1 mm phenylmethanesulfonate

Spermine, spermidine, putrescine, 1-phenylpropane-1,2-dione- 2-oxime (ISPF) and doxycycline were from Sigma. Human recombinant IFN type 1 was from Sigma (Oakville, Ontario, Canada). Z-VAD-FMK was from BioMol.

Cell culture and infection

rtTA-Neo-IRF-3

Cells were washed twice in NaCl ⁄ Pi and lyzed in 50 mm Tris ⁄ HCl, pH 7.4, containing 1% Nonidet P40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA supple- mented with fluoride, 5 lgÆmL)1 aprotinin and 5 lgÆmL)1 leupeptin (lysis buffer) for 15 min on ice. Mouse liver and kidney total protein extracts were prepared by Dounce homogenization of tis- sues in lysis buffer and centrifugation at 10 000 g for 30 min at 4 (cid:1)C. Supernatants were used as total protein extracts. Whole cell extracts (50 lg) or mouse tissue extracts (50 lg) were separated by SDS ⁄ PAGE and trans- ferred to nitrocellulose membrane (Bio-Rad, Mississauga, Ontario, Canada). The membrane was blocked in NaCl ⁄ Pi containing 0.05% Tween 20 and 5% nonfat dry milk for 1 h and incubated with primary antibody, anti-(IRF-3 FL- 425) Ig (1 lgÆmL)1; Santa Cruz), anti-ArgII (1 : 1000) Ig [52], anti-ArgI Ig (1 : 1000) [53], anti-(ODC sc-21515) Ig (1 lgÆmL)1; Santa Cruz), anti-ISG56 Ig (1 : 1000; a gift from Dr G. Sen, Lemer Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA) or anti-(a-actin) Ig (Chemicon) in blocking solution. After five 5-min washes in NaCl ⁄ Pi containing 0.05% Tween 20, the membranes were incubated for 1 h with horseradish peroxidase-conju- gated goat anti-rabbit, goat anti-mouse or rabbit anti-goat IgG (1 : 2000–1 : 10000) in blocking solution. Immunoreac- tive proteins were visualized by enhanced chemilumines- cence (Perkin-Elmer, Woodbridge, Ontario, Canada).

Measurement of arginase enzymatic activity

Jurkat cells (ATCC, Manassas, VA, USA) were grown in RPMI-1640 medium (wisent, St jean batiste de Roaville, Quebec, Canada) containing 10% heat-inactivated fetal bovine serum and antibiotics. Vero cells (ATCC) and RAW 264.7 (ATCC) cells were grown in DMEM medium (wisent) supplemented with 10% heat-inactivated fetal bovine serum and rtTA-IRF-3 5D and antibiotics. Jurkat cells [24] were grown in RPMI-1640 medium con- taining 10% heat-inactivated fetal bovine serum, glutamine, antibiotics, 2.5 lgÆmL)1 puromycin and 400 lgÆmL)1 G418 (Gibco, Burlington, Ontario, Canada). Twenty hours before stimulation, cells were seeded in fresh medium at 0.5 · 106 cellsÆmL)1. Induction with doxycycline was performed at 1 lgÆmL)1 for the indicated time in the presence of neutral- izing antibodies against type I IFNs as described [14]. Treatment with IFN-a was performed at 1000 UÆmL)1 for 16 h in complete medium. SeV infection (Cantell strain, 40 HAU per 106 cells) was carried out for 2 h in serum-free medium and further cultured for the indicated time in com- plete medium.

RT-PCR analysis

the addition of

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Arginase activity was measured by colorimetric assay [54]. Cells (105) were lyzed in 50 lL 0.1% Triton containing 5 lg antipain, 5 lg pepstatin, and 5 lg aprotinin. After 30 min at room temperature, 50 lL 10 mm MnCl2 ⁄ 50 mm Tris ⁄ HCl, pH 7.5 was added, and the lysate was activated at 55 (cid:1)C for 10 min. Arginine hydrolysis was performed at 37 (cid:1)C for 60 min by mixing 25 lL previously activated lysate with 25 lL 0.5 m arginine, pH 9.7. The reaction was stopped by 400 lL acidic mixture H2SO4 ⁄ H3PO4 ⁄ H2O (1 : 3 : 7, v ⁄ v ⁄ v). For quantification of urea produced, 25 lL 9% ISPF was added and incubated Total RNA from exponentially growing cells stimulated as described above and from mouse liver tissues was isolated using homogenization in TRIzol reagent (Gibco). Total RNA (1 lg) was reverse-transcribed in a final volume of 100 lL (Advantage RT-PCR kit; Clontech, Mountain View, CA, USA), and 20 lL was used for PCR amplification using the following primers: human and murine ArgII, 5¢-GAT CTGCTGATTGGCAAGAGACAA-3¢ and 5¢-CTAAATTC

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IRF-3-mediated antiviral response involves spermine

for 45 min at 100 (cid:1)C. After 10 min in the dark, A540 was measured. A standard curve was obtained by adding 100 lL urea (1.8–30 lg) to 400 lL acidic mixture and 25 lL ISPF. Proteins in the lysate were quantified using the Bradford assay (Bio-Rad). Arginase activity was determined as mUÆ(mg protein))1 [equivalent to lmol ureaÆmin)1Æ(mg protein))1].

VSV plaque assay

ric acid. After centrifugation at 3000 g for 10 min, perchlo- ric supernatants and protein precipitates were stored at )80 (cid:1)C until analyzed within 1 month. The dansylation pro- cedure was performed by a previously described method [55] using 1,10-diaminododecane as internal standard. Aliquots (200 lL) of the perchloric supernatants were allowed to react with 4 vol. dansyl chloride in acetone (5 mgÆmL)1) in the presence of solid sodium carbonate. After the dansyla- tion reaction (12 h at room temperature), excess dansyl chloride was removed by reaction with proline. The cyclo- hexane extract containing the dansyl derivatives was evapor- ated to dryness, and the residue resuspended in 200 lL acetonitrile.

Jurkat T cells were infected with VSV at a multiplicity of infection (m.o.i.) of 0.001 for 1 h in serum-free medium. After two washes in NaCl ⁄ Pi, infection was pursued in serum-free medium in the absence or presence of putrescine, spermidine or spermine, and supernatant was harvested at 14 h postinfection. In experiments where Z-VAD-FMK was used, the reagent was used at 100 lm for 1 h before infection, and maintained at this concentration during the infection. Serial dilutions of the supernatant were used to infect confluent plates of Vero cells in serum-free medium. After 1 h infection, the medium was removed and replaced by 3% methylcellulose. After plaques had formed, the meth- ylcellulose was removed and the cells were fixed with 4% formaldehyde for 1 h and stained with 0.2% crystal violet in 20% ethanol. Plaques were counted, averaged and multi- plied by the dilution factor to determine viral titer as pfuÆmL)1. Virus protein was detected in cells by immuno- blot as described above using antibodies against VSV (a gift from John Bell, Ottawa, CA, USA).

Detection of early and late apoptosis (annexin V/PI staining)

standard 1–10, diaminododecane.

The LC ⁄ MS was supplied with chem station 1100 soft- ware (Agilent Technologie; Massy-Palaiseau, Wilmington, DE, USA). Nitrogen gas was generated using a Jun-air model 2000–25M air compressor (Buffalo Grove, IL, USA) connected to a UHPLCMS Model nitrogen generator (Domnick Hunter France, S.A., Villefranche-sur-Saoˆ ne, France). Dansylated polyamine was analyzed by flow injec- tion analysis without performing a separation with a LC column [56]. For flow injection analysis ⁄ MS measurements, 30-lL samples were directly injected from the HP1100 ser- ies autosampler without LC separation into a stream of water ⁄ acetonitrile (9 : 1, v ⁄ v) at a flow rate of 0.5 mLÆ min)1. The following parameters were used for detec- tion: sec ⁄ scan cycle, 1.46; threshold, 150; step size, 0.35; ion mode positive; gain, 9.9; capillary voltage, +3000 V; cor- ona current, 6 lA; drying gas flow rate, 6 LÆmin)1; drying gas temperature, 300 (cid:1)C; nebulizer pressure, 30 psig; vapor- izer temperature, 400 (cid:1)C. Selected ion monitoring mode data masses were obtained with an atmospheric pressure chemical ionization source to monitor the protonated par- ent ions [M + H]+; at m ⁄ z 555.2 for bidansyl-putrescine, m ⁄ z 845.3 for tridansyl-spermidine, m ⁄ z 1135.4 for tetra- dansyl-spermine and m ⁄ z 639.3 for the bidansylated inter- nal Ionic intensities, deduced from the area under each selective peak, were cor- rected with respect to that of the internal standard. Poly- amine concentrations were determined by using calibration curves obtained from known amounts of a mixture contain- ing the four polyamines dansylated and extracted under the same conditions. Two independent polyamine-dansylation experiments were performed, and each polyamine measure- ment was performed in duplicate.

Acknowledgements

Jurkat T cells stimulated as described above were harvested at different time points and resuspended in 50 lL cold NaCl ⁄ Pi. Apoptosis was detected by reaction with fluorescein isothiocyanate (FITC)-conjugated annexin V and PI. Stain- ing was performed by the addition of cold staining mixture containing 500 lL binding buffer (10 mm Hepes, pH 7.4, 150 mm NaCl, 5 mm KCl, 1 mm MgCl2, 1.8 mm CaCl2), 1 lL FITC–annexin V and 1 lL PI (1 mgÆmL)1) for 5 min. Acquisition was performed on a FACScan flow cytometer (BD Biosciences, Mountain View, CA, USA) using FL-1 and FL-2 detectors. Analysis was performed using the cellquest software (BD Biosciences). Cells exhibiting annexin V– ⁄ PI+ staining were considered necrotic, those showing annex- in V+ ⁄ PI– staining were recognized as early apoptotic cells, and annexin V+ ⁄ PI+ cells were taken as late apoptotic.

Measurement of intracellular polyamine concentration

We thank Dr M. Mori and Dr J. Bell for reagents used in this study. We also thank Laurence Lejeune and Ste´ phanie Olie` re for excellent technical help with FACS analyses, and members of the Molecular Oncol- ogy Group of the Lady Davis Institute for helpful dis- cussions. This work was supported by grants to J.Hi.

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After treatment, cells were harvested, washed three times with NaCl ⁄ Pi, and disrupted by sonication in 0.2 m perchlo-

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IRF-3-mediated antiviral response involves spermine

regulatory factors 3 and 7. Mol Cell Biol 20, 6342– 6353. 12 Nicholl MJ, Robinson LH & Preston CM (2000) Acti-

vation of cellular interferon-responsive genes after infec- tion of human cells with herpes simplex virus type 1. J Gen Virol 81, 2215–2218.

from the Canadian Institutes of Health Research and CANVAC, the Canadian Network for Vaccines and Immunotherapeutics. N.G. was supported by a post- doctoral FRSQ fellowship, J.Ha. and B.R.T. by an NSERC studentship, R.L. by a FRSQ Chercheur Boursier, and J.Hi. by a CIHR Senior Scientist award.

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