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

Regulation of arginase II by interferon regulatory factor and the involvement of polyamines in the antiviral response Nathalie Grandvaux1,2, Francois Gaboriau3, Jennifer Harris1,4, Benjamin R tenOever1,2, ¸ Rongtuan Lin4 and John Hiscott1,2,4 Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, Montreal, Canada Department of Medicine and Oncology, McGill University, Montreal, Canada INSERM U522, Regulations des Equilibres Fonctionnels du Foie Normal and Pathologique, CHRU Pontchaillou, Rennes, France Department of Microbiology and Immunology, McGill University, Montreal, Canada Keywords antiviral response; arginase II; interferon regulatory factor (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 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-characterized interferon b (IFNB) gene is regulated through the cooperation of AP-1, NF-jB and interferon regulatory factor (IRF-3) transcription factors 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 concentrations 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 content revealed that expression of IRF-3 5D induces polyamine production, but that Sendai virus and vesicular stomatitis virus infections not These results show for the first time that the ArgII gene is an early IRF-3-regulated gene, which participates in the IFN-independent antiviral response through polyamine production and induction of apoptosis The establishment of an antiviral defense requires the co-ordinate activation of a multitude of signaling cascades in response to virus infection, ultimately leading to the expression of genes encoding cytokines, including type I interferons (IFNs), chemokines and proteins, that both impede pathogen replication and 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 recently described noncanonical IKK-related kinases, IKKe and tank-binding kinase (TBK)-1, which regulate IRF-3 phosphorylation and activation [6,7] IFNs are well-characterized components of the innate host defense, which act through engagement of specific cell surface receptors and trigger the activation of the janus kinase (JAK) ⁄ signal transducer and activator of transcription (STAT) signaling pathway Induction of the IFN-stimulated gene (ISG) factor (ISGF)-3 [ISGF3c(IRF-9) ⁄ STAT1 ⁄ STAT2] transcription 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 3120 FEBS Journal 272 (2005) 3120–3131 ª 2005 FEBS N Grandvaux et al antiviral ISGs through IFN-stimulated responsive element (ISRE) consensus sequences ([2,8]) Among the ISGs, IRF-7 contributes to the amplification of the IFN response [9–11] In addition to the IFN-dependent pathway, many antiviral ISRE-containing genes are 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 transcription 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 antiviral state, not only through induction of IFNb gene expression, but also through a specific IFN-independent 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) ArgII is the extrahepatic isoform of the arginase type enzymes, and ArgI is the hepatic-specific counterpart [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 pathway Subcellular localization of the two isoforms differs, with ArgI located in the cytoplasm and ArgII in the mitochondria [16] Whereas ArgI is well characterized as an essential enzyme of the urea cycle, the function of Arg II in extrahepatic tissues, which not possess urea cycle activity, is not well understood Inducible expression of active ArgII has been reported in macrophages upon stimulation with bacterial lipopolysaccharide (LPS), cAMP, and the ThII cytokine interleukin [17–19] Most importantly, induction of ArgII has been demonstrated in response to Helicobacter pylori infection, suggesting that it may be part of the host response to pathogen infection [20] Natural polyamines (spermine, spermidine and putrescine) regulate numerous processes, including cell growth and differentiation, immune response regulation, 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] In this study, we confirmed biochemically the DNA microarray results by demonstrating up-regulation of ArgII mRNA, protein and enzymatic activity in IRF3 FEBS Journal 272 (2005) 3120–3131 ª 2005 FEBS IRF-3-mediated antiviral response involves spermine 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 replication and mediates apoptosis Together, these results illustrate a new mechanism by which IRF-3 may contribute to the development of the IFN-independent antiviral state Results Induction of ArgII expression and activity by IRF-3 5D in Jurkat T cells Using DNA microarray analysis, we previously reported that the ArgII gene was up-regulated in the Jurkat T cell line following inducible expression of the constitutively active form of IRF-3, IRF-3 5D [14] Up-regulation of ArgII gene expression was observed after treatment of the tetracycline inducible cell line, rtTAIRF-3 5D-Jurkat, with doxycycline for 36 h, in the presence of neutralizing antibodies against IFNs [14] ArgII mRNA was strongly induced in IRF-3 5Dexpressing 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 up-regulation of ArgII was next studied in the context of SeV infection, a negative single-strand RNA paramyxovirus known to be a strong activator of IRF3 phosphorylation [23] ArgII protein expression and arginase activity were detected at 24 h and increased 5–10-fold between 48 and 60 h (Fig 2A) At the mRNA concentration, ArgII was induced h after SeV infection (Fig 4A), suggesting a delay between mRNA induction and protein detection Inducible ArgII expression has been previously described in macrophages [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 3121 IRF-3-mediated antiviral response involves spermine A N Grandvaux et al A B B C Fig 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 antibodies against ArgII Membranes were stripped and reprobed with antibodies against IRF-3 and actin (C) Cells were lyzed and analyzed 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 m(mg protein))1 This experiment is representative of three experiments and is expressed as mean ± SEM from triplicate determinations Fig Virus-inducible expression of ArgII in T lymphocytes and macrophages Jurkat cells (A) and Raw 264.7 cells (B) were infected 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 m(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 analyzed 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, ArgII induction in response to virus infection is IFN-independent 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 whether ArgII up-regulation could be amplified by IFN production, Jurkat cells were treated with type IFN (1000 mL)1) for 0–48 h ArgII protein concentra3122 Fig IFN-independent expression of ArgII Jurkat cells were treated with either SeV for 48 h or with type I IFN (1000 mL)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, membranes was reprobed with antibodies against ISG56 and actin FEBS Journal 272 (2005) 3120–3131 ª 2005 FEBS N Grandvaux et al A IRF-3-mediated antiviral response involves spermine Jurkat cells was studied Kinetic analysis of ODC mRNA by RT-PCR (Fig 4A) and ODC protein concentration by immunoblot (Fig 4C) revealed that ODC expression was not regulated at the mRNA or protein level after virus infection Similarly, in IRF3 5D-expressing Jurkat cells, ODC was not up-regulated at the protein level (data not shown) Spermine inhibits vesicular stomatitis virus (VSV) replication in Jurkat T cells B C Fig Induction of ArgII by SeV (A) Total RNA was extracted from Jurkat cells infected with SeV (40 HAmL)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) Wholecell 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 Membranes 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, respectively, were used as positive and negative control for ArgI expression [22] 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 polyamines (putrescine, spermidine and spermine) through conversion of l-arginine into l-ornithine [16] The latter is in turn used by ODC to produce putrescine, the precursor of spermidine and spermine To further analyze the regulation of the polyamine-synthetic pathway in virus infection, ODC expression in SeV-infected FEBS Journal 272 (2005) 3120–3131 ª 2005 FEBS To assess whether natural polyamines have a direct effect on viral replication, VSV, a negative singlestrand 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 replication using a sensitive, quantitative plaque assay (Fig 5A,B) In the absence of polyamine, the VSV titer reached 2.3 · 106 plaque-forming units (pfu)ỈmL)1, 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 pfmL)1, and at concentration of 100 lm, the virus titer was reduced more than logs, to 6.3 · 102 pfmL)1 In the presence of spermidine, the titer of VSV was slightly decreased to · 105 pfmL)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 matrix proteins (G, N, P and M) during the lytic cycle (Fig 5C) Spermine antiviral effect is dependent on apoptosis 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 apoptosis [21,22] Thus, the possibility that the antiviral effect of spermine is mediated by induction of apoptosis 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 and 3) However, when cells were pretreated with 3123 IRF-3-mediated antiviral response involves spermine N Grandvaux et al A B C Fig Spermine treatment inhibits VSV replication Jurkat cells were infected with VSV (m.o.i 0.001) for 14 h in serum-free medium 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 pfmL)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 Z-VAD-FMK, virus titer was comparable in the absence and presence of spermine (Fig 6, lanes and 5) This shows that activation of caspases is an essen3124 Fig The spermine antiviral effect requires caspase activation Jurkat cells were pretreated with Z-VAD-FMK (100 lM) or an equal volume of Me2SO for h before infection with VSV (m.o.i 0.001) for 14 h in serum-free medium in the absence or presence of spermine (50 lM) Supernatants were analyzed for VSV titer using a standard plaque assay Plaques were counted and titers calculated as pfmL)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) tial component of the antiviral effect triggered by spermine To directly demonstrate that spermine enhanced virus-induced apoptosis, annexin V ⁄ propidium iodide (PI) staining of apoptotic cells was quantified in VSVinfected Jurkat T cells in the absence or presence of spermine As shown in Fig 7, the presence of spermine during VSV infection strongly potentiated virusinduced apoptosis At 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+) Interestingly, 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 FEBS Journal 272 (2005) 3120–3131 ª 2005 FEBS N Grandvaux et al IRF-3-mediated antiviral response involves spermine 104 104 A 101 100 100 104 104 101 102 103 AnnexinY-FITC 104 NG050206.022 10 10 10 AnnexinY-FITC 10 100 101 Pl-FL2 102 Pl-FL2 102 103 103 NG050206.018 101 100 B Pl-FL2 102 103 Pl-FL2 102 101 100 101 102 103 AnnexinY-FITC 104 100 10 NG050206.021 103 NG050206.017 10 10 10 10 AnnexinY-FITC 10 Fig 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 h time point Data are representative of two independent experiments Spermine and spermidine are induced in IRF-3 5D-expressing, but not virus-infected, Jurkat cells Finally, to evaluate whether polyamines, and particularly 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 spermidine was significantly induced in IRF-3 5D-expressing Jurkat cells compared with control cells Intracellular polyamine content was also measured after virus infection, and polyamine production was not induced after SeV infection (Fig 8B) or VSV infection (data not shown) Thus, the final products of the polyaminebiosynthetic pathways, spermine and spermidine, are FEBS Journal 272 (2005) 3120–3131 ª 2005 FEBS Fig IRF-3 5D expression, but not SeV infection, triggers polyamine production in Jurkat cells (A) rt-TA-IRF-3 5D Jurkat cells were left uninduced (light-shaded bars) or induced with doxycycline (1 lgỈmL)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 perchloric acid extracts were used to quantify the intracellular concentration 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 produced in response to IRF-3 activation, but not during SeV or VSV infection Discussion 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 activation of IRF-3 stimulates the ArgII gene in an IFNindependent 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 3125 IRF-3-mediated antiviral response involves spermine polyamines were induced in response to the expression of a constitutively active form of IRF-3 This study shows for the first time that ArgII expression 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 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; pyrrolidine 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 dithiocarbamate [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 The role of polyamines in apoptosis is controversial; both induction of and protection against apoptosis by polyamines have been demonstrated [21,22] In agreement 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 upregulation and the polyamine-synthesis pathway in IRF-3 5D-induced apoptosis Although IRF-3 can stimulate 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 proliferation [33,34], it may participate in IRF-3-mediated apoptosis Another potential mechanism involves spermine, which induced apoptosis in Jurkat cells and enhanced virus-induced apoptosis at physiological concentrations [20,25,26] Polyamines are known to modulate DNA–protein interactions; specifically, spermine has been shown to induce NF-jB activation in breast cancer cells [35,36], whereas Oct-1 binding was inhib3126 N Grandvaux et al ited by polyamine [37] Polyamine depletion inhibited TNF-a-induced JNK activation and subsequently prevented caspase-3 activation in intestinal epithelial IEC6 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 Spermine and to a lesser extent spermidine inhibited VSV multiplication, but inhibition was abolished when cells were treated with the caspase inhibitor, Z-VADFMK, suggesting that spermine-mediated apoptosis may be part of the host antiviral response Furthermore, enhanced virus-induced apoptosis occurred in the presence of spermine (Fig 7) However, we cannot rule out the possibility that 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) A limited number of studies have examined the relationship between polyamine production and herpes virus replication Polyamine depletion was shown to block human cytomegalovirus replication [39,40], whereas inhibition of polyamine biosynthesis produced different effects on herpes simplex virus (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 fibroblasts [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 consumption of arginine by inducible nitric oxide synthase, which competes with arginase for the arginine substrate, to produce nitric oxide, an antiviral compound produced by macrophages [48,49] 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 surprising result, it is possible that SeV and VSV may have evolved strategies to antagonize polyamine synthesis and to evade the polyamine-mediated apoptotic FEBS Journal 272 (2005) 3120–3131 ª 2005 FEBS N Grandvaux et al response The molecular mechanisms used by viruses to block polyamine synthesis are under investigation 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-3dependent and IFN-independent manner Moreover, expression of a constitutively active form of IRF-3 leads to induction of spermine, which possesses proapoptotic and antiviral activities These results thus illustrate a potential new mechanism by which IRF-3 contributes to the development of the antiviral state IRF-3-mediated antiviral response involves spermine TCACACGTGCTTGATT-3¢ [50], 362 bp; human and murine ArgI, 5¢-ATTGGCTTGAGAGACGTGGACCCT-3¢ and 5¢-TTGCAACTGCTGTGTTCACTGTTC-3¢, 369 bp; human ODC, 5¢-TGTTGCTGCTGCCTCTACGTT-3¢ and 5¢-GCTGGCATCCTGTTCCTCTACTT-3¢, 138 bp [51]; 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 Immunoblot analysis Experimental procedures Reagents Spermine, spermidine, putrescine, 1-phenylpropane-1,2-dione2-oxime (ISPF) and doxycycline were from Sigma Human recombinant IFN type was from Sigma (Oakville, Ontario, Canada) Z-VAD-FMK was from BioMol Cell culture and infection 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 antibiotics rtTA-Neo-IRF-3 and rtTA-IRF-3 5D Jurkat cells [24] were grown in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum, glutamine, antibiotics, 2.5 lgỈmL)1 puromycin and 400 lgỈmL)1 G418 (Gibco, Burlington, Ontario, Canada) Twenty hours before stimulation, cells were seeded in fresh medium at 0.5 · 106 cellsỈmL)1 Induction with doxycycline was performed at lgỈmL)1 for the indicated time in the presence of neutralizing antibodies against type I IFNs as described [14] Treatment with IFN-a was performed at 1000 mL)1 for 16 h in complete medium SeV infection (Cantell strain, 40 HAU per 106 cells) was carried out for h in serum-free medium and further cultured for the indicated time in complete medium RT-PCR analysis 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 FEBS Journal 272 (2005) 3120–3131 ª 2005 FEBS 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, mm EDTA supplemented with mm phenylmethanesulfonate fluoride, lgỈmL)1 aprotinin and lgỈmL)1 leupeptin (lysis buffer) for 15 on ice Mouse liver and kidney total protein extracts were prepared by Dounce homogenization of tissues in lysis buffer and centrifugation at 10 000 g for 30 at °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 transferred 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 h and incubated with primary antibody, anti-(IRF-3 FL425) Ig (1 lgỈmL)1; Santa Cruz), anti-ArgII (1 : 1000) Ig [52], anti-ArgI Ig (1 : 1000) [53], anti-(ODC sc-21515) Ig (1 lgỈmL)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 h with horseradish peroxidase-conjugated goat anti-rabbit, goat anti-mouse or rabbit anti-goat IgG (1 : 2000–1 : 10000) in blocking solution Immunoreactive proteins were visualized by enhanced chemiluminescence (Perkin-Elmer, Woodbridge, Ontario, Canada) Measurement of arginase enzymatic activity Arginase activity was measured by colorimetric assay [54] Cells (105) were lyzed in 50 lL 0.1% Triton containing lg antipain, lg pepstatin, and lg aprotinin After 30 at room temperature, 50 lL 10 mm MnCl2 ⁄ 50 mm Tris ⁄ HCl, pH 7.5 was added, and the lysate was activated at 55 °C for 10 Arginine hydrolysis was performed at 37 °C for 60 by mixing 25 lL previously activated lysate with 25 lL 0.5 m arginine, pH 9.7 The reaction was stopped by the addition of 400 lL acidic mixture H2SO4 ⁄ H3PO4 ⁄ H2O (1 : : 7, v ⁄ v ⁄ v) For quantification of urea produced, 25 lL 9% ISPF was added and incubated 3127 IRF-3-mediated antiviral response involves spermine for 45 at 100 °C After 10 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 m(mg protein))1 [equivalent to lmol uremin)1Ỉ(mg protein))1] VSV plaque assay Jurkat T cells were infected with VSV at a multiplicity of infection (m.o.i.) of 0.001 for 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 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 h infection, the medium was removed and replaced by 3% methylcellulose After plaques had formed, the methylcellulose was removed and the cells were fixed with 4% formaldehyde for h and stained with 0.2% crystal violet in 20% ethanol Plaques were counted, averaged and multiplied by the dilution factor to determine viral titer as pfmL)1 Virus protein was detected in cells by immunoblot 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) 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 Staining was performed by the addition of cold staining mixture containing 500 lL binding buffer (10 mm Hepes, pH 7.4, 150 mm NaCl, mm KCl, mm MgCl2, 1.8 mm CaCl2), lL FITC–annexin V and lL PI (1 mgỈmL)1) for 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 annexin V+ ⁄ PI– staining were recognized as early apoptotic cells, and annexin V+ ⁄ PI+ cells were taken as late apoptotic Measurement of intracellular polyamine concentration After treatment, cells were harvested, washed three times with NaCl ⁄ Pi, and disrupted by sonication in 0.2 m perchlo- 3128 N Grandvaux et al ric acid After centrifugation at 3000 g for 10 min, perchloric supernatants and protein precipitates were stored at )80 °C until analyzed within month The dansylation procedure 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 vol dansyl chloride in acetone (5 mgỈmL)1) in the presence of solid sodium carbonate After the dansylation reaction (12 h at room temperature), excess dansyl chloride was removed by reaction with proline The cyclohexane extract containing the dansyl derivatives was evaporated to dryness, and the residue resuspended in 200 lL acetonitrile The LC ⁄ MS was supplied with chem station 1100 software (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-Saone, ˆ France) Dansylated polyamine was analyzed by flow injection 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 series autosampler without LC separation into a stream of water ⁄ acetonitrile (9 : 1, v ⁄ v) at a flow rate of 0.5 mLỈ min)1 The following parameters were used for detection: sec ⁄ scan cycle, 1.46; threshold, 150; step size, 0.35; ion mode positive; gain, 9.9; capillary voltage, +3000 V; corona current, lA; drying gas flow rate, LỈmin)1; drying gas temperature, 300 °C; nebulizer pressure, 30 psig; vaporizer temperature, 400 °C Selected ion monitoring mode data masses were obtained with an atmospheric pressure chemical ionization source to monitor the protonated parent ions [M + H]+; at m ⁄ z 555.2 for bidansyl-putrescine, m ⁄ z 845.3 for tridansyl-spermidine, m ⁄ z 1135.4 for tetradansyl-spermine and m ⁄ z 639.3 for the bidansylated internal standard 1–10, diaminododecane Ionic intensities, deduced from the area under each selective peak, were corrected with respect to that of the internal standard Polyamine concentrations were determined by using calibration curves obtained from known amounts of a mixture containing the four polyamines dansylated and extracted under the same conditions Two independent polyamine-dansylation experiments were performed, and each polyamine measurement was performed in duplicate Acknowledgements We thank Dr M Mori and Dr J Bell for reagents used in this study We also thank Laurence Lejeune ´ ` and Stephanie Oliere for excellent technical help with FACS analyses, and members of the Molecular Oncology Group of the Lady Davis Institute for helpful discussions This work was supported by grants to J.Hi FEBS Journal 272 (2005) 3120–3131 ª 2005 FEBS N Grandvaux et al from the Canadian Institutes of Health Research and CANVAC, the Canadian Network for Vaccines and Immunotherapeutics N.G was supported by a postdoctoral 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 IRF-3-mediated antiviral response involves spermine 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IRF -3 responsive, including the gene encoding arginase II (ArgII) ArgII is the extrahepatic isoform of the arginase type enzymes, and ArgI is the hepatic-specific counterpart [16] The two isoforms... 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. .. spermidine and spermine) through conversion of l-arginine into l-ornithine [16] The latter is in turn used by ODC to produce putrescine, the precursor of spermidine and spermine To further analyze the