Induction of the IFN-stimulated gene ISG factor ISGF-3 [ISGF3cIRF-9⁄ STAT1 ⁄ STAT2] transcrip-tion factor mediates the inductranscrip-tion of a network of Keywords antiviral response; ar
Trang 1and the involvement of polyamines in the antiviral
response
Nathalie Grandvaux1,2, Franc¸ois Gaboriau3, Jennifer Harris1,4, Benjamin R tenOever1,2,
Rongtuan Lin4and 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
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-teins, 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 regu-late 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 acti-vation 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] transcrip-tion factor mediates the inductranscrip-tion of a network of
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
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.
Trang 2antiviral 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
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)
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
of the host response to pathogen infection [20]
Natural polyamines (spermine, spermidine and
putres-cine) regulate numerous processes, 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]
In this study, we confirmed biochemically the DNA
microarray results by demonstrating up-regulation of
ArgII mRNA, protein and enzymatic activity in IRF3
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 Using DNA microarray analysis, we previously repor-ted that the ArgII gene was up-regularepor-ted 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-IRF-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 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 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 [2IRF-3] 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 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
Trang 3ArgII 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
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-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,
A
B
C
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 A540was 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.
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 106cells) 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.
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.
Trang 4regulation 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
Jurkat cells was studied Kinetic analysis of ODC mRNA by RT-PCR (Fig 4A) and ODC protein con-centration by immunoblot (Fig 4C) revealed that ODC expression was not regulated at the mRNA or protein level after virus infection Similarly, in
IRF-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
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 reached 2.3· 106plaque-forming units (pfu)ÆmL)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 pfuÆmL)1, and
at concentration of 100 lm, the virus titer was reduced more than 3 logs, to 6.3· 102pfuÆmL)1 In the pres-ence of spermidine, the titer of VSV was slightly decreased to 5· 105pfuÆmL)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)
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 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
A
B
C
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 Whole-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].
Trang 5Z-VAD-FMK, virus titer was comparable in the
absence and presence of spermine (Fig 6, lanes 4 and
5) This shows that activation of caspases is an
essen-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-virus-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
A
B
C
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 l M
putrescine, spermidine or spermine (C) Whole-cell extracts (20 lg)
from cells treated with 25 l M and 100 l M polyamine in (A) were
analyzed by immunoblotting using antibodies against VSV.
Fig 6 The spermine antiviral effect requires caspase activation Jurkat cells were pretreated with Z-VAD-FMK (100 l M ) 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 l M ) 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).
Trang 6Spermine and spermidine are induced in
IRF-3 5D-expressing, but not virus-infected,
Jurkat cells
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
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 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
AnnexinY-FITC
AnnexinY-FITC
AnnexinY-FITC
NG050206.017 NG050206.021
NG050206.022 NG050206.018
AnnexinY-FITC
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 l M 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.
A
B
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 10 6 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.
Trang 7polyamines were induced in response to the expression
of a constitutively active form of IRF-3
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 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
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-ited by polyamine [37] Polyamine depletion inhibinhib-ited 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
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 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 rela-tionship 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 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]
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 and to evade the polyamine-mediated apoptotic
Trang 8response 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-3-dependent and IFN-inIRF-3-dependent 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
Experimental procedures
Reagents
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
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
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)1for 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
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
TCACACGTGCTTGATT-3¢ [50], 362 bp; human and murine ArgI, 5¢-ATTGGCTTGAGAGACGTGGACCCT-3¢ and 5¢-TTGCAACTGCTGTGTTCACTGTTC-3¢, 369 bp; human ODC, 5¢-TGTTGCTGCTGCCTCTACGTT-3¢ and
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 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 1 mm phenylmethanesulfonate 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 4C 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 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 55C for 10 min Arginine hydrolysis was performed
at 37C 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 the addition of 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
Trang 9for 45 min at 100C 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
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)
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 stainStain-ing 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
After treatment, cells were harvested, washed three times
with NaCl⁄ Pi, and disrupted by sonication in 0.2 m
ric acid After centrifugation at 3000 g for 10 min, perchlo-ric supernatants and protein precipitates were stored at )80 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 reacdansyla-tion (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
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-Saoˆne, France) Dansylated polyamine was analyzed by flow injec-tion analysis without performing a separainjec-tion 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, 300C; nebulizer pressure, 30 psig; vapor-izer temperature, 400C 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 standard 1–10, diaminododecane 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
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
Trang 10from 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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