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Laboratories of
*
Viral Immunology and
Virology, Centre de recherche en Rhumatologie et Immunologie, and
Unit of Human ImmunoRetrovirology, Centre de recherche en Infectiologie, Centre de recherche du Centre Hospitalier de l’Université Laval, Université Laval, Québec, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and leukotrienes. EBV was also found to affect
phagocytosis of monocytes. In this study, we show that in addition to
these effects, EBV suppresses the biosynthesis of PGE2, a
pleiotropic immunomodulatory molecule that is synthesized by the
dioxygenation of arachidonic acid via the cyclooxygenase (COX) pathway.
This down-regulation of PGE2 formation involved the
inhibition of the inducible COX-2 isoform expression both at the
transcriptional and translational levels, whereas expression of the
constitutive COX-1 isoform was unaltered. Furthermore, exposure of
monocytes to EBV was found to impact on the NF-
B activation pathway,
which plays an essential role in the induction of COX-2 in monocytes.
The inhibition of PGE2 biosynthesis was relieved when the
experiments were conducted in presence of phosphonoacetic acid, an
inhibitor of herpesviruses DNA polymerase, indicating that viral
replication and/or neosynthesized viral proteins were involved in this
process. Thus, inhibition of PGE2 biosynthesis in monocytes
may represent an additional mechanism underlying EBV
pathogenicity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PGE2 is a potent vasodilator (4, 5) that also prevents overactivation of cellular immunity (6, 7, 8). It was also reported that PGE2 exerts antiviral activities. For example, in vitro treatment of monocyte-derived macrophages with PGE2 decreased HIV-1 virions penetration by suppressing expression of the HIV-1 coreceptor CCR5 (9). As well, addition of exogenous PGE2 was shown to reduce replication of adenoviruses types 1, 5, and 12, and parainfluenza virus presumably by increasing intracellular levels of cAMP (10, 11). PGE2 antiviral action has also been reported in vivo. Successive treatments of liver-transplanted patients suffering from recurrent hepatitis B virus infection, with i.v. PGE1 and oral PGE2, led to a significant decrease of viral replication (12).
The EBV, which belongs to the Herpesvirus family, has demonstrated its capabilities to adapt and evade host defense mechanisms. For instance, latent infection of B lymphocytes in vivo is one effective mean used by EBV to evade immune surveillance (13). The EBV BCRF-1 gene encodes a homologue of IL-10, which, like its cellular counterpart, possesses immunosuppressive properties (14). EBV is also able to interact with phagocytes such as neutrophils and monocytes and modulate synthesis of proinflammatory mediators (15, 16, 17). Moreover, our laboratory has previously shown that neutrophils are permissive to EBV viral entry and that EBV-infected neutrophils undergo premature cell death by apoptosis (18). More recently, we reported that EBV replicates in monocytes and reduces the ability of these cells to phagocytosis (19). Impairment of the phagocytosis machinery is likely to be advantageous for the viral outcome, because phagocytosis is involved in elimination of foreign organisms. In the present study, we show that EBV affects another cellular function of monocytes, the biosynthesis of PGE2. Because PGE2 is an important immunoregulatory mediator, this suppressive effect of EBV on its formation may represent an important event in the disruption of the immune response during the early stages of infection and may constitute another viral strategy to evade the immune surveillance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMCs obtained from healthy volunteers were isolated by centrifugation of heparinized venous blood over Ficoll-Hypaque gradient (Pharmacia, Uppsala, Sweden). PBMCs were next submitted to a Percoll density centrifugation to separate the monocytes from the lymphocyte populations, as previously described (20), and were further enriched using a cell-sorting procedure (Epics Elite ESP; Coulter Electronics Canada, Burlington, Ontario, Canada). This resulted in 99% pure monocyte suspensions, as assessed by flow cytometry using anti-CD14 mAb (Becton Dickinson, Mississauga, Ontario, Canada). Cell viability was tested by the trypan blue dye exclusion procedure and was superior to 99%.
Viral preparations
Preparations of EBV (strain B95-8) were produced as described previously (17). Briefly, B95-8 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, in presence of 20 ng/ml PMA, a known inducer of viral replication. When cell viability decreased to 20% or less, cell-free culture supernatants were filtered through a 0.45-µm pore size filter, and viral particles were concentrated by ultracentrifugation (16,000 x g, 2.5 h). Viral particles were resuspended in RPMI 1640 medium and kept at -80°C until use. Viral titers were evaluated and adjusted to 107 transforming units (TFU/ml), as previously described (21). For every experiment, cell-free supernatants collected from B95-8 cells that have not been exposed to PMA were processed as described above and served for mock-infected controls. When mentioned, EBV virions were inactivated by heating (1 h, 56°C), or by exposure to UV radiation (265 nm, 60 min), or by neutralization with the mAb 72A1 raised against the viral envelope gp350/220, as described elsewhere (16).
Culture conditions
Enriched monocytes (1 x 106 cells/ml) were preincubated for 1 h at 37°C with either infectious (105 TFU/ml) or inactivated EBV and then stimulated with 1 µg/ml of LPS (Sigma, St. Louis, MO) for time periods ranging from 0 to 24 h. In some cases, enriched monocytes were preincubated for 1 h with 200 µg/ml of phosphonoacetic acid (PAA), an inhibitor of viral DNA polymerase, before treatment with infectious EBV. Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, at 37°C in a humidified atmosphere with 5% CO2. Culture medium was found to contain less than 10 pg/ml of endotoxins, as evaluated by the Limulus amebocyte assays. Cell-free supernatants from each culture were harvested at the indicated times and stored at -80°C until used for PGE2 assays.
Enzyme immunoassays for PGE2, IL-1ß, and IL-6
Supernatants from mock-treated and EBV-infected monocyte cultures were harvested at the indicated times and tested for the presence of PGE2, IL-1ß, and IL-6, using commercially available enzyme immunometric assays with acetylcholine esterase as label (Cayman Chemical, Ann Arbor, MI). The detection limit for PGE2 was 29 pg/ml, with less then 0.01% cross-reactivity for other PGs. The detection limit for both IL-1ß and IL-6 was 1.5 pg/ml without significant cross-reactivity with other known cytokines.
Analysis of CD14 expression by flow cytometry
Monocytes (1 x 106 cells) were
infected with EBV particles (Fig. 2
) and, at the indicated times, cells
were washed twice with cold PBS (pH 7.4), followed by a preincubation
for 15 min at 4°C with 15 µg of human Igs (Miles Canada, Ontario,
Canada) in 100 µl of PBS to block Fc receptors. Cells were then
washed with PBS and incubated for an additional 30 min at 4°C with
either a fluoresceinated FITC anti-human CD14 mAb, or an IgG1
negative control FITC conjugate (BioSource International, Camarillo,
CA). After washing and fixation in 0.5% paraformaldehyde, cells were
analyzed with an EPICS-XL flow cytometer (Coulter Electronics) at an
excitation setting of 488 nm and emission setting of 540 nm.
Fluorescence was gated from monocytes, as judged by their forward and
side scatter.
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At the indicated times, untreated and LPS- and EBV-treated monocytes were washed twice with PBS (pH 7.4) and lysed in 100 µl of ice-cold HBSS containing 0.5% Triton X-100 and a cocktail of antiproteases consisting of 100 µM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Samples were diluted in 2x Laemmli buffer, boiled, electrophoresed on a 10% SDS-polyacrylamide gel, and immunoblotted, as described previously (16). Briefly, proteins were transferred for 3 h at 500 mA current setting onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA) pretreated in a blocking buffer containing 5% (w/v) dry milk and 0.15% Tween 20 in Tris-buffered saline (0.25 M Tris-HCl, pH 7.6, 0.19 M NaCl), and then incubated for 1 h at room temperature in the blocking solution containing anti-COX-1 (1/5000 diluted) or anti-COX-2 (1/2500 diluted) antisera (kindly provided by Dr. Jacques Maclouf, Institut National de la Santé et de la Recherche Médicale, Hôpital Lariboisière, Paris, France). Membranes were washed twice with TBS-Tween (0.15% v/v) and treated with a HRP-linked donkey anti-rabbit Ab. Bound Abs were visualized using the enhanced chemiluminescence (ECL) reagent according to the manufacturer’s protocol (Life Science, Boston, MA).
Northern blot analysis
Total RNA from unstimulated, EBV-treated, and LPS-stimulated monocytes (1 x 107 cells) was isolated using TRIzol reagent according to the manufacturer’s instructions (Life Technologies, Burlington, Ontario, Canada). Ten micrograms of RNA were loaded on a 1% agarose/formaldehyde gel and size fractionated by electrophoresis. RNA was then transferred overnight by capillary diffusion onto a Hybond-N nylon membrane (Amersham Canada Limited, Oakville, Ontario, Canada). The membranes were first preincubated for 3 h at 42°C, in a buffer containing 5x standard saline citrate phosphate/EDTA (SSPE), 2x Denhardt’s solution, 50% formamide, 5% dextran sulfate, and 100 µg/ml salmon sperm DNA. The hybridization was performed overnight in the same buffer containing 1 x 106 cpm/ml of the 32P-labeled cDNA probes (Prime-a-Gene labeling system) specific for COX-2 (1.3 kb), IL-1ß (1.3 kb), and IL-6 (1.2 kb) (the specific cDNA fragments were kindly provided by Dr. Patrice Poubelle, Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier de l’Université Laval, Québec, Canada). Membranes were subsequently washed twice in 0.5x SSC/0.1% SDS at 42°C for 30 min, followed by a more stringent wash at 65°C for 30 min in the same solution. Membranes were exposed to Kodak X-OMAT films at -80°C with intensifying screens. The GAPDH cDNA probe was used as internal control to demonstrate equal loading of DNA in each lane. The intensity of each autoradiographic signal was quantified by laser densitometry (FujiFilm, BAS-1800) and was normalized to its respective GAPDH signal.
Nuclear extract preparation and EMSA
Uninfected and infected monocytes were treated with LPS (1
µg/ml) for 30 min, and levels of nuclear NF-B were measured by
EMSA. Nuclear extracts were prepared as described elsewhere
(22). For EMSA, 5 µg of nuclear extracts were incubated
with 1 ng of 32P-labeled double-stranded
oligonucleotide, containing either the consensus binding site for
NF-B/c-Rel homodimeric and heterodimeric complexes (underlined),
5'-AGTTGAGGGGACTTTCCCAGGC-3', or as controls, the
mutated NF-B site 5'-AGTTGAGGCGACTTTCCCAGGC-3', or a
scrambled sequence that contains in this case the CREB DNA binding
motif 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3' (Santa Cruz
Biotechnology, Santa Cruz, CA). The oligonucleotides were 5' end
labeled with [
-32P]ATP using T4
polynucleotide kinase (Promega, Madison, WI), and unincorporated
32P labels were removed on a G-50 Sephadex
column. The incubation was performed at room temperature for 30 min in
1x binding buffer (100 mM HEPES, pH 7.9, 250 mM KCl, 5 mM EDTA, 250 mM
NaCl, 10% Ficoll, 40% glycerol, 10 mM DTT) containing 2 µg of
poly(dI-dC) and 10 µg of nuclease-free BSA. As an additional control,
nuclear extracts from LPS-stimulated monocytes were preincubated for 30
min with 100-fold excess of unlabeled NF-B oligonucleotide, which
completely abolished binding of the radiolabeled oligonucleotide to the
nuclear proteins. Samples were subjected to electrophoresis on a 4%
nondenaturing polyacrylamide gel for 1.5 h at 150 V in
Tris-borate-EDTA (TBE) 0.5x buffer. Gels were subsequently dried and
exposed to Kodak X-OMAT films at -80°C with intensifying
screens.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously reported that EBV affects arachidonate
metabolism in monocytes through priming for 5-lipoxygenase product
generation in response to stimulation with a second agonist
(16). To establish whether EBV can also interfere with the
COX pathway, we monitored both constitutive and LPS-induced
biosynthesis of PGE2 in uninfected and
EBV-treated monocyte cultures, over a 24-h period. As expected,
PGE2 levels increased gradually in response to
LPS stimulation, whereas the basal PGE2 levels in
unstimulated cells remained stable throughout the time course (Fig. 1). However, when monocytes were
preincubated for 1 h with EBV, and subsequently stimulated with
LPS, a reduction in PGE2 biosynthesis was
observed. Such a suppression of PGE2 biosynthesis
was significant from 12 h poststimulation with a 60–65%
reduction in PGE2 production (Fig. 1). In
contrast, the basal levels of PGE2, which are
mainly produced by the constitutive COX-1, remained low throughout the
time course and were not significantly affected by exposure to the
virus. Taken together, these results suggest that EBV may selectively
suppress PGE2 generation via the inducible COX-2
pathway.
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A, control monocytes or
monocytes that were incubated on ice with EBV to allow virus-cell
interactions showed similar levels of CD14 receptors, suggesting that
EBV does not bind to CD14, nor interferes with the
availability of CD14 receptors. Moreover, levels of CD14 in infected
cells remained comparable with those in control monocytes
even after 24- and 48-h exposure to EBV at 37°C (Fig. 2, B
and C), which allows expression of EBV viral genes in
monocytes (19). Effect of EBV on the expression of COX-2 in human monocytes
As a first step toward understanding the nature of the
EBV-mediated suppressive action, we evaluated its effect on COX-2
expression, an enzyme involved in PGE2
biosynthesis. This isoform of COX, normally absent in resting
monocytes, can be induced by a variety of stimuli, including LPS. As
depicted in Fig. 3, freshly isolated
monocytes do not constitutively express the COX-2 protein. However,
COX-2 levels increased rapidly following LPS stimulation, with maximal
expression at 12 h poststimulation with LPS. As expected,
levels of the noninducible COX-1 isoform were not affected by LPS
stimulation. The doublet observed in Western blot analysis is
characteristic of the COX-2 isoform and is due to glycosylation of the
protein (24). These results are consistent with previous
studies that have attributed the enhanced production of
PGE2 in stimulated monocytes/macrophages to the
inducible and transiently expressed COX-2 isoform (25).
Monocytes pretreated with EBV showed reduced levels of inducible COX-2
protein at 8 h poststimulation with LPS (Fig. 3), which
corresponds to the time point at which PGE2
levels began to be suppressed by EBV (Fig. 1). Maximal effects of EBV
were observed between 12 and 24 h, at which times expression of
COX-2 was barely detectable. This reduction of COX-2 protein levels
coincided with reduced levels of COX-2 mRNA transcripts by Northern
blot analysis. As shown in Fig. 4,
freshly isolated monocytes (0 h) express scarce amount of COX-2 mRNA.
Four hours after LPS stimulation, COX-2 mRNA was easily detectable with
minimal inhibition caused by EBV. However, by 8 h poststimulation
with LPS, COX-2 mRNA levels were reduced by EBV treatment by more than
half, as determined by densitometric analysis. Similar reduction in
COX-2 mRNA levels was observed at 12 h, and maximal inhibition was
observed at 24 h poststimulation. Thus, it appears that reduction
in PGE2 biosynthesis by EBV-treated monocytes is
directly related to a reduction in COX-2 mRNA levels.
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Additional experiments were then performed using inactivated
virions and an inhibitor of viral DNA polymerase to define the
mechanism through which EBV can diminish PGE2
formation in activated monocytes. Because maximum inhibitions were
observed 24 h after addition of LPS, we examined COX-2 expression
at this time point. Infectious EBV significantly reduced LPS-induced
COX-2 protein levels (Fig. 5). To prevent
specific binding of virions to the cell surface, viral particles were
either heat inactivated or neutralized with the mAb 72A1, which
recognizes the external viral envelope gp350/220. In both cases, EBV
lost its suppressive effect on COX-2 expression (Fig. 5). Since we
recently demonstrated EBV replication in monocytes (19),
we were also interested in the potential involvement of neosynthesized
viral proteins in the mediation of the EBV effects on
PGE2 biosynthesis. To assess this hypothesis, EBV
virions were inactivated by UV irradiation, which causes DNA damages
and prevents viral gene transcription with minimal effects on viral
particle structural integrity and infectivity. UV-treated EBV could not
suppress LPS-induced COX-2 expression (Fig. 5), suggesting that binding
to the cell surface and entry of virus into the monocytes are not
sufficient to cause COX-2 inhibition. These results strongly suggest
that viral gene transcription is needed to observe a reduction in COX-2
levels. In an attempt to determine whether early or late EBV proteins
are playing a role in the noticed COX-2 inhibition, we performed
experiments in the presence of PAA, a viral DNA polymerase inhibitor
that allows expression of early EBV proteins while preventing late
protein synthesis. As shown in Fig. 5, in the presence of PAA, EBV was
no longer able to suppress COX-2 protein expression, suggesting that a
late EBV protein is likely to be involved in COX-2 inhibition.
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To verify whether the suppressive effect of EBV on
PGE2 synthesis was merely due to a general
shutdown of host protein synthesis caused by the virus infection, we
examined the impact of EBV on other LPS-inducible proinflammatory
mediators such as the cytokines IL-1ß and IL-6. As shown in Fig. 6A, incubation of monocytes
with EBV stimulated secretion of IL-6, and to a lesser extent IL-1ß,
at 24 and 48 h postinfection. The potent IL-6 induction by EBV was
also observed at the transcriptional level, in which expression of IL-6
mRNA was increased by 2–3-fold in infected cells as compared with
untreated cells (Fig. 6B). These effects of EBV on IL-1ß
and IL-6 are in contrast to the PGE2 synthesis,
in which no induction was observed following the same time course (see
Fig. 1). The fact that EBV can specifically induce expression and
secretion of IL-1ß and IL-6 revealed that there is no overall
shutdown of host protein synthesis.
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). As expected, LPS stimulated expression
of IL-1ß and IL-6 both at the mRNA level (Fig. 6B) and
protein level (data not shown). However, in contrast to COX-2
expression, the presence of EBV did not affect LPS induction of those
cytokines (Fig. 6B), suggesting that LPS responsiveness of
monocytes is not overall affected. Therefore, EBV can differentially
modulate expression of proinflammatory mediators, presumably by
affecting different transductional pathways.
Inhibition of NF-B translocation in EBV-infected cells
Among the few transductional mechanisms that are currently known
to be implicated in COX-2 gene regulation, activation of the NF-B
pathway is of particular importance in monocytes/macrophages (26, 27). In resting cells, NF-B is normally present in the
cytoplasm in its inactivated form, complexed with its inhibitor,
I-B. Upon stimulation, NF-B is dissociated from I-B, and
translocates to the nucleus, where it mediates its transcriptional
activity through binding to consensus DNA sequences (GGGNNTNCCC)
found within the COX-2 promoter. We therefore investigated the
possibility that EBV may cause inhibition of COX-2 expression by
interfering with the nuclear translocation of NF-B. Thus, monocytes
infected or not with EBV for 1 h were stimulated with LPS, a
stimulus known to promote translocation of NF-B from the cytoplasm
to the nucleus. The presence of the transcription factor inside the
nuclei was determined by incubation of nuclear extracts with a
radiolabeled oligonucleotide probe, containing a NF-B recognition
sequence, followed by separation of NF-B probe complexes by EMSA, as
described in Materials and Methods. As shown in Fig. 7, low levels of activated NF-B could
be detected in control and EBV-infected monocytes, as witnessed by the
detection of two slower migrating complexes. The specificity of the
NF-B binding in EMSA was determined by cold competition with
unlabeled NF-B oligonucleotide, or by incubating nuclear extracts
with a labeled oligonucleotide containing a mutated NF-B site, or a
CREB site. In all cases, no specific NF-B complexes could be
detected. As expected, addition of LPS induced the levels of nuclear
proteins capable of binding to NF-B consensus sequence. However, LPS
stimulation of EBV-infected monocytes did not cause NF-B
translocation to the nucleus, suggesting that the inhibition of COX-2
expression could be attributable by the failure to effectively activate
NF-B in EBV-infected cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synthesis (29). More recently, we observed that
EBV could replicate and establish a complete lytic cycle in freshly
isolated monocytes (19). In the present study, we show
that EBV suppresses PGE2 biosynthesis in
LPS-activated monocytes. PGE2 inhibition by EBV
results from repression of COX-2 mRNA synthesis. As anticipated, COX-1
levels were not affected by either LPS or EBV treatment, confirming the
role of COX-2 as the major isoform involved in PG synthesis induced by
inflammatory stimuli. A specific interaction between EBV and the cell
surface of monocytes appears necessary for COX-2 inhibition, because
virions preincubated with the neutralizing mAb 72A1 (raised against the
viral envelope gp350/220) could not suppress COX-2 expression. Although
EBV binds to B lymphocytes via CD21, its receptor on monocytes remains
to be identified (15). We have investigated the
possibility that EBV might bind to CD14, which serves as a receptor for
LPS. However, treatment of monocytes with Abs against CD14 did not
prevent viral adsorption to the cell surface, suggesting that EBV
binding to monocytes does not involve CD14 (unpublished data). In the
present study, we show that EBV binding and infection do not interfere
with CD14 molecule. Therefore, that the inhibition of COX-2 expression
may be caused by occupancy of CD14 by EBV, which would prevent LPS
interaction with its receptor, is unlikely. Furthermore, preincubation
of monocytes with EBV does not affect LPS responsiveness of IL-6,
suggesting that EBV-infected monocytes possess functional LPS
receptors. Thus, the suppressive effect of EBV on
PGE2 synthesis is likely to occur at steps
downstream of the LPS receptor binding, possibly through interference
with signaling proteins such as protein tyrosine kinases and protein
kinase C, which are known to be involved in the induction of COX-2,
TNF-, and IL-1ß gene expression (30, 31, 32, 33).
Interestingly, we observed that PMA-induced PGE2
biosynthesis in monocytes is also inhibited by EBV, indicating that the
suppressive effect of the virus on PGE2 formation
is not specific to LPS-activated monocytes. Interestingly, EBV also lost its suppressive action on COX-2 expression when monocytes were incubated with viral particles inactivated by heat denaturation, UV irradiation, or when monocytes were preincubated with phosphonoacetic acid, an inhibitor of herpesviruses DNA polymerase. These results confirm that both structural integrity and infectivity are required to mediate EBV-induced suppression, and suggest that neosynthesized viral proteins are responsible for this process. That the suppression of both COX-2 expression and PGE2 release by EBV required a minimum of 8 h of infection might indicate that the suppression involves viral genes expressed late during infection. Which viral protein(s) is (are) responsible for the reduction in COX-2 transcription remains to be determined.
Although COX isozymes are essential components of the cellular enzymatic machinery involved in the biosynthesis of PGs, other enzymes, in particular the cytosolic phospholipase A2 (cPLA2) and/or the secreted PLA2, are also required for the formation of eicosanoids. We have previously shown that short-term (30–120 min) exposure of monocytes to EBV primes these cells for increased release of arachidonic acid and formation of leukotrienes, a process that appeared to involve the activation of the cPLA2, as observed by its increased phosphorylation (Ser505) and translocation (16). It is, however, distinctly possible that prolonged exposure (12–24 h) of monocytes to EBV impacts on the expression of cPLA2 and/or secreted PLA2 and consequently on PG biosynthesis. Studies are in progress to assess the effect of EBV on arachidonate release and PLA2 expression under the experimental conditions used in the present studies.
Our data suggest that EBV impairs the activation of the NF-B
transcription factor. Several studies have reported the importance of
NF-B in the induction of COX-2 gene transcription in both monocytes
and monocytic cell lines (34, 35). In fact, the COX-2 gene
was shown to possess two NF-B binding sites located within its
promoter (36, 37). In the present study, we show that EBV
decrease LPS-induced translocation of NF-B from the cytoplasm, where
it is found as an inactive complex with I-B, to the nucleus, where
it mediates transcriptional activation. Interestingly, p65, a component
of the NF-B dimer, was shown to physically interact with the EBV
immediate-early protein ZEBRA, which is essential to viral replication,
and inhibit its transactivating activities in B lymphocytes
(38). Such interaction between ZEBRA and p65 was recently
demonstrated in T lymphocytes, and it was suggested that the formation
of this complex is functionally analogue to the inactive I-B/NF-B
complex found in the cytoplasm. The occurrence of such inactive
NF-B/ZEBRA complex in EBV-infected monocytes remains to be
investigated. Another possible mechanism by which EBV could inhibit
translocation of NF-B is to prevent the inducible degradation of
I-B by interfering with targeting of the ubiquitinated I-B to the
proteasome. Interestingly, the EBV nuclear Ag 1 (EBNA-1) contains a
glycine-alanine repeat that is responsible for its resistance to
degradation through the ubiquitin-proteasome system (39).
Finally, another possible means of preventing NF-B activation that
has already been described for the African swine virus is to encode for
a homologue of I-B (40). Whether EBNA-1 or other EBV
proteins possessing a similar structural module interact with I-B
remains to be established.
NF-B is also involved in the transcriptional activation of several
monocyte/macrophage genes, including those that encode proinflammatory
cytokines (41). For example, TNF- gene possesses
multiple NF-B binding sites in its promoter, which makes its
expression highly induced following NF-B translocation to the
nucleus (42, 43). Regulation of TNF- gene is of
particular interest because our previous observations showed that EBV
strongly inhibits the transcription of this cytokine in monocytes
stimulated with either LPS or phorbol esters (29), two
known inducers of NF-B activation (44). Taken together,
it is tempting to speculate that regulation of COX-2 and TNF- genes
by EBV could result from impaired NF-B activation.
Other viruses, such as herpes simplex virus type 1 and human CMV, have
been shown to up-regulate PGE2 production in
infected monocytes (45, 46). Similarly, it was reported
that the formation of both 5-lipoxygenase and COX products derived from
arachidonic acid was enhanced in HIV-infected monocytes
(47). Thus, the effects of EBV on the arachidonate
metabolism are particular, in that while the 5-lipoxygenase pathway is
up-regulated, resulting in increased leukotriene synthesis, the COX-2
pathway is inhibited. To our knowledge, this is the first report of
PGE2 biosynthesis inhibition by a human virus. In
addition, our results indicate that expression of EBV late cycle
proteins is involved in such inhibition. Although the EBV-suppressive
effect reported herein is likely to result from an inhibition of
NF-B activation, the nature of EBV interactions with the
transductional events that trigger NF-B activation remains to be
established.
Arachidonate metabolites are important immunoregulatory mediators, and interference with the tightly regulated equilibrium between leukotriene and PG productions may represent an additional mean utilized by EBV to disturb the immune response and promote viral replication.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jean Gosselin, Laboratory of Viral Immunology, Centre de recherche en Rhumatologie et Immunologie, CHUQ, Pavillon du CHUL, Room T 1-49, 2705 boulevard Laurier, Sainte-Foy (Québec), G1V 4G2, Canada.
3 Abbreviations used in this paper: COX, cyclooxygenase; cPLA, cytosolic PLA; gp, glycoprotein; I-B, inhibitory protein that dissociates from NF-B; PAA, phosphonoacetic acid; PLA, phospholipase A; TFU, transforming unit.
Received for publication October 29, 1999. Accepted for publication March 30, 2000.
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 A. G. Granja, M. L. Nogal, C. Hurtado, V. Vila, A. L. Carrascosa, M. L. Salas, M. Fresno, and Y. Revilla The Viral Protein A238L Inhibits Cyclooxygenase-2 Expression through a Nuclear Factor of Activated T Cell-dependent Transactivation Pathway J. Biol. Chem., December 17, 2004; 279(51): 53736 - 53746. [Abstract] [Full Text] [PDF]
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N. Ray, M. E. Bisher, and L. W. Enquist Cyclooxygenase-1 and -2 Are Required for Production of Infectious Pseudorabies Virus J. Virol., December 1, 2004; 78(23): 12964 - 12974. [Abstract] [Full Text] [PDF]
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 A. O. Guerreiro-Cacais, L. Li, D. Donati, M. T. Bejarano, A. Morgan, M. G. Masucci, L. Hutt-Fletcher, and V. Levitsky Capacity of Epstein-Barr virus to infect monocytes and inhibit their development into dendritic cells is affected by the cell type supporting virus replication J. Gen. Virol., October 1, 2004; 85(10): 2767 - 2778. [Abstract] [Full Text] [PDF]
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S. Salek-Ardakani, S. A. Lyons, and J. R. Arrand Epstein-Barr Virus Promotes Human Monocyte Survival and Maturation through a Paracrine Induction of IFN-{alpha} J. Immunol., July 1, 2004; 173(1): 321 - 331. [Abstract] [Full Text] [PDF]
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T. L. Symensma, D. Martinez-Guzman, Q. Jia, E. Bortz, T.-T. Wu, N. Rudra-Ganguly, S. Cole, H. Herschman, and R. Sun COX-2 Induction during Murine Gammaherpesvirus 68 Infection Leads to Enhancement of Viral Gene Expression J. Virol., December 1, 2003; 77(23): 12753 - 12763. [Abstract] [Full Text] [PDF]
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M.-E. Janelle, A. Gravel, J. Gosselin, M. J. Tremblay, and L. Flamand Activation of Monocyte Cyclooxygenase-2 Gene Expression by Human Herpesvirus 6. ROLE FOR CYCLIC AMP-RESPONSIVE ELEMENT-BINDING PROTEIN AND ACTIVATOR PROTEIN-1 J. Biol. Chem., August 16, 2002; 277(34): 30665 - 30674. [Abstract] [Full Text] [PDF]
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M. Tardif, M. Savard, L. Flamand, and J. Gosselin Impaired Protein Kinase C Activation/Translocation in Epstein-Barr Virus-infected Monocytes J. Biol. Chem., June 28, 2002; 277(27): 24148 - 24154. [Abstract] [Full Text] [PDF]
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 S. Murono, H. Inoue, T. Tanabe, I. Joab, T. Yoshizaki, M. Furukawa, and J. S. Pagano Induction of cyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells PNAS, May 24, 2001; (2001) 121016998. [Abstract] [Full Text] [PDF]
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S. Murono, H. Inoue, T. Tanabe, I. Joab, T. Yoshizaki, M. Furukawa, and J. S. Pagano Induction of cyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells PNAS, June 5, 2001; 98(12): 6905 - 6910. [Abstract] [Full Text] [PDF]
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