Download Original article Inhibition of lytic reactivation of Kaposi`s sarcoma

Antiviral Therapy 2011; 16:17–26 (doi: 10.3851/IMP1709)
Original article
Inhibition of lytic reactivation of Kaposi’s sarcomaassociated herpesvirus by alloferon
Naeun Lee1, Seyeon Bae1, Hyemin Kim1, Joo Myung Kong1, Hang-Rae Kim1, Byung Joo Cho 2, Sung Joon
Kim 3, Seung Hyeok Seok4, Young-il Hwang1, Sooin Kim5, Jae Seung Kang1,6*, Wang Jae Lee1*
Department of Anatomy and Tumor Immunity Medical Research Center, Seoul National University College of Medicine, Seoul, Korea
Department of Ophthalmology, Konkuk University, School of Medicine, Konkuk University Hospital, Seoul, Korea
3
Department of Physiology and Ischemia/Hypoxia Disease Institute, Seoul National University College of Medicine, Seoul, Korea
4
Department of Microbiology and Immunology and Institute for Experimental Animals, Seoul National Univerisy College of Medicine,
Seoul, Korea
5
EntoPharm Co. Ltd, Seoul, Korea
6
Institute of Complementary and Integrative Medicine Medical Research Center, Seoul National University, Seoul, Korea
1
2
*Corresponding author e-mails: [email protected]; [email protected]
Background: Alloferon, an immunomodulatory ­peptide,
has antiviral capability against herpesvirus. In this
research, we aimed to investigate the effect of alloferon
on the regulation of the life cycle of Kaposi’s sarcomaassociated herpesvirus (KSHV), and its mechanisms.
We also assessed the antiviral activity of alloferon on
natural killer (NK) cells as an early antiviral immune
responder.
Methods: We first examined the change in cell proliferation and the expression of the viral genes in a
KSHV-­infected cell line, body-cavity-based B lymphoma
(BCBL)-1, under the lytic cycle by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. To elucidate the
antiviral mechanism of alloferon, we tested calcium
influx and the activation of the extracellular signal-regulated kinase (ERK) pathway. Furthermore, we evaluated
the cytotoxicity of NK cells against BCBL-1 by alloferon.
Results: Alloferon effectively recovered the suppressed
proliferation of BCBL-1 by TPA, which was achieved by
the down-regulation of lytic-cycle-related viral genes,
RTA, K8 and vIRF2. To clarify the signal transduction
pathways related to the regulation of the viral genes
by alloferon, we confirmed that the calcium influx into
BCBL-1 was apparently inhibited by alloferon, which preceded the suppression of the phosphorylation of ERK and
the activation of AP-1 by TPA. Moreover, when NK cells
were exposed to alloferon, their cytolytic activity was
improved, and this was mediated by the enhancement of
perforin/granzyme secretion.
Conclusions: The results of this study suggest that alloferon
can be used as an effective antiviral agent for the regulation of the KSHV life cycle by the down-regulation of AP-1
activity and for the the enhancement of antiviral immunity
by up-regulation of NK cell cytotoxicity.
Introduction
Kaposi’s sarcoma-associated herpesvirus (KSHV) is
a double-stranded DNA virus and is classified formally as human herpesvirus 8 in the genus Rhadinovirus of the subfamily Gammaherpesvirinae. Since
the identification of KSHV, this virus has been known
as the aetiological agent of Kaposi’s sarcoma (KS),
primary effusion lymphoma (PEL) and multicentric
Castleman’s disease (MCD) [1–5]. KS is a tumour of
endothelial cell origin that is found most frequently
in immunosuppressed patients, especially those who
are HIV-infected and not receiving treatment. PEL is
a rare, aggressive, non-Hodgkin’s B-cell lymphoma
©2011 International Medical Press 1359-6535 (print) 2040-2058 (online)
AVT-10-OA-1518_Lee.indd 17
that develops as a malignant effusion in the peritoneal, pleural or pericardial space. Unlike PEL, MCD
is a lymphoproliferative disorder characterized by
enlarged lymph nodes, infiltration of plasma cells and
vascular proliferation [6]. These pathologies are all
caused by KSHV infection.
It has been reported that the viral genome of KSHV
is approximately 170 kb and encodes >81 open reading frames (ORFs) [7–9]. Following primary infection,
KSHV establishes a persistent infection and takes up
one of the two alternative genetic life cycle programmes
upon infection of host cells [6]. When a host cell is
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N Lee et al.
infected with KSHV, the virus genome is translocated to
the nucleus, then enters a latent cycle. During the latent
cycle, the viral episome is maintained but no viral progeny is produced. In order to maintain the latent cycle,
a small number of viral proteins are expressed, which
enable them to escape the antiviral immune responses
of the host cells.
Upon specific physiological stimulation, such as
hypoxia or coinfection with HIV type-1, latently
infected cells are induced to enter the lytic cycle. The
expression of the KSHV lytic genes is tightly regulated
and induced in regular sequence: immediate-early, early
and late genes. Of various lytic proteins, replication and
transcriptional activator (RTA; encoded ORF50) is necessary for the lytic reactivation of KSHV [10,11]. RTA
is a transcriptional transactivator that binds directly to
the DNA of several KSHV promoters with high affinity and function as a lytic switching protein [9,12–15].
Following RTA activation, other immediate-early, early
and late genes of the lytic cycle are expressed, including
Kb-ZIP (also known as K8), viral interferon-regulatory
factor (vIRF2) and modulator of immune recognition-2
(MIR-2 or K5). Kb-ZIP (K8) interacts and colocalizes
with the transcriptional coactivator cyclic AMP responsive element binding protein, which modulates p300
transcriptional activity [6]. It has recently been reported
that K8 is also essential for the lytic gene expression
and virion production [16]. vIRF2 suppresses interferon regulatory factor (IRF)1- and IRF3-driven activation, and ORF45 interacts with IRF7 and prevents
its phosphorylation [7,17]. Activation of K8, vIRF2
and ORF45 results in the inhibition of type I interferon
(IFN)-induced signalling. K5 proteins are part of a large
family of membrane-bound E3 ubiquitin ligases that
efficiently down-regulate the expression of major histocompatibility complex (MHC) class I molecules on
the surface of infected cells [6]. It is well known that
the lytic reactivation of KSHV plays important roles in
its pathogenesis [18]; therefore, the control of latent to
lytic reactivation in KSHV-infected cells might regulate
the development of the KSHV-associated diseases. As
described above, recent studies for the regulation of
KSHV life cycle have been focused on the regulation of
the viral life-cycle-related genes.
Alloferon is an immune-modulating peptide which
is isolated from bacteria-challenged larvae of the blow
fly Calliphora vicina and consists of 13 amino acid
sequences: HGVSGHGQHGVHG [19]. Chernysh et
al. [20], colleagues based in Russia, have demonstrated
that alloferon stimulates a natural cytotoxicity of human
peripheral blood lymphocytes and enhances their antitumoural and antiviral activities through the induction
of IFN synthesis in mouse and human models [20].
Several types of modified alloferons are already developed and clinically used in Russia. Interestingly, they
18 AVT-10-OA-1518_Lee.indd 18
are highly effective in the prevention of recurrent genital
infection of herpesviruses; however, their specific mechanisms related to the regulation of the viral life cycle
from the latent to lytic cycle still need to be clarified. To
evaluate the effect of alloferon on the regulation of the
viral life cycle from the latent to lytic cycle, we used a
KSHV-infected cell line, body-cavity-based B lymphoma
(BCBL)-1, in the present study. Because KSHV is latent
in BCBL-1 and its lytic reactivation can be induced by
12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment, it is suitable for the evaluation of the effect of
alloferon on the regulation of the viral life cycle. Therefore, we evaluated the effect of alloferon as an antiviral
agent for the regulation of the life cycle of KSHV and
examined its specific related mechanism using a KSHVinfected cell line, BCBL-1.
Methods
Cell culture
BCBL-1 (a KSHV-positive Epstein–Barr virus negative
PEL cell line) was maintained in RPMI 1640 medium
(Gibco BRL, Grand Island, NY, USA) supplemented
with 10% heat-inactivated fetal bovine serum, 100 U/ ml
penicillin and 100 µg/ml streptomycin (HyClone,
Logan, UT, USA). For the lytic reactivation of KSHV
in BCBL-1 cells, cells were treated with TPA (Sigma, St
Louis, MO, USA) at a concentration of 20 ng/ml.
Measurement of the cellular cytotoxicity of alloferon
A quantity of 10,000 BCBL-1 cells were seeded onto
a 96-well plate and treated with alloferon according to the indicated dose, with or without TPA. After
a 12-h incubation, cell proliferation and cytotoxicity
were determined by using a cell counting kit, CCK-8
(Dojindo Laboratory, Kumamoto, Japan).
Assessment of cell proliferation
To examine the effect of alloferon on the proliferation
of BCBL-1 cells with or without treatment with TPA,
[3H]-thymidine incorporation assays were performed as
follows: BCBL-1 cells were incubated in the presence
or absence of TPA for 12 or 24 h after the addition of
[3H]-thymidine (2.5 µCi/ml). Then, the proliferation of
each group was monitored through the measurement of
radioactivity by a scintillation counter (Wallac, Boston,
MA, USA). Data are expressed as mean ±sd and analysed using the Student’s t-test.
RNA isolation and real-time reverse transcription PCR
Total RNAs of BCBL-1 cells from each group were
isolated using Trizol Reagent (Invitrogen, Carlsbad,
CA, USA) and were reverse-transcribed into complementary DNA using oligo (dT) primers and avian
myeloblastosis virus reverse transcriptase (iNtRON,
©2011 International Medical Press
9/2/11 15:56:39
Antiviral effect of alloferon
Daejeon, Korea). The real-time reverse transcription
(RT)-PCR was processed with SYBR Green PCR master mix (MBI Fermentas, St Leon-Rot, Germany) and
performed using an ExiCycler™ (Bioneer, Daejeon,
Korea). To ascertain changes in the expression of the
gene of interest, the differences between expression of
the housekeeping gene (GAPDH) and the gene of interest were calculated using the 2-∆(∆CT) method [21].
Western blot analysis
BCBL-1 cells were lysed in lysis buffer. Cell extracts
were subjected to 10% SDS-PAGE and transferred to
nitrocellulose membranes (Millipore, Bedford, MA,
USA). After blocking with 5% skim milk in 0.1% Tween
20-phosphate-buffered saline, membranes were incubated with the primary antibodies: anti-K8 antibody,
anti-LANA antibody (ABI, Columbia, MD, USA), antipERK-antibody (Santa Cruz Biotechnology, Santa Cruz,
CA, USA), anti-ERK-antibody (Santa Cruz Biotechnology) and anti-human β-actin (Cell Signaling Technology, Beverly, MA, USA) overnight at 4°C. Anti-rabbit or
anti-mouse IgG horseradish peroxidase (Cell Signaling
Technology) was used as the secondary antibody. Bands
were visualized with a chemiluminescence detection kit
(ECL; Amersham Biosciences, Little Chalfont, UK).
Measurement of intracellular calcium level
BCBL-1 cells were resuspended in Tyrode’s buffer and
were labelled with 5 µM cell-permeable fluorescent calcium indicator Fura-2 (Molecular Probes Inc., Eugene,
OR, USA). The cells were washed with phosphate-buffered saline and resuspended in fresh Tyrode's buffer.
Then, fluorescence was monitored at 530 nm using
a Perkin–Elmer Fluorescence Spectrometer (LS-50B;
Perkin–­Elmer, Wellesley, MA, USA). Each sample was
pretreated with 2 mM EGTA (Sigma) to prevent the
influx of external calcium. After 10 min of baseline
recordings, all samples were treated with 2 µM thapsigargin (Sigma) to deplete calcium from the endoplasmic
reticulum and then treated with 5 mM Ca2+ to induce
the store-operated calcium entry.
Transfection and luciferase reporter gene assay
For electroporation, cells were washed once and resuspended in 500 µl of serum-free RPMI. The cells were
electroporated with pAP-1/luciferase (Clontech, Palo
Alto, CA, USA) and pRL-TK/luciferase (Promega, Madison, WI, USA) using a Bio-Rad Electroporator (BioRad Laboratories, Inc., Richmond, CA, USA). The cells
were incubated for 24 h and then alloferon and/or TPA
were added to each group. After incubation for another
12 h, luciferase assays were performed on cell lysates
with a Dual-Luciferase assay kit (Promega) according
to the manufacturer's instructions. Luminescence was
measured on a GloMax luminometer (Promega).
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AVT-10-OA-1518_Lee.indd 19
Isolation of natural killer cells and flow cytometric
analysis
Peripheral blood mononuclear cells were purified from
peripheral blood obtained from normal healthy volunteers by standard Ficoll-Hypaque density-gradient centrifugation. Natural killer (NK) cells were isolated from
peripheral blood mononuclear cells using a human NK
cell isolation kit and an AutoMACS (Miltenyi Biotechnology GmbH, Bergisch, Gladbach, Germany) according to the manufacturer’s instructions. Purified NK cells
were incubated with or without 4 µg/ml alloferon for
12 h. After 12 h of incubation, NK cells were stained
with each antibody. Allophycocyanin-­labelled anti­human NKG2D antibody (BD Pharmigen, San Diego,
CA, USA) and FITC-labelled anti-human 2B4 antibody
(BD Pharmigen) were used as the markers of NK cellactivating receptors. Fluorescein-conjugated anti-human KIR antibody (R&D Systems, Minneapolis, MN,
USA) and fluorescein-conjugated anti-human CD94
antibody (R&D Systems) were used as the markers of
NK cell inhibitory receptors. Cell surface fluorescence
was analysed with a Becton Dickinson FACScalibur
(BD Biosciences, San Jose, CA, USA).
51
Cr release assay
Cytotoxicity was measured by means of 51Cr release
assays using KSHV-infected BCBL-1 cells as target cells
and human NK cells as effector cells [19]. Target cells
were radio-labelled with 100 µCi of Na251CrO4 (Amersham Pharmacia Biotech, Uppsala, Sweden) at 37°C for
1 h. After being washed twice, the labelled target cells
were resuspended and dispensed onto 96-well U-bottomed microtitre plates. Effector cells were added to the
appropriate wells to give the effector:target cell ratios
of 5:1, 25:1 and 50:1 in a total of 150 µl of the culture
medium. After 4 h of incubation, 100 µl of the culture
supernatants were obtained and counted in a gamma
counter. Maximum isotope release was measured by
incubating the targets in 3% NP-40 (Sigma). Spontaneous isotope release was measured by incubating the
targets in the culture medium. The following formula
was used to calculate cytotoxicity: specific lysis value
(%)=100× (experimental release value [cpm]- spontaneous release value [cpm])/(maximum release value
[cpm]- spontaneous release value [cpm]). Each assay
was performed in triplicate.
Detection of granzyme B, soluble Fas ligand and
tumour necrosis factor-α
Purified NK cells were cocultured with BCBL-1 cells as
described in the section on 51Cr release assay. After 4 h of
incubation, cell culture supernatants were collected and
the productions of granzyme B, soluble Fas ligand (sFasL)
and tumour necrosis factor (TNF)-α were detected using
a granzyme B ELISA kit (Bender MedSystems, Vienna,
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Figure 1. Effects of alloferon on cell proliferation and cytotoxicity of BCBL-1 cells
B
1
Cell proliferation, cpm/well, ×104
Absorbance at 450 nm
A
0.8
0.6
0.4
0.2
0
TPA
Alloferon
concentration,
ng/ml
-
-
-
-
+
+
+
+
0
20
80
320
0
20
80
320
14
12 h
24 h
12
10
P=0.001
8
6
4
2
0
TPA
Alloferon,
20 ng/ml
-
+
+
-
-
+
(A) Cytotoxicity of body-cavity-based B lymphoma (BCBL)-1 cells was determined by cell counting kit assays. BCBL-1 cells were incubated in the presence or absence
of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) according to the concentration of alloferon (0, 20, 80 and 320 ng/ml). After a 12-h incubation, absorbance was
measured at 450 nM. Experiments were performed in triplicate. (B) Cell proliferation of BCBL-1 cells was assessed by [3H]-thymidine incorporation assays. Each group
was incubated in the presence or absence of TPA or alloferon as indicated. Alloferon was administered 3 h after TPA treatment.
Austria), sFasL ELISA kit (BioSource, Camarillo, CA,
USA) and a TNF-α ELISA kit (eBioscience, San Diego,
CA, USA) according to the manufacturers’ instructions.
Results
Alloferon does not affect cell viability of BCBL-1 cells
but recovers suppressed proliferation by TPA
To examine the direct cellular cytotoxicity of alloferon
on BCBL-1 cells, cells were incubated in the presence
of various doses of alloferon (0–320 ng/ml). As shown
in Figure 1A, alloferon did not have a direct cytotoxic
effect and did not affect cell proliferation of BCBL-1
cells. It has been documented that TPA suppresses the
proliferation of BCBL-1 cells, whereas it induces lytic
cycle-associated gene expression of KSHV. We therefore
investigated whether alloferon recovers the suppression
of TPA-induced BCBL-1 cell proliferation. As shown in
Figure 1B, we confirmed the suppression of BCBL-1 cell
proliferation by treatment with TPA. When alloferon was
added to BCBL-1 cells 3 h after treatment with TPA, cell
proliferation was recovered and sustained for 12 h in the
presence of alloferon. This suggests that alloferon might
have a regulatory effect on lytic reactivation of KSHV.
Expressions of the immediate-early viral genes are
suppressed by treatment with alloferon
There have been some reports indicating that a viral
gene activates the lytic cycle expression of KSHV [22].
20 AVT-10-OA-1518_Lee.indd 20
Based on the results shown in Figure 1, we investigated
the regulatory effect of alloferon on the expression of
the viral genes involved in the immediate-early phase of
the KSHV lytic cycle. Changes in viral gene expression
by alloferon were examined by RT-PCR. Among several genes, the expressions of five genes involved in the
immediate-early lytic phase, RTA, K8, ORF45, vIRF2
and K5, were increased by TPA, but their expressions
were significantly suppressed for 3 h in the presence of
alloferon (Figure 2A). Next, we performed real-time
RT-PCR to clarify the expressions of RTA, vIRF2, K5
and K8 by treatment with TPA and/or alloferon. The
expressions of these genes, except K5, were up-regulated by treatment with TPA, but the TPA-mediated
up-regulation was dramatically suppressed by alloferon
(Figure 2B). K8 was increased by treatment with TPA
but suppressed in the presence of alloferon at the protein level as well (Figure 2C). The expression of another
lytic phase-related gene, vIL-6, was also examined, but
there was no significant change.
Alloferon suppresses viral protein expression by
suppression of Ca2+ influx
Some reports have demonstrated that agents mobilizing intracellular calcium, such as ionomycin, reactivate
KSHV in latently infected BCBL-1 cells and that calcium-mediated virus reactivation in KSHV-infected cells
could be inhibited by cyclosporine [23–25]. It has been
proposed that a calcium-mediated signalling pathway
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Antiviral effect of alloferon
Figure 2. Changes in the mRNA expression level of the KSHV lytic genes in BCBL-1 upon TPA and alloferon treatment
TPA
Alloferon
1
-
2
+
-
3
+
4
+
+
18
16
Fold induction
RTA
B
K8
vIRF2
ORF45
2
18
P<0.01
RTA
14
Fold induction
A
12
1
0.8
0.6
0.4
K5
0.2
0
vIL-6
TPA
Alloferon -
β-Actin
+
+
-
-
-
+
+
1.4
Alloferon
-
+
-
+
K8
β-Actin
3.5
P<0.01
vIRF2
3
1.2
Fold induction
-
Fold induction
TPA
16
14
12
1
0.8
0.6
0.4
0.2
0
+
+
4
C
2.5
2
1.5
1
P<0.01
0.4
0
+
+
+
+
0.6
0
+
-
K5
+
-
0.8
0.2
+
+
1
0.5
TPA
Alloferon -
P<0.01
K8
-
+
+
-
+
+
(A) Reverse transcription (RT)-PCR was performed as follows: control (lane 1), 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment (lane 2), alloferon (20 ng/ml)
treatment (lane 3), pretreatment with TPA for 3 h prior to alloferon treatment (lane 4). Cells in each group were incubated for 3 h. (B) Real-time RT-PCR was carried
out as described in (A). Data are represented with fold induction for a standard value of the control group in the absence of TPA and alloferon. (C) Western blot
analysis was followed against Kaposi’s sarcoma-associated herpesvirus (KSHV) K8 protein. Each group was the same as in (B) and was incubated for 6 h. Western blot
analysis was performed as described in Methods. ORF, open reading frame; RTA, replication and transcriptional activator; vIRF2, viral interferon-regulatory factor.
could lead to regulation of the life cycle of KSHV. In
this study, we confirmed that the expression of the lyticcycle-associated gene, RTA, was tightly regulated by the
Ca2+ ion through the blockade of Ca2+ influx using a
calcium chelator, EGTA (Figure 3A). That is, when Ca2+
influx was blocked by EGTA, the expression of RTA
was down-regulated. This result means that lytic reactivation of KSHV was inhibited in BCBL-1 cells. We
therefore investigated whether Ca2+ influx was inhibited
in the presence of alloferon. When BCBL-1 cells were
treated with only alloferon in the absence of TPA, there
was no detectable change in Ca2+ influx (NL et al., data
not shown). As shown in Figure 3B, when cells were
preincubated with alloferon and TPA for 10 min prior
to the addition of 5 mM Ca2+ ion, it was found that
intracellular calcium influx was decreased. This result
implies that alloferon might regulate lytic-cycle-associated viral gene expression through the down-regulation
of the calcium-mediated signalling pathway.
Antiviral Therapy 16.1
AVT-10-OA-1518_Lee.indd 21
Alloferon inhibits TPA-induced activation of ERK
and AP-1
There have been several reports regarding the reactivation of KSHV from latency. It is reported that KSHV
reactivation from latency has been shown to be modulated, not only by the MEK/ERK pathway, but also
by JNK and p38 pathways through the activation of
AP-1, which binds to the promoter of RTA [26,27]. To
investigate the intracellular signalling pathway related
to the suppressed expression of the lytic-cycle-related
gene upon alloferon treatment, changes in the phosphorylation of ERK and activation of AP-1 were assessed.
As shown in Figure 4A, alloferon effectively suppressed
the TPA-induced phosphorylation of ERK. In addition,
we investigated the activity of AP-1 through the luciferase reporter gene assay containing the AP-1 binding
site in the promoter of the luciferase gene (Figure 4B).
Luciferase activity was increased by treatment with
TPA, but was suppressed in the presence of alloferon.
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N Lee et al.
statistically significant. With regard to TNF-α, there
was no remarkable change. These findings suggest that
alloferon improves the cytolytic activity of NK cells and
increases granzyme B production.
We performed the same experiment after transfection
of the phosphorylated nuclear factor-κB/luc gene construct, containing the nuclear factor-κB binding site in
the promoter of the luciferase gene, but there was no
significant change (NL et al., data not shown).
Discussion
Cytolytic activity of NK cells is increased by alloferon
and mediated by increased granzyme B secretion
Alloferon has been shown to have preventive effects on
recurrent herpes simplex virus (HSV) infection. It has
been successfully developed through clinical trials in
Russia. However, the mechanism by which this occurs
has not yet been completely elucidated. We designed this
study to elucidate the antiviral effects of alloferon on
BCBL-1 cells, which are a persistently KSHV-­infected
PEL cell line.
In this study, it was found that alloferon did not
induce cell apoptosis in either the latent or lytic phases
of KSHV; however, cell proliferation assays demonstrated that alloferon rescued the suppression of cell
proliferation following lytic reactivation. This action is
closely related to the regulatory effect of alloferon on the
lytic reactivation of KSHV in infected cells. The KSHV
RTA plays an important role in controlling the switch
from latency to lytic replication in KSHV latently-infected cells sufficiently to drive the viral lytic cascade to
completion and induce the production of encapsidated
virions [13–16]. The regulation of RTA and other lytic
cycle-related gene expressions by alloferon suggests
We have so far described the direct regulatory effect of
alloferon on KSHV-infected cells. By contrast, because
alloferon has been identified as an immunomodulating
peptide derived from the immune system of insects, we
considered its modulatory effect on the immune susceptibility of KSHV-infected cells. NK cells were representative of antiviral immune cells in an innate immune
system [28,29]. We investigated the effect of alloferon
on the enhancement of antiviral activity of NK cells. As
shown in Figure 5A, NK cells by treatment with alloferon enhanced the immune susceptibility of BCBL-1
cells; however, there were no changes in the surface
expression of stimulatory and inhibitory receptors on
NK cells (Figure 5B). To clarify the mechanism of the
enhanced killing effect of NK cells, granzyme B, sFasL
and TNF-α, their concentrations in coculture media
of NK and BCBL-1 cells were measured by respective ELISA. As shown in Figure 5C, the production of
granzyme B was significantly up-regulated and that of
sFasL was also up-regulated, but the difference was not
Figure 3. Change in the intracellular Ca2+ ion level in BCBL-1 upon TPA and alloferon treatment
A
B
BCBL-1, 1.3 mM Ca2+ buffera
500
Control
TPA
TPA+ alloferon
EGTA
-
+
+
TPA
-
-
+
RTA
β-Actin
Ca 2+ concentration, nM
450
400
350
300 EGTA, 2 mM
Ca2+, 5 mM
250
Thapsigargin, 2 µM
200
150
100
50
80
0
1,
00
0
1,
20
0
1,
40
0
1,
60
0
1,
80
0
60
0
40
0
0
20
0
0
Time, s
(A) Body-cavity-based B lymphoma (BCBL)-1 cells were incubated in the presence or absence of EGTA for 3 h. Complementary DNA from each group were synthesized,
and reverse transcription-PCR for Kaposi’s sarcoma-associated herpesvirus replication and transcriptional activator gene (RTA) was performed. (B) The intracellular
calcium level of 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-treated BCBL-1 cells in the presence or absence of alloferon was measured as described in Methods.
The dotted line represents Ca2+ influx following store-operated calcium entry by 2 mM thapsigargin treatment. aBCBL-1 cells were labelled with 5 µM cell-permeable
fluorescent calcium indicator Fura-2 (Molecular Probes Inc., Eugene, OR, USA) at 30 min after treatment of alloferon.
22 AVT-10-OA-1518_Lee.indd 22
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Antiviral effect of alloferon
that alloferon can be used as an efficient drug in KSHVmediated disease. Wang et al. [30] have described that
the activation of RTA promoter is mediated by AP-1
activation. Accordingly, we demonstrated the suppressive effect of alloferon on TPA-induced AP-1 activation in this study. Therefore, the modulatory effect of
alloferon on lytic reactivation of KSHV was achieved
through the suppression of AP-1 activation followed
by the suppression of RTA expression. Moreover, there
has been an interesting report regarding RTA and HSV
type-1 [31]. Finally, our findings suggest that alloferon
can also be used as a therapeutic agent for HSV-related
disease through the regulation of RTA expression. It
also supports the finding that alloferon is effectively
used as a therapeutic agent for the prevention of recurrent genital herpes infection in Russia.
To eradicate a virus and prevent the development
of disease by viral infection, it is important that direct
prevention of viral replication or reactivation should
be achieved by an antiviral agent and that the antiviral
immunity mediated by NK cells or virus-specific T-cells
be enhanced. The increased NK cytotoxicity by alloferon
treatment is already reported by Chernysh et al. [20];
however, they did not investigate the specific mechanism
of alloferon on the increase of NK cytotoxicity. It is generally known that granzyme B and sFasL play important roles in the cytolytic activity of NK cells. As shown
in Figure 5, alloferon enhances NK cell activity against
BCBL-1 via increased production of granzyme B and
sFasL. This is the first report regarding the specific mechanism of alloferon on the enhancement of NK activity.
Alloferon had an overt effect on the regulation of
K3 and K5, which play crucial roles in the suppression of MHC class I expression. Based on this finding, the effect of alloferon was primarily directed at
the enhancement of innate immunity mediated by NK
cells. As expected, alloferon successfully enhanced
NK cell cytotoxicity against BCBL-1 cells and was
mediated by an increase in granzyme B. It is generally
known that NK cell cytotoxicity is mediated by granule’s exocytosis, nitric oxide production, Fas–FasL
interaction and TNF-α activity [29]. In our study,
alloferon did not show the effect on nitric oxide production, although granzyme B is a major cytotoxic
effector molecule for NK cell-mediated cytotoxicity.
In addition, there were no changes in stimulatory and
inhibitory receptors on the surface expression of NK
cells. Based on our finding that the immune susceptibility of BCBL-1 cells against NK cells is increased in
the presence of alloferon, changes in the expression
of corresponding ligands for NK cell-activating receptors on BCBL-1 cells, such as MICA and MICB, upon
alloferon treatment should be further investigated.
Regarding the enhancement of antiviral immunity,
the humoural immune response also plays an important
role through the production of the viral antigen-specific
Figure 4. Effects of alloferon on the ERK pathway in TPA-treated BCBL-1
A
B
TPA
-
-
+
+
Alloferon
-
+
-
+
AP-1
2
pERK
β–Actin
Fold induction
ERK
1.6
1.2
0.8
0.4
TPA
0
Alloferon
-
-
+
+
-
+
-
+
(A) Western blot analysis was performed for phosphorylated extracellular signal-regulated kinase (ERK) and ERK in body-cavity-based B lymphoma (BCBL)-1 cells
upon 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and alloferon treatment. BCBL-1 cells were preincubated with TPA for 3 h prior to alloferon treatment, and were
incubated for an additional 1 h after alloferon treatment. Western blot analysis was performed as described in Methods. (B) BCBL-1 cells were electroporated with the
pAP-1/luciferase and pRL-TK/luciferase reporter systems. Then, alloferon was treated for 12 h. Luciferase activity was measured from these cell lysates using a DualLuciferase assay kit (Promega, Madison, WI, USA).
Antiviral Therapy 16.1
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N Lee et al.
There have been no specific drugs licensed for controlling the KSHV life cycle so far. Highly active antiretroviral therapy, CHOP-like therapy (comprised of
cyclophosphamide, doxorubicin, vincristine and prednisone), rituximab and autologous stem cell therapy
are currently used, but these therapies are for the
treatment of PEL or non-Hodgkin’s lymphoma caused
by KSHV infection. They are types of chemotherapy,
antibody, which is involved in antibody-dependent cellmediated cytotoxicity and the engulfment of antibodycoated virus by phagocytes. Although we could not
show the effect of alloferon on the production of the
virus-specific antibody in this experiment, it is under
investigation in an in vivo system using murine gammaherpesvirus-68, a homologous murine virus strain to
human herpesviruses.
Figure 5. Cytolytic activity of NK cells by alloferon treatment
A
B
2B4
70
P<0.05
0
100 101 102 103 104
FL1-H
60
50
KIR
128
40
30
0
100 101 102 103
FL1-H
10
0
1:25
1:5
0
100
1:50
Events
20
104
CD94
128
Events
Specific release value, %
80
Events
Alloferon 0 µg/ml
Alloferon 4 µg/ml
NKG2D
128
Events
128
101
Target:effector ratio
102 103
FL1-H
Control
104
0
100 101
102 103
FL1-H
104
Alloferon,
4 µg/ml
Alloferon,
0 µg/ml
C
400
300
200
100
0
Alloferon,
4 µg/ml
-
+
1,200
TNF-α concentration, pg/ml
sFasL concentration, pg/ml
Granzyme B
concentration, pg/ml
P<0.05
1,000
800
600
400
200
0
-
+
500
400
300
200
100
0
-
+
(A) The cytotoxic effect of natural killer (NK) cells was assessed by 51Cr release assays. Human NK cells were treated with 4 µg/ml alloferon. After 12 h incubation,
NK cells (effector cells) were cocultured for 4 h with 51Cr-labelled body-cavity-based B lymphoma (BCBL)-1 cells (target cells) according to the indicated ratios
(target:effector =1:5, 1:25 and 1:50). (B) Isolated NK cells were treated with 4 µg/ml alloferon for 12 h. Then, flow cytometric analysis was carried out using the
antibodies against the stimulatory (2B4 and NKG2D) and inhibitory (KIR and CD94) receptors of the NK cells. (C) The products of cytolytic granules (granzyme B,
soluble Fas ligand [sFasL] and tumour necrosis factor [TNF]-α) of NK cells were detected with a respective ELISA kit under the same conditions as (A).
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Antiviral effect of alloferon
and not suitable for the regulation of the life cycle
of KSHV from the latent to the lytic cycle [32]. Acyclovir and ganciclovir are the most popular antiviral
drugs because they can control the life cycle of herpesviruses from the latent to the lytic cycles; however, it
has recently been reported that resistance against these
drugs increases gradually. We therefore believe that
alloferon can be used effectively as a potential antiviral
drug that has dual functions, including one for the
regulation of KSHV and HSV life cycles and another
for the enhancement of the immune system, because
complete eradication of viruses can be achieved with
the aid of the cell-mediated immune system by NK cells
or virus-specific T-cells.
Acknowledgements
This study was funded by the Korea Science and
Engineering Foundation, Tumor Immunity Medical
Research Center, Seoul National University College
of Medicine (grant number R13-2002-025-02001-0)
and the Science Research Center Program, Research
Center for Women’s Disease (grant number R112005-017-03001).
Disclosure statement
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Accepted 3 June 2010; published online 21 December 2010
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