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Biochemical and Biophysical Research Communications 301 (2003) 1062–1068
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Inhibition of Epstein–Barr virus lytic cycle
by ())-epigallocatechin gallate
Li-Kwan Chang,a Ta-Tung Wei,b Ya-Fang Chiu,b Chao-Ping Tung,b
Jian-Ying Chuang,b Shang-Kai Hung,b Ching Li,c and Shih-Tung Liub,*
a
Department of Biology, Kaohsiung Medical University, 100, Shih-Chuan 1st Rd., Kaohsiung 807, Taiwan
Molecular Genetics Laboratory, Department of Microbiology and Immunology, Chang-Gung University, 259, Wen-Hwa 1st Rd.,
Kwei-Shan, Taoyuan 333, Taiwan
Department of Microbiology and Immunology, Chung-Shan Medical University, 110, Sec. 1, Chien-Kuo N. Rd., Taichung 402, Taiwan
b
c
Received 6 January 2003
Abstract
())-Epigallocatechin gallate (EGCG), abundant in green tea, is a potent anti-microbial and anti-tumor compound. This investigation used immunoblot, flow cytometry, microarray, and indirect immunofluorescence analyses to show that at concentrations
exceeding 50 lM, EGCG inhibits the expression of Epstein–Barr virus (EBV) lytic proteins, including Rta, Zta, and EA-D, but does
not affect the expression of EBNA-1. Moreover, DNA microarray and transient transfection analyses demonstrated that EGCG
blocks EBV lytic cycle by inhibiting the transcription of immediate-early genes, thus inhibiting the initiation of EBV lytic cascade.
Ó 2003 Elsevier Science (USA). All rights reserved.
Catechin, a major component of green tea, contains
several isomers, including ())-epigallocatechin gallate
(EGCG), ())-epicatechin, ())-epicatechin gallate, ())epigallocatechin, and (+)-catechin [13]. These compounds have a broad spectrum of anti-microbial activity
against bacteria, fungi, and viruses. For example,
EGCG not only inhibits the growth of Vibrio cholerae,
Escherichia coli O157:H7, Staphylococcus aureus, and
Trichophyton sp. [24,32], but also protects against infections by rotaviruses and enteroviruses [19]. Additionally, Nakayama [21] showed that EGCG binds to
hemagglutinin of influenza virus, thus blocking the attachment of the viral particles to the target cell receptors
and preventing infection [21]. Nakane et al. [20] and
Yamaguchi et al. [34] also identified EGCG as an antihuman immunodeficiency virus type 1 (HIV-1) agent;
EGCG not only prevents HIV attachment and cell entry
but also destroys viral particles and virus production.
This effect is attributed to the inhibition of reverse
*
Corresponding author. Fax: +886-3211-8292.
E-mail address: [email protected] (S.-T. Liu).
transcriptase, viral transcription, and HIV-1 proteases
[20,34]. Moreover, EGCG exhibits anti-tumor activities.
Jankun et al. [14] reported that EGCG inactivates tumor-related proteases. In addition, Lin and Lin [17]
identified EGCG as an inhibitor of nitric oxide synthase
(NOS). The effect of EGCG in inhibiting NOS and decreasing levels of inducible NOS (iNOS) ultimately inhibits the signal transduction pathway of NF-jB.
Furthermore, Balasubramanian et al. [1] showed that
EGCG activates the MAPK signaling pathway and
increases the levels of Fra-1, Fra-2, FosB, JunB, Jun-D,
c-Jun, and c-Fos in human keratinocytes, causing AP1-dependent gene expression.
Epstein–Barr virus (EBV) is a human herpesvirus.
EBV infection causes infectious mononucleosis and is
closely associated with BurkittÕs lymphoma, nasopharyngeal carcinoma, T-cell lymphoma, HodgkinÕs disease, and posttransplant lymphoproliferative diseases
[10,15, 18,22,23,28,29,33]. Among these diseases, infectious mononucleosis and posttransplant lymphoproliferative diseases are closely related to EBV lytic
activation, causing the virus to spread through the body
[12,27]. Notably, a recent investigation using indirect
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0006-291X(03)00067-6
L.-K. Chang et al. / Biochemical and Biophysical Research Communications 301 (2003) 1062–1068
1063
immunofluorescence showed that EGCG inhibits the
expression of EBV diffused early antigen (EA-D) in 12O-tetracdecanoylphorbol-13-acetate (TPA)- and n-butyric acid-treated Raji cells [31]. This investigation
builds upon this earlier study by demonstrating that
EGCG inhibits the expression of not only EA-D but
also the immediate-early proteins of EBV, thus, blocking the EBV lytic cascade.
GAPDH gene were used as internal standards. The values of dot intensities obtained from three independent experiments were normalized and averaged.
Transfection and luciferase assay. Plasmids used for transfection
studies were prepared by CsCl gradient centrifugation. Transfection
luciferase activity was measured according to a method described
elsewhere [4].
Materials and methods
Inhibition of the expression of EBV lytic proteins by
EGCG
Cell line and induction of EBV lytic cycle. An EBV-positive BurkittÕs lymphoma cell line, P3HR1, was cultured in RPMI 1640 medium
supplemented with 10% fetal calf serum. EBV lytic cycle was induced
with 300 nM trichostatin A (TSA) (Upstate Biotechnology, Lake
Placid, NY) [3].
Immunoblot analysis. Proteins resolved by SDS–polyacrylamide gel
were electrotransferred to Hybond C membrane (Amersham Pharmacia Biotech) at 90 V for 1 h and probed with the appropriate antibodies. Proteins were finally detected, using an ECL detection kit
(Amersham). Monoclonal anti-Rta and anti-Zta antibodies were purchased from Argene (Varilhes, France). Monoclonal anti-EA-D antibody was purchased from Advanced Biotechnologies (Columbia,
MD). Polyclonal anti-EBNA-1 antibody was provided by M. Chao,
Chang-Gung University.
Flow cytometry analysis. P3HR1 cells (2 106 ) were washed with
phosphate-buffered saline (PBS), followed by fixing with 4% paraformaldehyde for 30 min, and finally by treatment with PBS containing
0.1% Triton X-100 for 5 min. Cells were then washed with PBS, treated
with 1% BSA in PBS for 1 h, and incubated with 1:200-diluted
monoclonal anti-Rta antibody, monoclonal anti-Zta antibody,
monoclonal anti-EA-D antibody, or polyclonal rabbit anti-EBNA-1
antibody for 1 h at 37 °C. Next, the cells were washed with PBS, treated
with 0.5% Tween 20 in PBS, and incubated with 1:200-diluted FITCconjugated goat anti-mouse immunoglobulin G (KPL, Guildford,
UK) for 1 h at 37 °C to detect EA-D, Rta, and Zta. Alternatively, cells
were incubated with 1:200-diluted rhodamine-conjugated goat antirabbit immunoglobulin G (KPL) to detect EBNA-1. Finally, cells were
resuspended in 1% paraformaldehyde and analyzed on a FACScan
cytofluorometer (Becton–Dickinson, USA).
Indirect immunofluorescence analysis. P3HR1 cells were treated
with antibodies as described for the flow cytometry analysis, except
that the cells were plated on poly-L -lysine (Sigma Chemical)-coated
coverslips and fixed with 4% paraformaldehyde. Finally, fluorescence
was observed with a Zeiss Axioskop 20 fluorescence microscope. Images were captured with a charge-coupled device camera and processed
using the Image-Pro Plus, version 4.5 software (Media Cybernetics the
Imaging Experts, Maryland).
Hybridization analysis. EBV DNA-chips with PCR-amplified DNA
spanning the entire EBV genome were used to analyze EBV gene
transcription. The EBV DNA in each dot, fabricated on the DNAchip, with a length of around 2 kb except for the W-repeat region, was
amplified by PCR and confirmed by DNA sequencing. To examine
EBV gene transcription, 1 lg mRNA, which was isolated from P3HR1
cells using an Oligotex mRNA isolation kit (Qiagene, Hilden, Germany), was reverse transcribed with 3 lg random hexamers and 200 U
MMLV reverse transcriptase (Promega, Madison, WI), in a substrate
mixture containing dNTPs and biotin-16-dUTP. Finally, the biotinlabeled cDNAs were used to hybridize the DNA on the DNA-chips
following a method described elsewhere [5]. The EBV DNA-chips were
scanned using a UMAX PowerLook 3000 scanner at a resolution of
3000 dpi. The intensities of the dots on the scanned images were
measured using a program available at the website, http://www.microarrays.org/, and the dots that contained the b-actin gene and the
Results and discussion
As is generally known, EBV has two life cycles. After
infecting B lymphocyte cells, the virus is maintained
under latent conditions. However, EBV must complete a
lytic productive cycle to proliferate. Using antiserum
from a nasopharyngeal carcinoma patient, Taniguchi
et al. [31] showed that EGCG treatment can reduce EAD expression in TPA-treated Raji cells, suggesting that
EGCG inhibits EBV lytic replication. To investigate the
mechanism through which EGCG influences the lytic
cycle of EBV, P3HR1 cells were first treated with EGCG
for 1 h prior to a treatment with 300 nM TSA to activate
the EBV lytic cycle [3]. The inhibitory effect of EGCG
on EA-D expression was assessed by immunoblot
analysis, which was performed using a 1:2000-diluted
polyclonal anti-EA-D antibody [6]. The results revealed
that although EA-D expression was induced by 300 nM
TSA (Fig. 1), TSA-induced EA-D expression was fully
inhibited by 100 lM EGCG (Fig. 1), a concentration
which inhibits HIV proliferation [34] and prevents cells
from entering the S phase of the cell cycle [16]. To
confirm that the lack of EA-D expression by P3HR1
cells was not related to cell death, this work stained the
Fig. 1. Effect of EGCG treatment on the expression of EA-D, Rta, Zta,
and EBNA-1 during the lytic cycle. P3HR1 cells were treated with
EGCG for 1 h and then untreated (A) or treated (B) with 300 nM TSA
to induce EBV lytic cycle. Cells were then lysed 24 h after the TSA
treatment. Immunoblot analysis was subsequently performed to determine the expression of EA-D, Rta, Zta, and EBNA-1.
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cells using trypan blue and it was found that after 24 h of
treatment, although cells ceased to grow, neither 100 lM
EGCG nor 300 nM TSA treatment caused cell death in
P3HR1 cells. On the other hand, a combination of
300 nM TSA and 100 lM EGCG was toxic to cells and
caused roughly 40–50% cell death after 24 h of treatment. Therefore, a lack of EA-D expression could be
partly attributed to a reduction in viable P3HR1 cells.
We also observed that the toxic effect of EGCG-TSA
treatment was reduced when the concentration of
EGCG decreases; after 24 h of treatment, less than 20%
cell death was observed when the concentration of
EGCG decreased to 70 lM, and almost no cell death
was observed when the concentration of EGCG decreased to 50 lM. Meanwhile, at 50 and 70 lM EGCG
significantly reduced the expression of EA-D (Fig. 1).
On the other hand, EGCG at concentrations below
50 lM did not inhibit TSA-induced EA-D expression
(Fig. 1). Since the transcription of the EA-D gene,
BMRF1, requires two EBV immediate-early proteins,
Rta and Zta [11], we further investigated whether the
lack of EA-D expression was attributed to the inhibition
of Rta and Zta expression by EGCG. Examination with
1:1000-diluted monoclonal anti-Rta antibody and
monoclonal anti-Zta antibody revealed that TSAinduced Rta and Zta expression had inhibited by EGCG
at concentrations exceeding 50 lM EGCG (Fig. 1),
suggesting that the lack of EA-D expression was probably resulted from the inhibition of these two immediate-early proteins. Notably, although TSA-induced Zta
and EA-D expression was fully inhibited by 100 lM
EGCG, low level of Rta expression persisted, with a
Fig. 2. Flow cytometry analysis of the expression of Rta, Zta, EA-D, and EBNA-1. P3HR1 cells were treated with 70 lM (EGCG-70) or 100 lM
(EGCG-100) EGCG for 1 h and then with 300 nM TSA. Following 24 h of culturing, cells were incubated with monoclonal anti-Rta, and monoclonal
anti-Zta, monoclonal anti-EA-D antibodies, and finally stained with secondary anti-mouse IgG-fluorescein isothiocyanate (FITC)-conjugated antibody. Cells were also incubated with polyclonal anti-EBNA-1 antibody and finally stained with secondary anti-rabbit IgG-rhodamine-conjugated
antibody. The percentage of positive cells was measured with linear gates M2 set at 0.5% on unstained P3HR1 cells and M1 corresponding to a
fluorescence signal exceeding this percentage.
L.-K. Chang et al. / Biochemical and Biophysical Research Communications 301 (2003) 1062–1068
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faint Rta band still being detectable by immunoblot
(Fig. 1). Consequently, EGCG at 100 lM may be insufficient to totally repress the expression of Rta.
Effect of EGCG on the synthesis of EBNA-1
The effects of EGCG-TSA treatment on the expression of an EBV latent protein, EBNA-1, were also
examined by using a 1:10000-diluted polyclonal antiEBNA-1 antibody. Immunoblot analysis revealed that
treating the P3HR1 cells with TSA did not alter the
amount of EBNA-1 in the cells (Fig. 1). Moreover,
adding both EGCG and TSA to the culture also failed
to influence the expression of EBNA-1. The level of
EBNA-1 remained constant and was independent of the
concentration of EGCG used (Fig. 1).
Quantitative flow cytometry analysis of the inhibitory
effects of EGCG on expression of EBV proteins
Flow cytometry analysis was performed for TSA and
EGCG treated cells, based on immunostaining of Rta,
Zta, and EA-D with primary antibody and subsequently
detecting the primary antibody with fluorescein isothiocyanate (FITC) or rhodamine-conjugated secondary
antibody. The analysis revealed that only low percentage of P3HR1 cells untreated with TSA expressed Rta,
Zta, and EA-D (Fig. 2). Moreover, the population expressing Rta, Zta, and EA-D increased to 23.4%, 24%,
and 17.3%, respectively, following TSA treatment (Fig.
2). On the other hand, following treatment with 70 lM
EGCG, the percentage of cell population expressing Rta
and Zta decreased to 9.8% and 4.9%, respectively, while
the percentage expressing EA-D reduced to 10.7%. Finally, after treatment with 100 lM EGCG, the percentage of P3HR1 cells expressing Rta, Zta, and EA-D
reduced to 0.5%, 1.7%, and 4.9%, respectively. Additionally, EGCG had little influence on the expression of
EBNA-1 (Fig. 2). These results are consistent with the
observations from immunoblot analysis (Fig. 1).
Fig. 3. Indirect immunofluorescence analysis of the inhibitory effects of
EGCG on the expression of EBV lytic genes. P3HR1 cells were treated
with TSA to activate the EBV lytic cycle or were left untreated to
maintain EBV under latency. TSA-treated cells were also pre-exposed
to 70 lM (EGCG-70) and 100 lM (EGCG-100) EGCG to examine the
inhibitory effect of EGCG on EBV lytic expression. Finally, at 24 h
after incubation, cells were processed for indirect immnofluorescence
using anti-Rta, anti-Zta, anti-EA-D, and anti-EBNA-1 antibodies.
Each panel in this figure contains approximately equal number of cells.
Indirect immunofluorescence analysis of EBV protein
expression
The inhibitory effects of EGCG onEBV protein expression were also examined by indirect immunofluorescence analysis. The staining results revealed that Rta,
Zta, and EA-D were expressed at 24 h after TSA treatment (Fig. 3). On the other hand, EGCG at 70 lM and
100 lM markedly reduced the expression of these three
proteins at 24 h after treatment (Fig. 3). Furthermore,
EBNA-1 expression was observed in all the cells examined, regardless of whether the cells were treated with
TSA (Fig. 3). Meanwhile, the presence of 70 and 100 lM
EGCG had little effect on EBNA-1 expression (Fig. 3).
Fig. 4. Analysis of inhibitory effects of EGCG on the transcription of
EBV lytic transcription. mRNA was isolated from P3HR1 cells untreated (A and B) or treated with 300 nM TSA (C and D), 300 nM TSA
and 70 lM EGCG (E and F), and 300 nM TSA and 100 lM EGCG (G
and H) for 24 h (A, C, E, G) and 48 h (B, D, F, H). Subsequently, cDNA
probes were prepared and labeled with biotin-16-dUTP, and the membranes were reacted with streptavidin-b-galactoside and X-gal. Finally,
the membrane was scanned using a UMAX PowerLook 3000 scanner
under a resolution of 3000 dpi. Dot 1h, EBER-1 and EBER-2; 2b, b-actin
gene; 2g, W repeats; 3b, GAPDH gene; 3g, BHRF1; 5h, BMRF1; 6b,
BMLF1; 6e, BLLF2; 7d, BRLF1; 7c, BZLF1; and 7g, EBNA-1 gene.
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Analysis of the effect of EGCG on EBV lytic gene
transcription
Although changes in dot intensities are not directly
correlated with changes in the exact amount of mRNA,
the results reveal whether a gene is expressed or repressed during lytic cycle. Hybridization results revealed that the W repeat region (Fig. 4A, dot 2g) and
the EBNA-1 gene (Fig. 4A, dot 7g) were transcribed
under latent conditions. Notably, the dot that contained EBER-1 and EBER-2 DNAs (dot 1h) also reacted positively to the probes (Fig. 4A). This result was
unexpected since EBER-1 and EBER-2 transcripts
should not have been isolated by the method employed
here. EBERs may be abundant in cells; the mRNA
isolation procedure used here probably did not completely remove the two EBER species which were
subsequently reverse transcribed and amplified by RTPCR. Additionally, the expression of EBV lytic genes
was generally repressed under latent conditions (Fig.
4A). However, a different expression profile was noted
following TSA treatment; EBV lytic genes were expressed 24 and 48 h after the treatment (Figs. 4C and
D). Notably, the expression of EBV immediate-early
genes increased after TSA treatment; the dots that
contained BRLF1 and BZLF1 (dots 7d and 7c) (Fig.
4C) became visible after 24 and 48 h of TSA treatment.
Expression of early genes was also noticed; BMRF1
(dot 5h), BHRF1 (dot 3g), and BMLF1 (dot 6b) were
detected 24 and 48 h after TSA treatment (Figs. 4C and
D). Moreover, the expression of EBV late genes, including BcLF1 (dots 9d and 9e) and the gene that encodes gp350/220, BLLF2 (dot 6e), was undetected
under latent conditions (Figs. 4A and B), slightly visible
at 24 h, and clearly visible at 48 hr after TSA treatment
(Figs. 4C and D). After the cells were treated with
300 nM TSA and 70 or 100 lM EGCG for 24 h, the
expression of these lytic genes was repressed (Figs. 4E
and G); the intensities of dots 7c, 7d, 3g, 5h, 6b, 9d, 9e,
and 6e declined by 30%, 50%, 80%, 20%, 30%, 90%,
60%, and 50%, respectively. However, the expression of
these genes became detectable 48 h after treatment
(Figs. 4F and H). The amount of EGCG in the culture
medium may decrease over a 48 h period to a level insufficient to repress the EBV lytic expression since TSAinduced EBV lytic gene expression remains repressed at
48 hr if EGCG is replenished 24 hr after TSA treatment
(data not shown).
Inhibition of the transcription of EBV immediate-early
genes
A transient transfection assay was performed to examine the effects of EGCG on the transcription of EBV
immediate-early genes. P3HR1 cells (5 106 ) were
transfected by electroporation with 10 lg of pRluc [3], a
reporter plasmid containing a firefly luciferase gene (luc)
transcribed from the BRLF1 promoter. The cells were
then treated with 300 nM TSA and 70 or 100 lM EGCG
immediately following transfection. Luciferase activity
expressed from the plasmid was subsequently analyzed
using a method described previously [4]. Since TSA
treatment induces the EBV lytic cycle and activates the
BRLF1 promoter [3], the luciferase activity exhibited by
the cells increased by around 3.5-fold at 24 h following
transfection (Fig. 5A). On the other hand, treating the
Fig. 5. Transient transfection assay of the activity of the BRLF1 and the BZLF1 promoter. Reporter plasmids pRluc and pZluc (10 lg) were
transfected into P3HR1 cells (white bars). Next, cells were treated with 300 nM TSA (gray bars), with 70 lM (A) or 100 lM (B) of EGCG, and finally
with 300 nM of TSA (black bars). Moreover, the luciferase activity exhibited by the plasmids was monitored at 24 h after treatment. Plasmid pGL2Basic (Promega) was used as as a vector control. Each transfection experiment was repeated three times and each sample was prepared in duplicate in
the experiment.
L.-K. Chang et al. / Biochemical and Biophysical Research Communications 301 (2003) 1062–1068
cells with TSA and 70 lM EGCG reduced the luciferase
activity to the background level (Fig. 5A). EGCG also
inhibited the transcription of the BZLF1 promoter in a
transient transfection assay. The transcription from a
luciferase reporter plasmid, pZluc, containing a luc gene
transcribed from the BZLF1 promoter, was activated by
the TSA treatment by 9.4-fold at 24 h after transfection
(Fig. 5A). However, the transcription decreased to the
background level if the cells were also treated with
70 lM EGCG (Fig. 5A). Transcription from these two
promoters was also inhibited by 100 lM EGCG
(Fig. 5B).
Acyclovir, [9-(2-hydroxyethoxymethyl)guanine]triphosphate, and its nucleoside analogs are frequently used to
treat EBV infections, such as infectious mononucleosis
[8], EBV-induced posttransplant lymphoproliferative
disorders [25], and EBV-associated B cell lymphoma
[2]. The antiviral activity of acyclovir can be attributed
to a preferential inhibition of EBV-associated DNA
polymerase but does not affect host DNA polymerases
[7]. Notably, Takase et al. [30] demonstrated that addition of acyclovir to anti-IgG-stimulated Akata cells
inhibits the productive replication of EBV DNA but
does not affect the expression of BZLF1, BRLF1,
BMRF1, and BHRF1. Unlike acyclovir, this study
shows that EGCG, rather than inhibiting EBVencoded DNA polymerase and EBV DNA replication
directly, inhibits the transcription of EBV immediateearly genes, thus causing a block of EBV lytic cascade.
On the other hand, Liberto and Cobrinik [16] showed
that EGCG treatment arrests epidermal growth factorstimulated MCF10A breast epithelial cells at the midG1 phase and prevents cells from entering the S phase,
suggesting that EGCG may regulate EBV immediateearly gene via cell cycle control. However, Rodriguez
et al. [26] showed that initiation of Zta expression and
EBV lytic cascade occurs preferentially in the G0 =G1
phases, which occurs prior to the Mid-G1 phase.
Therefore, whether the lack of Zta expression is attributed to the G1 phase arrest remains unclear. Since
the intensive use of nucleoside analogs to treat herpesvirus infections inevitably creates viruses resistant to
these drugs [9], EGCG has potential as an alternative
of acyclovir for treating such infections.
Acknowledgments
The authors thank Professor M. Chao for providing anti-EBNA-1
antibody. Kuan-Yin Shen is also appreciated for assisting in the flow
cytometry analysis. This work was supported by a Medical Research
Grant, CMRP720-VI, from Chang-Gung Memorial Hospital, a research grant from the National Health Research Institute of the ROC
(NHRI-EX91-8901SL), and grants (NSC91-2320-B-037-014 and
NSC91-2320-B-182-048) from the National Science Council of the
ROC, and a research fund (CSMU 90-OM-B-018) from Chung-Shan
Medical University.
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