The Epstein–Barr virus (EBV) is a human herpesvirus that infects lymphoid and epithelial cells.1)Infection by this virus causes infectious mononucleosis and is closely related to several malignant diseases, including Burkitt’s lymphoma,2)
T-cell lymphoma,3) Hodgkin’s disease,4) gastric cancer5) and
nasopharyngeal carcinoma.6)After infecting B lymphocytes, the virus is typically latent.7)However, the virus has to enter a lytic cycle to proliferate.8) At the onset stage of the EBV
lytic cycle, the virus expresses two immediate-early genes, BRLF1 and BZLF1, which encode transcription factors, Rta and Zta, respectively.9)These two transcription factors often cooperate or act alone to activate various sets of lytic genes, including BMRF1 and BALF5, which encode diffused early antigen (EA-D) and DNA polymerase,10)respectively. With-out Rta or Zta, the virus cannot complete its lytic cycle.11)As it is generally known, acyclovir and ganciclovir, two nucleo-side analogs that specifically inhibit the function of her-pesvirus DNA polymerases, have been used clinically to treat herpes infections.12)Compounds isolated from Artemisia
lac-tiflora, Camelia sinesis, Glycyrrhiza radix13) and Oryzae sativa also inhibit the EBV lytic cycle.14—17) Of these
com-pounds, epigallocatechin gallate (EGCG) from green tea, C.
sinesis, has been shown to inhibit the transcription of BRLF1
and BZLF1.15)
Andrographis paniculata is a medicinal plant that is
com-monly used to treat a range of illnesses, including bacterial infections, inflammations and high blood pressure. Earlier works have identified many important ingredients in the plant, including diterpenes, flavonoids and stigamasterols. One of the diterpenoids, andrographolide, is particularly im-portant because of its anti-inflammatory,18)antithrombotic,19) anticancer20) and immunostimulatory activities.21)
Further-more, andrographolide exhibits a neutralizing activity against the human immunodeficiency virus (HIV)22,23) and herpes simple virus 1 (HSV-1).24) This investigation demonstrates that the ethanolic extract from A. paniculata (EEAP) and an-drographolide inhibit the transcription of EBV immediate-early genes and the production of EBV virions. Andro-grapholide is potentially useful as an anti-EBV drug.
MATERIALS AND METHODS
Materials A. paniculata was collected from the San Dei
Men area in the Pingtung County, Taiwan and verified by Prof. C. S. Kuoh. The specimen of this plant is deposited in the Herbarium of National Cheng Kung University, Tainan, Taiwan. Andrographolide (Aldrich Chemical Company) was dissolved in dimethylsulfoxide (DMSO) before use.
Extraction of A. paniculata Ten grams of dried powder from the aerial parts of A. paniculata was extracted three times in 100 ml of ethanol by refluxing at 85 °C for 2 h. Fol-lowing each extraction, the ethanol fraction was collected by filtration. The ethanol was then removed by rotary evapora-tion and yield was 0.53%. The resulting residues were dis-solved in DMSO for in vitro studies.
Cell Culture and EBV Lytic Induction P3HR1 cells, a Burkitt’s lymphoma cell line that is latently infected by EBV, were cultured in RPMI 1640 medium that was supplemented with 10% fetal calf serum (Biological Industries, Israel). After culturing for 24 h, the cells were treated with EEAP and andrographolide. After incubating for 1 h, cells were treated with 30 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 3 mM of sodium butyrate (SB) to induce the EBV lytic cycle.25,26)
Analysis of Expression of EBV Lytic Proteins Cell lysate was prepared from 3106P3HR1 cells with 100ml of lysis buffer that contained 50 mMTris–HCl, pH 7.8, 150 mM NaCl, 5 mMEDTA, 0.5% Triton X-100 and 0.5% NP40 using a method described elsewhere.15) SDS-polyacrylamide gel
electrophoresis and immunoblot analysis with Rta, anti-Zta and anti-EA-D antibodies, which were purchased from Argene (Varilhes, France), were performed using methods described previously.27)
Indirect Immunofluorescence Analysis P3HR1 cells were plated on poly-L-lysine-coated overslips and fixed with 4% paraformaldehyde at 4 °C for 15 min, followed by treat-ment with PBS containing with 0.5% Triton X-100 for 10 min at room temperature. After blocking with 1% BSA in PBS at room temperature for 30 min, monoclonal anti-Rta, Taiwan University; Taipei 106, Taiwan.
Received June 2, 2008; accepted August 14, 2008; published online August 19, 2008
Andrographis paniculata NEESis a medicinal plant that is commonly used in Asia. This work demonstrates that 25mmg/ml of ethanolic extract from A. paniculata (EEAP) and 5 mmg/ml of andrographolide, a bioactive
com-pound in EEAP, effectively inhibit the expression of Epstein–Barr virus (EBV) lytic proteins, Rta, Zta and EA-D, during the viral lytic cycle in P3HR1 cells. Transient transfection analysis revealed that the lack of expression of Rta, Zta and EA-D is caused by the inhibition of the transcription of BRLF1 and BZLF1, two EBV immediate-early genes that encode Rta and Zta, respectively. This study finds that the inhibition prevents the virus from producing mature viral particles. Meanwhile, andrographolide is not toxic to P3HR1 cells when the concentra-tion is below 5mmg/ml, indicating that the compound is potentially useful as an anti-EBV drug.
Key words Epstein–Barr virus; antiviral activity; andrographolide; Andrographis paniculata
© 2008 Pharmaceutical Society of Japan ∗ To whom correspondence should be addressed. e-mail: tplin007@mail.chna.edu.tw
anti-Zta, and anti-EA-D antibodies were applied at a dilution of 1 : 200 and incubated at room temperature for 1 h. Next, the cells were washed with PBS and incubated with the FITC-conjugated goat anti-mouse IgG for 1 h. After counter-staining with 4,6-diamidio-2-phenylindole (DAPI), the cells were examined under a fluorescence microscope.
Flow Cytometry Analysis P3HR1 cells were treated with antibodies as described for indirect immunofluores-cence analysis, except that the cells were fixed with 4% paraformaldehyde. Finally, cells were resuspended in 1% paraformaldehyde and analyzed with a flow cytometer (model FACScanTO, BD Biosciences).
Transient Transfection Assay P3HR1 cells (5106)
were transfected with 10mg of pRLUC and pZLUC with a Bio-Rad electroporator using the method of Chang et al.28,29) Lysate was prepared at 24 h after transfection and luciferase activity was determined using a luminometer (Berthold, Ger-many) according to a method described elsewhere.28)
Determining the Number of EBV Particles Formed by P3HR1 Cells After lytic induction, P3HR1 cells were cul-tured for 5 d. EBV particles released into the culture medium were harvested by treatment with polyethylene glycol (PEG) 6000 and centrifugation at 9400g for 1 h. The pellet was treated with DNase I and proteinase K, before being ex-tracted with phenol. EBV in the aqueous fraction was precip-itated with ethanol and the amount of EBV DNA was deter-mined by real-time PCR, using the method of Chiu et al.30)
Cell Viability Test A 5 mg/ml 3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) solution in RPMI 1640 medium was added into 6105 P3HR1 cells. The dehydrogenase activity of the viable cells was measured using the method of Carmichael et al.31)
Statistical Analysis Data were analyzed statistically by one-way analysis of variance (ANOVA) using the SAS JMP 6.0 software package. Data are presented as meansS.D. and a p value of 0.05 was regarded as significant.
RESULTS
Inhibition of the Expression of EBV Lytic Proteins P3HR1 cells (3106) were treated with TPA and SB for 24 h
to induce the EBV lytic cycle. As expected, immunoblot analysis revealed that three EBV lytic proteins, Rta, Zta and EA-D, were expressed after lytic induction (Figs. 1A, B). Meanwhile, adding 6.25mg/ml of EEAP prior to lytic induc-tion had little effect on the synthesis of these three proteins (Fig. 1A). However, the synthesis of Rta and EA-D was
par-tially inhibited by 12.5mg/ml of EEAP (Fig. 1A), although that of Zta was unaffected at this concentration (Fig. 1A). At 25mg/ml, EEAP significantly reduced the expression of Zta and completely suppressed the expression of Rta and EA-D (Fig. 1A); at 50mg/ml, the EEAP completely inhibited the expression of Zta (Fig. 1A). Meanwhile, 5mg/ml of andro-grapholide significantly decreased the expression of Rta and entirely suppressed the expression of Zta and EA-D (Fig. 1B). None of these three proteins was expressed after 10mg/ml of andrographolide was added to the culture medium (Fig. 1B). These results indicated that EEAP and an-drographolide inhibit the expression of EBV lytic proteins.
Indirect Immunofluorescence Analysis of EBV Proteins Expression The inhibitory effects of EEAP and andro-grapholide on the expression of EBV proteins were also as-sessed by indirect immunofluorescence analysis. The staining results showed that Rta, Zta and EA-D were expressed at 24 h after TPA and SB treatment (Fig. 2). The treatment with 25mg/ml EEAP or 5 mg/ml andrographolide prior to lytic in-duction significantly inhibited the expression of these three proteins at 24 h after treatment (Fig. 2).
Quantitative Flow Cytometry Analysis of the In-hibitory Effects of EEAP and Andrographolide on EBV Proteins Expression The presence of lytic proteins was further confirmed by flow cytometry analysis. The popula-tion of P3HR1 cells that expressed Rta, Zta and EA-D was 52.8%, 60. 8%, and 53.5%, respectively, following TPA and SB treatment (Fig. 3A). Adding 25mg/ml EEAP before lytic induction decreased the population that expressed Rta, Zta and EA-D decreased to 21.1%, 27.1% and 8.6%, respectively (Fig. 3A); treating with 2.5mg/ml andrographolide, 18.6%, 22.5% and 15.5%, respectively. Moreover, treating the cells with 5mg/ml andrographolide decreased the expression to 1.2%, 3.1% and 0.3%, respectively (Fig. 3B). The expression of these proteins was undetected if the cells were treated with 25mg/ml EEAP or 5 mg/ml andrographolide.
Inhibition of the Transcription of EBV Immediate-Early Genes Transient transfection assay was performed to determine whether EEAP and andrographolide influenced the transcription of BRLF1 and BZLF1. Reporter plasmids, pRLUC and pZLUC, which contained a firefly luciferase gene transcribed from the BRLF1 and BZLF1 promoter, were transfected into P3HR1 cells after EBV lytic induction. Analyzing the luciferase activity exhibited by the cells indi-cated that adding 12.5mg/ml of the EEAP into the culture medium inhibited the activity of the BRLF1 promoter by 58%. However, the same concentration of the extract did not Fig. 1. Inhibition of Rta, Zta and EA-D Expression by EEAP and Andrographolide
EEAP (Extract) (A) or andrographolide (Adgp) (B) was added to P3HR1 cells 1 h before lytic induction with TPA and sodium butyrate (SB). Syntheses of Rta, Zta and EA-D by EBV was studied by immunoblot analysis at 24 h after lytic induction with anti-Rta, anti-Zta, anti-EA-D and anti-b-actin antibodies.
influence the activity of the BZLF1 promoter (Fig. 4). At 25mg/ml, the EEAP inhibited the activity of the BRLF1 and BZLF1 promoter by 79% and 84% respectively (Fig. 4). Meanwhile, andrographolide at 5mg/ml inhibited the activity of the BRLF1 and BZLF1 promoter by 62% and 92%; at 2.5mg/ml, by 39% and 83%, respectively (Fig. 4).
Inhibition of EBV Virion Production Numbers of EBV virion produced by P3HR1 cells were determined at the fifth day after lytic induction. As expected, treating P3HR1 cells with TPA and SB produced about 4.2107viral parti-cles (Fig. 5). Treating the cells with 25mg/ml of EEAP or 5mg/ml of andrographolide reduced the numbers of viral particles to the background level (Fig. 5), indicating that the EEAP and andrographolide inhibited EBV lytic replication.
Effect of EEAP and Andrographolide on the Viability
of P3HR1 Cells The toxicity of EEAP and andro-grapholide to P3HR1 cells was determined using the MTT method.31) The EEAP at concentrations of 25mg/ml and 50mg/ml killed 10% and 22% of P3HR1 cells after 24 h of culturing (Fig. 6A). The extract at 100mg/ml killed 60% of the cells (Fig. 6A). This study also demonstrated that andro-grapholide did not influence the cell viability at concentra-tions under 5mg/ml. The compound killed about 20% of the cells at 10mg/ml (Fig. 6B).
DISCUSSION
As it is generally known, many clinical symptoms of her-pesvirus infections, for example, cold sores, genital herpes, chicken pox, infectious mononucleosis, are associated with Fig. 2. Indirect Immunofluorescence Analysis of the Inhibitory Effects of EEAP and Andrographolide on the Expression of EBV Lytic Genes
P3HR1 cells were treated with TPA and SB to stimulate the expression of lytic genes, including Rta, Zta, and EA-D. Cells untreated with TPA and SB (Latent) were used as a negative control. Cells treated with TPA and SB were also pre-treated with 12.5 and 25mg/ml EEAP (Extract-12.5 and Extract-25) (A) or 2.5 and 5 mg/ml andrographolide (Adgp-2.5, Adgp-5) (B) to examine the inhibitory effects of EEAP and andrographolide on the expression of EBV lytic proteins. At 24 h after lytic induction, proteins expressed by the cells were detected by indirect immunofluorescence analysis using anti-Rta, Zta, and EA-D antibodies.
Fig. 3. Determining the Expression of Rta, Zta and EA-D in P3HR1 by Flow Cytometry Analysis
P3HR1 cells were treated with EEAP or andrographolide before lytic induction. At 24 h after lytic induction, cells were incubated with monoclonal Rta, Zta and anti-EA-D antibodies and stained with secondary anti-mouse IgG-FITC-conjugated antibody. M2 represents stained P3HR1 cells.
viral lytic reactivation.32,33)Hence, anti-herpes drugs that are commonly used such as acyclovir and ganciclovir, are nucle-oside analogs, suppress the function of herpes DNA poly-merase and viral lytic DNA replication.12,34,35) An earlier
study demonstrated that EBV virions that are produced by B lymphocytes exhibit a tropism by which they preferentially
ent in the plant, andrographolide, since this compound at 5mg/ml (14 mM) also effectively inhibits the expression of these proteins (Figs. 1B, 2B, 3B). An earlier study showed that EGCG from green tea inhibits the EBV lytic cycle at a concentration of 50—70mM,15)which is substantially higher than the dose of andrographolide that effectively inhibits the EBV lytic cycle at 14mM(5mg/ml) (Figs. 1, 2, 3, 4), suggest-ing that andrographolide is more effective than EGCG in in-hibiting EBV reactivation.
This investigation establishes that EEAP and andro-grapholide inhibit the transcription of BRLF1 and BZLF1 (Fig. 4), explaining why the EBV cannot complete its lytic cycle to produce EBV particles (Fig. 5). Exactly how andro-grapholide inhibits the transcription of BRLF1 and BZLF1 is currently unknown. It is plausible that andrographolide in-hibits the functions of the transcription factors that activate the transcription of BRLF1 and BZLF1. The transcription of BRLF1 and BZLF1 is also known to be affected by the acti-vation of various signaling pathways, including p38 and JNK37); further work must be conducted to determine
whether andrographolide inhibits the signaling pathways that are known to activate the transcription of EBV immediate-early genes. Chen et al.38) found that andrographolide not only suppresses the mitochondrial pathway of apoptosis but also activates the phosphatidyl inositol-3-kinase/Akt signal-ing pathway. However, whether this pathway influences the expression of BRLF1 and BZLF1 is unknown.
As it is generally known, EBV in P3HR1 cells does not enter the lytic cycle unless the cells are treated with com-pounds such as TPA and SB.25,26)However, this work identi-fied a low level of EBV DNA in the culture medium after the cells had been cultured for 5 d (Fig. 5). EBV is known to re-activate spontaneously, especially under stress conditions.39)
After culturing for 5 d, important growth factors in the cul-ture medium may be depleted and insufficient to support nor-mal cell growth. Under such stress conditions, EBV may enter the lytic cycle, explaining why low numbers of EBV particles were detected (Fig. 5).
A. paniculata, which is rich in andrographolide, is
exten-sively used as a remedy with anti-inflammatory, antipyretic and antimicrobial activities.18,22—24,40,41)This study finds that
andrographolide has an anti-EBV activity. Furthermore, the result also revealed that andrographolide has low cytotoxicity toward P3HR1 cells under the concentration of 5mg/ml. Thus, it is potentially useful in preventing the lytic develop-ment of EBV and other related herpes viruses.
Acknowledgements This work was supported by re-search grants NSC-92-2622-B-041-002-CC3 from the Na-tional Science Council, R.O.C., and CNBT 94-01 from Chia-Nan University of Pharmacy and Science.
Fig. 6. Toxicity of EEAP and Andrographolide to P3HR1 Cells Cell viability was determined using the MTT method.31)
P3HR1 cells were treated with EEAP (Extract) and andrographolide after lytic induction. The data are presented as meanS.D. from three independent experiments. ∗ p0.05, which represents a sig-nificant difference from the values of the control group.
Fig. 4. Inhibition of BRLF1 and BZLF1 Transcription by EEAP and An-drographolide
EEAP or andrographolide (Adgp) was added to P3HR1 cells 1 h before transfection with pRLUC (A) and pZLUC (B) and lytic induction with sodium butyrate (SB). Lu-ciferase activity exhibited by the cells was monitored using a luminometer at 24 h after lytic induction. Each transfection experiment was performed three times and each sam-ple in the experiments was prepared in duplicate. The data are presented as means S.D. from three independent experiments.
Fig. 5. Inhibition of EBV Virion Production by EEAP and Andro-grapholide
P3HR1 cells were treated with 25mg/ml of EEAP and 5 mg/ml of andrographolide 1 h before lytic induction. EBV particles that were released into the cultured were har-vested by PEG6000 precipitation. The amount of EBV DNA was determined by real-time PCR after the DNA was extracted from the viral particles. Cells that had not been treated with TPA and sodium butyrate were used as a negative control (NC). The data are presented as meanS.D. from three independent experiments.
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