Biochemical and Biophysical Research Communications 405 (2011) 186–191

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier .com/locate /ybbrc

Autophagy is involved in anti-viral activity of pentagalloylglucose (PGG)against Herpes simplex virus type 1 infection in vitro

Ying Pei a, Zhen-Ping Chen a, Huai-Qiang Ju a, Masaaki Komatsu b, Yu-hua Ji c, Ge Liu d, Chao-wan Guo d,Ying-Jun Zhang e, Chong-Ren Yang e, Yi-Fei Wang a,⇑, Kaio Kitazato d,⇑a Biomedicine Research and Development Center of Jinan University, Guangzhou, Guangdong 510632, Chinab Laboratory of Frontier Science, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japanc Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangzhou 510632, Chinad Division of Molecular Pharmacology of Infectious agents, Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, NagasakiUniversity, Nagasaki 852-8521, Japane Kunming Institute of Botany, the Chinese Academy of Sciences, Yunnan, Kunming 650204, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 24 December 2010Available online 7 January 2011

Keywords:AutophagyPentagalloylglucose (PGG)HSV-1 infectionsAnti-viral activity

0006-291X/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.bbrc.2011.01.006

⇑ Corresponding author. Fax: +81 95 819 2898.E-mail addresses: peiying-19802@163.com (Y. Pe

Chen), 344464448@qq.com (H.-Q. Ju), komatsu-ms@tjyh@jnu.edu.cn (Y.-h. Ji), lggege_15@hotmail.com (mail.com (C.-w. Guo), zhangyj@mail.kib.ac.cn (Y.-J. Z(C.-R. Yang), twang-yf@163.com (Y.-F. Wang), kkholi@

Pentagalloylglucose (PGG) is a natural polyphenolic compound with broad-spectrum anti-viral activity,however, the mechanisms underlying anti-viral activity remain undefined. In this study, we investigatedthe effects of PGG on anti-viral activity against Herpes simplex virus type 1 (HSV-1) associated withautophagy. We found that the PGG anti-HSV-1 activity was impaired significantly in MEF-atg7–/– cells(autophagy-defective cells) derived from an atg7–/– knockout mouse. Transmission electron microscopyrevealed that PGG-induced autophagosomes engulfed HSV-1 virions. The mTOR signaling pathway, anessential pathway for the regulation of autophagy, was found to be suppressed following PGG treatment.Data presented in this report demonstrated for the first time that autophagy induced following PGGtreatment contributed to its anti-HSV activity in vitro.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

HSV-1 is the first human herpesvirus discovered and is the pro-totype virus of alphaherpes-viridae subfamily [1]. HSV-1 is nor-mally associated with orofacial infections and can establishlatent infections in sensory neurons that can reactivate at or nearthe point of entry into the body, resulting in lesions [2]. Clinicallyeffective anti-herpesvirus drugs, in the form of nucleoside analogs,have been developed over the past 25 years. Acyclovir (ACV) hasbeen the standard therapy for HSV infections, however, the wide-spread use of ACV has led to the emergence of ACV-resistantstrains since the early 1980s [3]. Therefore, new anti-viral agentsexhibiting different mechanisms of action are urgently needed totreat infections with HSV and related viruses.

Autophagy is a conserved catabolic process that involves thesequestration of organelles and long-lived proteins residing in

ll rights reserved.

i), 530670663@qq.com (Z.-P.igakuken.or.jp (M. Komatsu),G. Liu), chaovan_kwok@hot-hang), cryang@mail.kib.ac.cnmsn.com (K. Kitazato).

the cytoplasm into a unique organelle, the autophagosome, whichserves important roles in both the maintenance of cellular homeo-stasis and survival under stress conditions [4]. Autophagy is an in-nate defense mechanism associated with the elimination ofintracellular pathogens via a process known as xenophagy bydegrading the viruses in autolysosomes [5,6]. However, it is well-known that herpesviruses have developed defense strategies forevading autophagic defense processes [7]. HSV-1 counteracts theinduction of autophagy via HSV-1 ICP34.5 protein, and which actsby two separate mechanisms: binding to Beclin 1, and antagoniz-ing autophagy-stimulating protein kinase R (PKR) signaling [8,9].

Pentagalloylglucose (PGG) is an active compound present inmany traditional medicinal herbs exhibiting multiple biologicalactivities. It has been reported that PGG has several activitiesincluding anti-mutagenic properties [10], anti-inflammatory [11],anti-cancer activity [12–14], antioxidant [15] and anti-viral effectsagainst HSV-1 [16], respiratory syncytial virus (RSV) multiplication[17] and hepatitis B virus (HBV) [18]. Recently, PGG has been re-ported that can induce autophagy in particular prostate cancer celllines [14]. In this study, we investigated the anti-viral action ofautophagosomes induced by PGG as a novel means of inducinganti-viral activity in vitro.

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2. Materials and methods

2.1. Pentagalloylglucose (PGG)

PGG used in this study was extracted from Phyllanthus emblicaand its molecular structure previously defined [19]. PGG puritywas over 98% and dissolved in dimethyl sulfoxide (DMSO) beforeuse. The final concentration of DMSO was less than 0.1%.

2.2. Cells and viruses

Human male diploid fetal lung fibroblasts (MRC-5 [CCL-171],ATCC, Manassas, VA) were cultured in Dulbecco’s Modified EagleMedium (DMEM; Invitrogen, Carlsbad, CA) supplemented with10% fetal bovine serum (FBS; Invitrogen). HSV-1 strain F (ATCCVR733) was propagated in Vero cells and stored at �80 �C untiluse. Virus titers were determined by the plaque assay. Immortal-ized, wild type and ATG7-deficient mouse embryonic fibroblastcells (MEF and MEF-atg7–/–) were kindly provided by Dr. MasaakiKomatsu (Tokyo Metropolitan Institute of Medical Science)[20,21].

2.3. Cytotoxicity assays and anti-viral activity assays

PGG cytotoxicity was assessed using the MTT reduction assay[22]. MRC-5 cells were cultured in 96-well plates and incubatedin the presence of serial twofold dilutions (3.125–100 lM) ofPGG in 100 ll of medium. After 3 days, 12 ll MTT (5 mg/ml inphosphate-buffered saline, PBS) were added to each well and sub-sequently incubated for 4 h to allow cells to convert MTT into for-mazan. After removing the medium, 100 ll DMSO was added todissolve formazan crystals and after 30 min the OD (optical den-sity) was determined using a microplate spectrophotometer usingdual wavelengths of 540 and 630 nm. The 50% cytotoxic concentra-tion (CC50) was calculated by regression analysis of the dose–re-sponse curves generated from the MTT assay [23].

Virus-induced cytopathic effect was also determined by theMTT method. Confluent cells grown in a 96-well plate were inocu-lated with HSV-1 at 100 TCID50 (50% tissue culture infective dose)per well for 2 h and then overlaid with medium containing serialtwofold dilutions of PGG (3.125–50 lM). Plates were incubatedfor 24 h at 37 �C and analyzed using the MTT assay as above. Per-cent cytopathic effect inhibition by different PGG concentrationswas calculated and the 50% effective concentration (EC50) was cal-culated by regression analysis of the dose–response curves gener-ated from the MTT assay.

2.4. Labeling of autophagic vacuoles with monodansylcadaverine(MDC)

MDC labeling was carried out as previously described [24] withminor modifications. Analysis of autophagy induction was assessedas a function of dose and time, respectively. Specifically, MRC-5cells were treated with various concentrations of PGG (0–20 lM)for 10 h or 10 lM PGG for various lengths of time (0–20 h) andthen incubated with MDC at 50 lM for 10 min at 37 �C. Cells werethen washed four times with PBS, trypsinized, and collected bycentrifugation and resuspended in 10 mM Tris–HCl, pH 7.2 andthen subjected to FACS analysis (excitation wavelength 380 nm,emission filter 525 nm) using a FACS Calibur flowcytometer (BDFACSAria).

To determine if autophagosome formation increased in MEFand MEF-atg7–/– cells after PGG treatment (10 lM), cells were trea-ted with PGG for 20 h and then subjected to MDC staining and fluo-rescence photometry as described previously [24].

2.5. Western blot analysis

Uninfected MRC-5 cells or MRC-5 cells infected with HSV-1 at amultiplicity of infection (moi) of 5 for 2 h were treated with orwithout PGG (10 lM) and incubated for 20 h. Cells were lysed inRIPA buffer (1� PBS, 1% Nonidet P-40, 0.5% sodium deoxycholateand 0.1% SDS). Lysate protein concentrations were determinedusing the Bradford assay. Fifty micrograms of protein were loadedper lane and subjected to 12% SDS–PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis). Separated proteins weretransferred onto PVDF membranes and blocked with 5% nonfatdry milk in 10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween 20. Afterwashing, the membranes were incubated with either anti-LC3(MBL International, Woburn, MA) or anti-GAPDH (Millipore, Bille-rica, MA), respectively. After three 10 min washes in Tris-bufferedsaline with Tween 20 (TBS-T), the membranes were incubated for2 h with horse radish peroxidase-conjugated secondary antibodies(Millipore). Protein bands were detected by ECL or ECL plus andimaged by autoradiography. GAPDH protein levels were used as acontrol for protein loading.

To detect mTOR pathway regulation induced by PGG-treatment,confluent MRC-5 cells were treated with or without PGG (10 lM)for 20 h. Cells were lysed in RIPA buffer, subjected to SDS–PAGEand analyzed by Western blot analysis using anti-mTOR antibodies(anti-phospho-mTOR [Ser2448]) antibodies, anti-p70 S6 kinaseantibodies, (anti-phospho-p70 S6 kinase [Thr389]), anti-4E-BP1antibodies (anti-phospho-4E-BP1 [Thr37/46]), anti-S6 ribosomalprotein antibodies, anti-phospho-S6 ribosomal protein (Ser235/236) antibodies (Cell Signaling Technology, Danvers, MA) andanti-GAPDH antibodies.

2.6. Transmission electron microscopy (TEM)

Uninfected MRC-5 cells or MRC-5 cells infected with HSV-1 atan moi of 5 for 2 h, were treated with or without 10 lM PGG for20 h and then harvested by trypsinization. Cells were fixed in3.0% glutaraldehyde (pH 7.2) for 1.5 h, post-fixed in 1% osmiumtetroxide for 1 h followed by dehydration and then embedded inSpurr (Sigma–Aldrich, St. Louis, MO). Ultrathin sections were cutout and stained with aqueous uranyl acetate and lead citrate andexamined by TEM (JEM1400).

2.7. Indirect immunofluorescence assay

MRC-5 cells were grown on coverslips and treated with or with-out PGG (10 lM) for 20 h. Cells were fixed with 4% paraformalde-hyde (PFA) and permeabilized with 0.02% Triton X-100 (both inPBS) and subsequently incubated with anti-LC3 antibodies(1:1000 dilution) (Abcam) for 60 min followed by an alexa flour488 secondary antibody (Molecular Probes, Invitrogen) incubationfor 60 min. After each step, the slides were washed repeatedly withPBS. Fluorescence was observed and recorded using a confocal la-ser scan microscope (LSM 510 meta, Zeiss).

2.8. Virus titration

To compare effects of PGG on HSV-1 yields in MEF cells andMEF-atg7–/– cells, virus titers were analyzed by measuring tissueculture infectious dose (TCID50, 50% tissue culture infective dose).MEF cells and MEF-atg7–/– cells were infected with HSV-1 at anmoi of 5 for 2 h and treated with or without PGG (10 lM) for18 h. Cultures were frozen, thawed and harvested for TCID50 mea-surements. Tenfold dilutions were utilized to inoculate Vero cellscultured in wells of a 96-well plate and maintained for 7 days.Virus titers were calculated using the Reed-Muench method andexpressed as TCID50 per 0.1 ml of supernatant. PGG inhibitory rates

188 Y. Pei et al. / Biochemical and Biophysical Research Communications 405 (2011) 186–191

were calculated according to virus titers. Inhibitory rate (%) = [(vir-al titers of viral control groups � viral titers of PGG-treatmentgroups)/viral titers of viral control groups] � 100.

2.9. Statistical analysis

Data were evaluated using a Welch t-test when only two valuesets were compared. Each experiment was performed at least threetimes. Results are expressed as mean ± SD (standard deviation)with significance at P < 0.05 or P < 0.01.

3. Results

3.1. Cytotoxicity and anti-viral activity of PGG

The cytotoxicity and anti-viral activity of PGG on MRC-5 cellswas determined using the MTT assay. The CC50 (the 50% cytotoxicconcentration) of PGG was 611.33 ± 6.48 lM and the PGG EC50

(50% effective concentration) for inhibiting HSV-1 was7.96 ± 0.74 lM.

3.2. Induction of autophagy by PGG

Autophagic vacuoles were visualized in cells using autofluores-cent MDC labeling [24]. PGG treatment increased autophagic vac-uole formation in a dose- and time-dependent manner (Fig. 1Aand B). Western blot analysis confirmed that PGG treatment en-hanced the conversion of LC3-I to LC3-II; a patent feature ofautophagy (Fig. 1C). Furthermore, morphological and ultrastruc-

Fig. 1. PGG induces autophagosome generation. (A) MRC-5 cells were incubated with diffflow cytometric analysis demonstrating that PGG treatment increased autophagic vacuthree similar experiments. (B) MRC-5 cells were incubated with 10 lM PGG for variousincreased autophagic vacuole formation in a time-dependent manner. Data are expresGAPDH were analyzed by Western blot analysis using anti-LC3 and anti-GAPDHimmunofluorescence analyses of MRC-5 cells treated with or without PGG (10 lM) ftreatment group (b) but not in the untreated control (a). Compared to the control (c), LC3treated group (d). (For interpretation of the references to colour in this figure legend, th

tural cellular changes were observed using transmission electronmicroscopy (TEM). Compared to the control (Fig. 1D a), more auto-phagosomes (arrows) were observed in the cytoplasm of PGG-trea-ted cells (Fig. 1D b). Immunofluorescence staining using anti-LC3antibodies to monitor autophagosome formation was also per-formed. Compared to the control (Fig. 1D c), LC3-positive fluores-cence (arrows) was induced by PGG treatment (Fig. 1D d).

3.3. Anti-HSV-1 activity of PGG-induced autophagosomes

To confirm whether the significant anti-viral activity of PGGwas associated with PGG-induced autophagy, we first determinedthat PGG induced the conversion of LC3-I to LC3-II in infected cells(Fig. 2A) compared to non-infected cells (Fig. 1C), suggesting thatPGG also induced autophagosome formation in the infected cells.To evaluate the anti-HSV-1 effect of PGG-induced autophago-somes, we chose MEF cells (normal mouse embryonic fibroblastcells) and MEF-atg7–/– (atg-deficient cells cultured from a knockoutmouse embryo). Atg7 is essential for ATG conjugation and auto-phagosomes in MEF-atg7–/– cells form at lower levels comparedto wild type controls [20], which was confirmed in our study thatdemonstrated (using MDC labeling) that the PGG-mediated in-creases in autophagosome levels was less in MEF-atg7–/– cells(5.33 ± 1.15) compared to MEF cells (22.33 ± 3.06) (Fig. 2B). Theanti-viral activity of PGG on MEF and MEF-atg7–/– cells followingvirus titration was used in evaluating the effects of PGG-inducedautophagosomes on viral clearance. These experiments demon-strated that HSV-1 yields in PGG-treated MEF-atg7–/– cells weresignificantly lower (0.70 ± 0.03) than in PGG-treated MEF cells

erent concentrations (0–20 lM) of PGG for 10 h. MDC fluorescence was measured byole formation in a dose-dependent manner. Results are expressed as mean ± SD oflengths of time (0–20 h) and MDC fluorescence measured as above. PGG treatmentsed as mean ± SD results of three experiments. (C) Expression of LC3-I, LC3-II and

antibodies to probe PGG-treated or untreated cells. (D) TEM and indirector 20 h. The structures of autophagosomes (arrows) were detected in the PGG-green puncta (arrows) were detected by immunofluorescence staining in the PGG-

e reader is referred to the web version of this article.)

Fig. 2. Autophagosomes induced by PGG-treatment have potent anti-viral activity. MRC-5 cells were infected with HSV-1 at a multiplicity of infection of 5 for 2 h and treatedwith or without PGG (10 lM). At 20 h post infection, cell samples from the different treatment groups were harvested. (A) Western blot analysis revealed that PGG-treatmentdecreased LC3-I and increased LC3-II levels. (B) MDC labeling revealed that autophagosome levels increased in MEF cells following PGG-treatment (10 lM) and that theselevels were higher than in MEF-atg7–/– cells treated in similar fashion. (C) Mean inhibitory rates against HSV-1 of PGG (10 lM) in MEF and MEF-atg7–/– cells. (D) TEM analysisof PGG-induced autophagosome-engulfed HSV-1 virions in PGG-treated cells (a: virus infected cells; b: PGG-treated infected cells). Virus egressed from infected cells (a),whereas PGG-induced autophagosomes engulfed HSV-1 virions 20 h post infection in PGG-treated cells (b). Bars, 1 lM. Data are expressed as mean ± SD of three experiments.⁄P < 0.05, ⁄⁄P < 0.01.

Y. Pei et al. / Biochemical and Biophysical Research Communications 405 (2011) 186–191 189

(0.89 ± 0.08) (Fig. 2C). TEM verified that PGG-induced autophago-somes engulfed HSV-1 virions at 20 h post infection (Fig. 2D b)whereas HSV-1 virions egressed from cells at 20 h post infectionin infected cells not treated with PGG (Fig. 2D a), supporting the di-rect involvement of autophagy in HSV-1 degradation.

3.4. Autophagy induction by PGG was associated with down-regulation of the mTOR-pathway

Currently, the most commonly used and specific inducer ofautophagy is rapamycin; a direct mTOR inhibitor [25–28]. Theactivity of mTOR, a protein kinase, can be determined by assessingthe phosphorylation levels of its substrates e.g., ribosomal S6 pro-tein kinase (p70S6K), eukaryotic initiation factor 4E-binding pro-tein 1 (4E-BP1) and S6P (a p70S6K1 substrate). Since it wasdemonstrated that rapamycin induced autophagy by reducingphosphorylation of p70S6K, S6P and 4E-BP1 [29], we determinedif PGG-induced autophagy was independent of this mTOR-regu-lated pathway. As shown in Fig. 3, PGG-treatment for 20 h severelydecreased phosphorylation of mTOR on Ser2448 and p70S6K onThr389, but not phosphorylation of S6p on Ser235/236 and 4E-BP1 on Thr37/46 in MRC-5 cells. Interestingly, we also observedthat PGG-treatment decreased the total levels of mTOR, p70S6Kand S6P but not 4E-BP1. Similar results were obtained in infectedcells in the presence or absence of PGG-treatment (data notshown). Western-blot results suggested that the inhibition of

mTOR pathway is an important mechanism of induction of autoph-agy by PGG.

4. Discussion

Based on recent data, it appeared that intrinsic autophagy didnot in fact have an impact on the HSV-1 replication cycle in cellcultures [30], because it was shown that HSV-1 interfered withthe cell’s autophagic defense mechanisms [8,9]. However, in thisstudy, comparisons between PGG-mediated anti-HSV-1 activityin MEF cells and MEF-atg7–/– cells suggested that PGG-inducedautophagy was required for its anti-HSV-1 activity. Furthermore,electron microscopy results provided direct proof that PGG-in-duced autophagosomes engulfed HSV-1 virions. These results sug-gested that PGG may enhance the intrinsic, anti-viral activity byenhancing autophagosome formation and xenophagic degradationof virions.

Moreover, the mammalian target of rapamycin (mTOR) path-way was shown to be crucial in the activation of autophagy, mTORinhibition induces autophagy formation in various systems [31].Inhibition of mTOR by rapamycin stimulates protein degradationin some mammalian cells [25,27,32–34]. Recent reports have sug-gested that rapamycin may have potential applications in HIV-1prevention and treatment [35,36]. The present results demon-strated that PGG treatment severely decreased phosphorylationof mTOR on Ser2448 and of p70S6K on Thr389 without any effects

Fig. 3. PGG-treatment impairs mTOR signaling. MRC-5 cells treated with or without PGG (10 lM) for 20 h were analyzed for mTOR activity by Western blot analysis. PGGinduced dephosphorylation of mTOR and p70S6K and downregulated the levels of T-mTOR, T-p70S6K and T-S6P. Results are expressed as mean ± SD of three independentexperiments. ⁄⁄P < 0.01.

190 Y. Pei et al. / Biochemical and Biophysical Research Communications 405 (2011) 186–191

on the phosphorylation of S6p on Ser235/236 or of 4E-BP1 onThr37/46 in MRC-5 cells. Also, we observed that PGG treatmentdownregulated the total mTOR, p70S6K and S6P levels but notthose of 4E-BP1 suggesting that PGG impaired mTOR signaling atboth total protein levels and phosphorylation levels and supporteda mechanism whereby PGG-induced autophagy by inhibitingmTOR.

Taken together, this study for the first time demonstrated thatautophagy may be an important mechanism mediating PGG anti-viral activities, suggesting that PGG is a promising compound thatcan be adapted for use against HSV-1 infections. In addition, inhib-iting of mTOR signal pathway may be an important mechanism ofinduction of autophagy by PGG.

Acknowledgments

This study was supported by grants from the Joint Funds of Na-tional Science Foundation of China (U0632010), the State Key Lab-oratory of Phytochemistry and Plant Resources in West China,Kunming Institute of Botany, Chinese Academy of Sciences(P2008-KF07, P2008-ZZ08), ‘‘211 Grant of the Ministry of Educa-tion (MOE)’’, and ‘‘the Fundamental Research Funds for the CentralUniversities’’. This work was supported partly by Grant-in-aid fromthe Tokyo Biochemical Research Foundation and a Grant-in-Aid forScientific Research (C) from the Ministry of Education, Culture,Sports, Science and Technology of Japan (No. 22590274 to K.K.).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bbrc.2011.01.006.

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