Mechanism of action of the suppression of herpes simplex virus type 2 replication by pterocarnin A

Microbes and Infection 6 (2004) 738–744 www.elsevier.com/locate/micinf

Original article

Mechanism of action of the suppression of herpes simplex virus type 2 replication by pterocarnin A Hua-Yew Cheng a, Ta-Chen Lin b, Chien-Min Yang a, Kuo-Chih Wang c, Chun-Ching Lin a,* a

Graduate Institute of Pharmaceutical Sciences, College of Pharmacy, Kaohsiung Medical University, No. 100, Shin-Chuan 1st Road, 807 Kaohsiung, Taiwan, ROC b Department of Pharmacy, Tajen Institute of Technology, 907 Ping-Tung, Taiwan, ROC c Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, 807 Kaohsiung, Taiwan, ROC Received 17 February 2004; accepted 29 March 2004 Available online 18 May 2004

Abstract The purpose of this study was to investigate the in vitro antiviral properties of pterocarnin A, extracted from the bark of Pterocarya stenoptera (Juylandaceae). Results showed that pterocarnin A exhibited anti-herpes simplex virus (HSV) activity. It had a low selectivity index (SI) value and only possessed some level of cell cytotoxic effect at high antiviral concentrations. Mechanism studies demonstrated that pterocarnin A inhibited herpes simplex virus type 2 (HSV-2) from attaching and penetrating into cells. It also actively suppressed HSV-2 multiplication in Vero cells even when added 12 h after infection. This observation indicated that pterocarnin A affected the late stage(s) of HSV-2 infection cycle. Pterocarnin A also significantly reduced viral infectivity at high concentrations. From these observations, it was concluded that pterocarnin A suppressed both early and late in the replication cycle of HSV-2. The various modes of action of pterocarnin A in interfering with certain steps of viral infection thus merit further investigation. © 2004 Elsevier SAS. All rights reserved. Keywords: Antiviral activity; HSV-2; Pterocarnin A; Modes of action

1. Introduction Herpes simplex virus (HSV) causes a variety of diseases in humans, with different degrees of severity, ranging from mild to severe, and in certain cases, it may even lead to life-threatening conditions, especially in immunocompromised patients [1,2]. According to epidemiological surveys, the HSV infection rate has continuously increased in most countries [3–5]. For example, the age-adjusted seroprevalence of HSV-2 in the United States has risen from 16.0% in the second National Health and Nutrition Examination Surveys (NHANE II) to 20.8% in NHANE III, a relative increase of 30% [5]. Nucleoside analogs have been extensively investigated and have been commonly used as anti-herpesvirus agents [6]. Usually, acyclovir (ACV), famciclovir and foscarnet are ef* Corresponding author. Tel.: +886-7-312-1101x2122; fax: +886-7-313-5215. E-mail address: [email protected] (C.-C. Lin). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2004.03.009

fective in the treatment of HSV infections, but the failure of their treatment is reportedly due to infections by antiviralresistant HSV, which is most common among immunocompromised patients and also due to the recurrence of latent viruses [7–12]. According to literature, the prevalence of ACV-resistant HSV among immunocompromised patients is about 5%, and reaches 14% among bone marrow transplant recipients [10–12]. Although some non-nucleoside inhibitors of herpesviruses have been developed in the laboratory [13–15], none of them are officially approved for HSV therapy [16,17]. Consequently, there is still a need in the future to search for new and more effective antiviral agents that can substitute or complement currently used antiviral medicine. Many plants have been traditionally used as treatment for virus infection. Recently, some of them have been reported in the literature to exhibit anti-HSV activity [18,19]. These findings indicate that herbal medicines might be employed as an alternative therapy for HSV infection in the future.

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OH HO O HO

C

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OH OH HO O

O O C

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OH OH HO

for the plaque assay consisted of DMEM plus 2% FCS, 1% methylcellulose and antibiotics as described above. HSV-2 strain 196 was kindly provided by Dr. Lien-Chai Chiang (Department of Microbiology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan). Its titer was determined by plaque assay and was expressed as plaque-forming units (PFU) per ml. Virus stocks were stored at –80 °C until use. 2.3. Antiviral assays

HO HO

OH

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HO OH

OH

Fig. 1. Structure of pterocarnin A isolated from bark of P. stenoptera.

Pterocarya stenoptera C. DC. (Juylandaceae), also known as Chinese Wingnut, is traditionally used as an insecticide and also in an infusion as a wash to remove scabies, eczema, and abscesses. The leaves and bark of this plant are said to be carminative and anthelmintic. In this study we evaluated the in vitro anti-HSV-2 properties of pterocarnin A, a pure compound extracted from the bark of P. stenoptera. This is the first report on the antiviral activity of pterocarnin A.

2. Materials and methods 2.1. Test compounds Pterocarnin A (Fig. 1) was isolated from the bark of P. stenoptera as described previously [20]. Briefly, the bark of P. stenoptera was extracted at room temperature with acetone–water (4:1, v/v). The extract was concentrated under reduced pressure and then eluted with acetone through celite column. The acetone eluate was chromatographed on a Sephadex LH-20 to give two fractions. Fraction 2 was further chromatographed on a Sephadex LH-20 and MCI-gel CHP 20P to obtain pterocarnin A. The structure and purity of pterocarnin A was determined by its spectroscopic and physical data as described previously [21]. ACV was purchased from Sigma Company (USA). Pterocarnin A and ACV were dissolved in sterile de-ionized water before use.

2.3.1. XTT assay The antiviral activity of pterocarnin A was assayed using XTT (sodium 3′-[1-(phenylamino-carbonyl)-3,4tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid) as described by Weislow et al. [22]. Briefly, 104 cells per well were seeded into 96-well culture plates (Falcon). After 4 h of incubation at 37 °C with 5% CO2, cells were infected with HSV-2 at multiplicity of infection (MOI) = 0.5 or 5.0, and then various concentrations of pterocarnin A were added. The infected cells were incubated at 37 °C for another 72 h. The medium was then aspirated and XTT reagent was added. The plate was reincubated for an additional 2 h to allow the production of formazan. Optical densities were measured with EIA reader (Lab Systems) at a test wavelength of 492 nm and a reference wavelength of 690 nm. The antiviral activity of pterocarnin A and its minimal concentration required to inhibit 50% HSV-2 growth (IC50) were evaluated according to Cheng et al. [23]. 2.3.2. Plaque reduction assay Vero cells were seeded into 24-well culture plates (Falcon) at a density of 105 cells per well and incubated at 37 °C with 5% CO2 until reaching at least 95% confluency. The cell monolayer was then infected with 100 PFU HSV-2 in the absence or presence of pterocarnin A and further incubated at 37 °C for 1 h with 5% CO2. After 1 h of adsorption, the cell monolayer was overlaid with overlay medium. The overlay medium was removed 2 days later, and the infected cell monolayer was fixed and stained with 10% formalin and 1% crystal violet, respectively. The minimal concentration of pterocarnin A required to reduce the 50% plaque number (IC50) was calculated according to Cheng et al. [23]. 2.4. Cytotoxicity assay and selectivity index (SI)

2.2. Cells and virus All reagents and medium for cell culture were purchased from Gibco BRL (Grand Island, NY). African green monkey kidney cells (Vero) (ATCC CCR-81) were obtained from the hospital of Kaohsiung Medical University (Kaohsiung, Taiwan). Cells were propagated in Dulbeco’s modified Eagle medium (DMEM) supplemented with 5% fetal calf serum (FCS), 200 U/ml penicillin G sodium, 200 µg/ml streptomycin sulfate and 0.5 µg/ml amphotericin B. Overlay medium

The cytotoxic effect of pterocarnin A on proliferating cells was assayed with an XTT-based method [24]. It was performed with the procedures similar to XTT assay, except that HSV-2 was not inoculated. The initial seeding cell number was 5.0 × 103 cells per well, and after 72 h incubation, the cell number was increased to around 3.0 × 104 cells per well. The 50% cell cytotoxic concentration (CC50) of pterocarnin A was calculated according to Cheng et al. [23], and the SI was evaluated as the ratio of CC50 to IC50.

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Table 1 Anti-HSV-2 activity, cytotoxic effect and SI of pterocarnin A on Vero cells a Compound

Antiviral activity (µM) XTT b IC50

Pterocarnin A ACV

MOI = 0.5 5.4 ± 0.3 0.8 ± 0.1

MOI = 5.0 6.6 ± 0.4 1.2 ± 0.2

PRA b IC50 2.3 ± 0.2 0.4 ± 0.1

IC90 3.8 ± 0.2 1.1 ± 0.2

SI c

Cytotoxic effect (µM) XTT b CC50 31.7 ± 1.6 >1000

XTT MOI = 0.5 5.9 >1250

MOI = 5.0 4.8 >833.3

PRA

13.8 >2500

a

Antiviral activity was determined by XTT and plaque reduction assays. Cytotoxicity was determined by XTT assay. Values represent the mean ± S.D. of three independent experiments. c SI was the ratio of CC50 to IC50.

b

2.5. Virucidal assay

2.8. Penetration assay

Virucidal activity of pterocarnin A was evaluated as described by Carlucci et al. [25]. A virus suspension containing 2 × 107 PFU HSV-2 was mixed with or without various concentrations of pterocarnin A for 6 h at 26 °C. The sample was then diluted and its residual infectivity was determined by plaque assay.

The procedures for virus penetration assay have been described elsewhere [28,29]. The Vero monolayer was grown in 24-well culture plate and pre-chilled at 4 °C for 1 h. The cell monolayer was then infected with 200 PFU HSV-2 and incubated at 4 °C for another 3 h to allow the attachment of HSV-2 to the cell monolayer. After 3 h of incubation, 5.0 µM pterocarnin A was added. The control group contained no pterocarnin A. The infected cell monolayer was then incubated at 37 °C to maximize the penetration of viruses. At 10 min intervals, the infected cell monolayer was treated with PBS at pH 3 for 1 min to inactivate unpenetrated virus. PBS at pH 11 was then added immediately to neutralize acidic PBS (pH 3). The neutral PBS was removed and the cell monolayer was overlaid with overlay medium. After further 48 h of incubation at 37 °C, the cell monolayer was fixed and stained. Plaques were counted and the percentage of inhibition of penetration was calculated.

2.6. Time of addition studies The antiviral activity of test samples was evaluated at various time periods up to 24 h according to procedures described by Boulware et al. [26]. Briefly, Vero cells were seeded into 12-well culture plates (Nunc) at a density of 2 × 105 cells per well and incubated at 37 °C with 5% CO2 for 24 h. The cell monolayer was then infected with 1 × 105 PFU HSV-2/well. Five micromolar of pterocarnin A or ACV was added into wells at intervals of 0, 2, 4, 7 and 12 h postinfection. At 24 h post-infection, infected cells were scraped and viruses were released from cells by freeze–thawing for three times. Cell pellets were removed by centrifugation at 1100 × g for 10 min. The virus titer of each supernatant was determined by plaque assay. The percent of inhibition was calculated as the reduction in virus titer observed in infections containing compound compared to that of infections containing de-ionized water as a solvent control.

2.9. Statistical analysis Results were expressed as mean ± standard deviation (S.D.). Each experiment was performed at least three times. Student’s unpaired t-test was used to evaluate the difference between the test sample and solvent control. A P value of less than 0.05 was considered statistically significant.

2.7. Attachment assay

3. Results

The attachment assay described by Logu et al. [27] was used in this study with minor modification. Briefly, the Vero cell monolayer, which was grown in 24-well culture plate, was pre-chilled at 4 °C for 1 h. The medium was aspirated and the cell monolayer was then infected with 200 PFU HSV-2 in the absence or presence of serial dilution of pterocarnin A. After further incubating the infected cell monolayer at 4 °C for another 3 h, the medium was aspirated to remove unabsorbed virus. The cell monolayer was then washed with PBS three times and overlaid with 1% methylcellulose medium. Cell monolayer was incubated at 37 °C for another 48 h before it was fixed and stained. The inhibitory percentage of pterocarnin A for HSV-2 attachment to Vero monolayers was calculated.

3.1. Effect of pterocarnin A on virus replication and cell growth Table 1 demonstrates that pterocarnin A exhibited antiHSV-2 activity with IC50 of 5.4 ± 0.3 µM in XTT assay. The IC50 value shifted from 5.4 ± 0.3 to 6.6 ± 0.4 µM as the MOI changed from 0.5 to 5.0. For plaque reduction assay, the IC50 and IC90 value were 2.3 ± 0.2 and 3.8 ± 0.2 µM, respectively. Pterocarnin A showed some level of cytotoxic effect on growing Vero cells at high antiviral concentrations with a CC50 of 31.7 ± 1.6 µM. About 90% of cells survived under the treatment with 20.0 µM pterocarnin A (data not shown). The SI values for XTT and plaque reduction assays were 5.9 and 13.8, respectively (Table 1).

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Fig. 2. Virucidal activity of pterocarnin A (●) and ACV (C) against HSV-2. The test sample was pre-incubated with HSV-2 at 26 °C for 6 h. The residual infectivity was then determined by plaque assay. Each point represents the mean ± S.D. for three independent experiments. The asterisk (*) indicates a significant difference between the test sample and solvent control (P < 0.05).

3.2. Effect of pterocarnin A on viral infectivity Fig. 2 shows that pterocarnin A failed to affect the viral infectivity at concentration of 5.0 µM or less. However, 10.0 µM or higher concentrations of pterocarnin A significantly reduced the virus infectivity. The percentage of residual infectivity for 0.0, 2.5, 5.0, 10.0, 20.0 and 50.0 µM pterocarnin A-treated virus was 100.0 ± 14.7%, 96.4 ± 13.7%, 103.6 ± 25.4%, 6.1 ± 0.7%, 0.6 ± 0.1% and 0.0 ± 0.0%, respectively. Since 5.0 µM or lower concentrations of pterocarnin A showed no virucidal activity, these concentrations were, therefore, selected and applied in all of the following mode of action studies. 3.3. Time course studies of pterocarnin A In order to investigate the stage in which pterocarnin A affected the viral life cycle, an experiment on the time of addition was performed. Results demonstrated that the antiviral activity of pterocarnin A was unaffected by the time of addition (Table 2). Pterocarnin A remained active in inhibiting the viral replication even when added 12 h post-infection. A similar result was observed when the time of addition experiment of ACV, a known inhibitor of viral DNA replicaTable 2 Effect of pterocarnin A treatment time on anti-HSV-2 activity Time period of incubation (h) 0–24 2–24 4–24 7–24 12–24

Percent of inhibition Pterocarnin A ACV 99.9 ± 0.0 * 100.0 ± 0.0 * 88.9 ± 16.3 * 100.0 ± 1.2 * 82.0 ± 27.5 * 100.0 ± 0.6 * 88.1 ± 17.5 * 99.9 ± 4.9 * 81.6 ± 31.8 * 99.9 ± 14.9 *

The experimental concentration of pterocarnin A and ACV was 5 µM. Each value represents mean ± S.D. of three independent experiments. The asterisk (*) indicates a significant difference between the test sample and solvent control (P < 0.05).

0 0

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Fig. 3. Effect of pterocarnin A (●) and ACV (C) on HSV-2 attachment to Vero cells. The Vero monolayer was pre-chilled at 4 °C for 1 h. Two hundred PFU HSV-2 was then inoculated to the cell monolayer in the absence or presence of test compound and incubated for another 3 h. The percentage of inhibition of the test compound was evaluated by plaque assay. Each point represents the mean ± S.D. for three independent experiments. The asterisk (*) indicates significant a difference between the test sample and solvent control (P < 0.05).

tion, was performed. This result indicated that pterocarnin A affected the late stage (12 h or later) of HSV-2 infection. 3.4. Effect of pterocarnin A on viral attachment and penetration Pterocarnin A was effective in inhibiting viral infection when it was added concurrently with HSV-2 to cells (Table 2). This observation suggested that pterocarnin A might also exhibit its effect at any time during the first 12 h event(s) of HSV-2 infection, including viral attachment, viral penetration, the entering of viral DNA into cell nucleus, etc. These steps precede the late event(s) of HSV-2 infection, and therefore, the effect of pterocarnin A on viral attachment and penetration was investigated. Results demonstrated that pterocarnin A blocked HSV-2 attachment to cells and its inhibitory effect was dependent on the dosage levels (Fig. 3). Pterocarnin A blocked 52.2% and 100.0% of the HSV-2 from attaching to the cells at a concentration of 1.0 and 4.0 µM, respectively. In contrast, ACV, which is commonly known to be only active in affecting HSV replication, failed to significantly inhibit virus attachment up to 10.0 µM. Besides the attachment, pterocarnin A was also shown to inhibit HSV-2 penetration into the Vero cell (Fig. 4). A significant inhibitory effect was noted as early as 10 min after pterocarnin A was added (P < 0.05). The percentage of blocking of penetration by 5.0 µM pterocarnin A in the first 10 min was 94.4%. 4. Discussion Natural products from plants and microorganisms have provided the pharmaceutical industry with one of its most

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Fig. 4. Effect of pterocarnin A (●) and ACV (C) on the penetration of HSV-2. The Vero monolayer was pre-chilled at 4 °C for 1 h and then infected with 200 PFU HSV-2 at 4 °C for 3 h. After 3 h incubation, 5 µM pterocarnin A was added. At 10 min intervals, extracellular virus was inactivated by PBS at pH 3 for 1 min. PBS at pH 11 was then added to neutralize acidic PBS. The neutral PBS was removed and overlay medium was added. Each point represents the mean ± S.D. for three independent experiments. The asterisk (*) indicates significant a difference between the test sample and solvent control (P < 0.05).

important sources of “lead” compounds in the search for new drugs and medicines. It is estimated that at least 75% of antiviral medicines approved from 1981 to 2002 are either of natural origin or derived from the knowledge gained from studies of natural products [30]. Many reports indicate that compounds isolated from medicinal plants can suppress the multiplication of HSV [19,31,32]. An example is the approval of n-docosanol, a naturally occurring antiherpetic agent, by US. Food and Drug Administration as a topical treatment for herpes labialis [33–35]. Thus, research in medicinal plants and ethnopharmacology can serve as an alternative approach for the discovery of novel antiviral agents. Our present study demonstrated that pterocarnin A inhibited HSV-2 multiplication in an MOI-dependent manner. Results generated from the cytotoxicity assay showed that even if pterocarnin A had a low SI value (Table 1), it only affected the level of growth and viability of Vero cells at high antiviral concentrations. As a result, it was concluded that the inhibitory action of pterocarnin A on HSV-2 multiplication was not directly a result of its cytotoxic effect toward the cells. The pre-treatment of uninfected cells with pterocarnin A and then washing it out did not prevent cells from acquiring virus infection (data not shown). Also, similar antiviral activity was observed between pterocarnin A pre-treated and non pre-treated groups (data not shown). These observations indicated that the pre-treatment of pterocarnin A did not affect HSV-2 infection in Vero cells. During HSV attachment, the virus envelope glycoprotein C (gC) will bind to heparin sulfate residues present on the proteoglycans on the surface of the target cells [36]. Initial binding is ensued by a stable attachment, a process which is

dependent on the presence of glycoprotein D (gD) [37]. Membrane fusion between virion envelope and plasma membrane of the target cell requires glycoproteins D, B, H and L, which probably act in combination [38,39]. Our results demonstrated that pterocarnin A inhibited HSV-2 attachment to cells. This finding is consistent with the previous studies [40]. Tannins have been reported to inhibit HSV infection [41,42]. It is believed that the inhibitory effect is derived from the binding of tannin molecules to the protein coat of the virus and/or to the host cell membrane. Virus adsorption and eventually virus penetration are arrested [40]. Since pterocarnin A is a hydrolysable tannin, it is not surprising that it can inhibit HSV-2 attachment. In addition to virus attachment, pterocarnin A also inhibited the penetration of HSV-2. During the penetration experiment, HSV-2 was allowed to attach to the cells; however, it did not penetrate into the cells. The addition of pterocarnin A was in fact observed to be able to block HSV-2 penetration into the target cells. These observations suggested that pterocarnin A affects the virus penetration process possibly through detaching the already bound virus–cell complex and/or through the disturbance of viral glycoproteins. Results of the time of addition studies revealed that pterocarnin A still actively suppressed HSV-2 multiplication in Vero cells even when added 12 h post-infection. Since the duration of these experiments was 24 h, only one round of virus replication could occur. It was thus suggested that pterocarnin A affected other step(s) of the HSV-2 infectious cycle, in addition to the virus attachment and penetration. Previous studies showed that samarangenin B, a condensed tannin, can suppress HSV-1 replication in Vero cells [19]. The anti-HSV-1 activity of samarangenin B is mediated through inhibition of viral a gene expression, blocking b gene transcription, and arresting viral DNA synthesis and structural protein expression. Although both pterocarnin A and samarangenin B are tannins, further studies are required to find out whether similar effects are observed for pterocarnin A, as well as its underlying mechanisms of action in blocking other steps in HSV-2 replication. Pterocarnin A also significantly reduced HSV-2 infectivity at concentrations higher than those used in mechanism studies. However, it did not affect infectivity at low concentrations. These observations indicated that the mechanisms of action of pterocarnin A in blocking viral attachment and penetration, and also interfering with the late stage of virus infection were not related to its virucidal ability. Cassady and Whitley have suggested that future antiherpesvirus agents will probably target enzymes or viral factors essential for infection or inhibiting other steps in the viral infection cycle, such as viral entry, protein synthesis or capsid assembly [43]. In this study, pterocarnin A was found to reduce viral infectivity at high concentrations (Fig. 2), inhibit virus attachment to cells (Fig. 3), block virus penetration into cells (Fig. 4) and also affect the late stage of virus infection (Table 2). The various modes of action make ptero-

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carnin A an interesting candidate for further investigation of its potential in inhibiting the HSV-2 life cycle.

Acknowledgements We thank Professor Itsuo Nishioka, Dr. Gen-ichiro Nonaka and Dr. Azuma Ryutaro (Faculty of Pharmaceutical Sciences, Kyushu University, Japan) for their help and for providing pterocarnin A. We also thank Dr. Lien-Chai Chiang (Department of Microbiology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan) for providing HSV-2 strain 196.

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