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Anti-Infectious Bronchitis Virus (IBV) Activity of 1,8-cineole: Effect on Nucleocapsid (N) ProteinZhiwei Yang a b , Nan Wu a b , Yujie Fu a b , Gang Yang a b , Wei Wang a b , Yuangang Zu a b &Thomas Efferth ca Key Laboratory of Forest Plant Ecology , Ministry of Education, Northeast ForestryUniversity , Harbin , 150040 , PR Chinab Engineering Research Center of Forest Bio-preparation , Ministry of Education, NortheastForestry University , Harbin , 150040 , PR Chinac Department of Pharmaceutical Biology , Institute of Pharmacy and Biochemistry, Universityof Mainz , Mainz , 55099 , GermanyPublished online: 15 May 2012.

To cite this article: Zhiwei Yang , Nan Wu , Yujie Fu , Gang Yang , Wei Wang , Yuangang Zu & Thomas Efferth (2010) Anti-Infectious Bronchitis Virus (IBV) Activity of 1,8-cineole: Effect on Nucleocapsid (N) Protein, Journal of Biomolecular Structureand Dynamics, 28:3, 323-330, DOI: 10.1080/07391102.2010.10507362

To link to this article: http://dx.doi.org/10.1080/07391102.2010.10507362

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Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 28, Issue Number 3, (2010) ©Adenine Press (2010)

*Phone: +86-451-82190535Fax: +86-451-82190535E-mail: fuyujie1967@yahoo.com.cn

Zhiwei Yang1,2,#

Nan Wu1,2,#

Yujie Fu1,2,*Gang Yang1,2 Wei Wang1,2

Yuangang Zu1,2,*Thomas Efferth3

1Key Laboratory of Forest Plant Ecology,

Ministry of Education, Northeast Forestry

University, Harbin 150040, PR China2Engineering Research Center of Forest

Bio-preparation, Ministry of Education,

Northeast Forestry University, Harbin

150040, PR China3Department of Pharmaceutical Biology,

Institute of Pharmacy and Biochemistry,

University of Mainz, Mainz 55099,

Germany

#These two authors contribute equally to

this work.

Anti-Infectious Bronchitis Virus (IBV) Activity of 1,8-cineole: Effect on Nucleocapsid (N) Protein

http://www.jbsdonline.com

Abstract

In the present study, anti-IBV (infectious bronchitis virus) activity of 1,8-cineole was stud-ied by MTT assay, as well as docking and molecular dynamic (MD) simulations. The CC50 of 1,8-cineole was above 10 mM. And the maximum noncytotoxic concentration (TD0) of 1,8-cineole was determined to be 3.90 ± 0.22 mM, which was much higher than that of riba-virin (0.78 ± 0.15 mM). 1,8-cineole could inhibit IBV with an IC50 of 0.61 mM. MTT assay showed that the inhibition of IBV by 1, 8-cineole appears to occur moderately before enter-ing the cell but much strongly after penetration of the virus into the cell. In silico simulations indicated that the binding site of 1,8-cineole was located at the N terminus of phosphorylated nucleocapsid (N) protein, with interaction energy equaling -40.33 kcal mol-1. The residues TyrA92, ProA134, PheA137, AspA138 and TyrA140 had important roles during the bind-ing process and are fully or partially conserved in various IBV strains. Based on spatial and energetic criteria, 1,8-cineole inerfered with the binding between RNA and IBV N-protein. Results presented here may suggest that 1,8-cineole possesses anti-IBV properties, and therefore is a potential source of anti-IBV ingredients for the pharmaceutical industry.

Key words: 1,8-cineole; Anti-IBV activity; MTT; Docking; Active site.

Introduction

Infectious bronchitis virus (IBV), the prototype species of the family Coronaviri-dae, is one of the primary causes of respiratory disease in domestic fowl. IBV primarily and initially infect the trachea though they can also infect the kidney and oviduct and other epithelial surfaces. IBV is the causative agent of infectious bron-chitis (IB), an acute and highly contagious disease of chickens. IB affects respira-tory, kidney and oviduct tissues, resulting in respiratory disease, retarded growth and reduced egg production (1-2). A number of IBV serotypes have been identi-fied worldwide, and vaccines containing strains of IBV from multiple serotypes are routinely used in commercial chicken flocks. However, antigenically different serotypes and newly emerged genetic variants makes the control of IBV infection a challenging task, even sometimes cause ‘vaccine breaks’. Therefore, IB some-time breaks out and remains one of the most important poultry diseases in many countries of the world (3-5). Consequently, study and exploration of an effective anti-IBV medicine have significant value and broad foreground.

IBV has a single stranded RNA genome, approximately 27 kb in length, of positive polarity specifies the production of three major structural proteins: the phospho-rylated nucleocapsid (N) protein, the membrane (M) glycoprotein and the surface (S) glycoprotein (spike protein). The N protein is a highly immunogenic phos-phoprotein also implicated in viral genome replication and in modulating cell sig-naling pathways. The N-terminal of N-protein (NTD) serves as a functional unit

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critical for the specific interaction with RNA, which exhibits a U-shaped structure, with two arms rich in basic residues (6). In addition, the key functional residues of NTD are highly conserved among coronaviruses between different antigenic groups (7-8), according to the spike (S) protein which contributes to the distinc-tive peplomers on the viral surface and contains neutralizing and group-specific epitopes. The N protein has been a major protein target in the development of anti-IBV medicine (6-10).

Recently, the essential oils and various extracts of plants have provoked interest as sources of natural products. They have been screened for their potential uses as alternative remedies for the treatment of many infectious diseases. Particularly, 1,8-cineole, also called eucalyptol, is a major component of camphor-scented essential oils found in eucalyptus leaves, bay leaves and other aromatic plant foli-age. Recent clinical research has shown that 1,8-cineole presents anti-inflammatory and pain release properties and may promote leukemia cell death (11-16).

To the best of our knowledge, the anti-IBV activities of 1,8-cineole have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-IBV activity of 1,8-cineole by MTT assay and to explore the geometry and energetics of binding using molecular dynamics and molecular simulations which allow to explore and understand target structure, stability and protein-ligand interactions as has been demonstrated by several recent publications in this Journal (17-42). In this paper, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode of 1,8-cineole and IBV N protein, on the base of spatial and energetic criteria. We anticipate that the insight into the understanding of binding mechanism will be of value in the rational design of IBV inhibitors.

Materials and Methods

Materials

1,8-cineole and ribavirin were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and was stored in glass vials with Teflon sealed caps at -20 ± 0.5°C in the absence of light.

Cell Cultures

Vero-E6 (African green monkey kidney cells) was purchased from Harbin Veteri-nary Research Institute (Harbin, P. R. China). The cells were grown in monolayer culture with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The mono-layers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-IBV assays, and propagated at 37°C in an atmosphere of 5 % CO2.

Viruses

The IBV Gray strain was purchased from National Control Institute of Veterina-tory Bioproducts and Pharmaceuticals (Beijing, P. R. China). Virus was routinely grown on Vero-E6 cells. IBV-Gray stock cultures were prepared from supernatants of infected cells and stored at -80°C.

Cytotoxicity Assay

The cellular toxicity of 1,8-cineole on Vero-E6 cells was assessed by MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of vari-ous concentrations (10 mM – 0.078 mM) of 1,8-cineole for eight replicates and incubated at 37°C in a humidied atmosphere of 5% CO2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was

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added to each well, after incubated at 37°C for 4 h, remove the supernatants, then 200 μL DMSO was added and incubated at 37°C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD0) and 50% cytotoxic concentration (CC50) were calculated by linear regression analysis of the dose-response curves generated from the data.

Anti-IBV Activity

Inhibition of virus replication was measured by MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37°C. The residual inoculum was discared and infected cells were added with DMEM containing 2% FCS. Each assay was performed in eight replicates. After incubation for 72 h at 37°C, the cul-tures were measured by MTT method as described above. The concentration of 1,8-cineole and ribavirin which inhibited virus numbers by 50% (IC50) was determined from dose-response curves.

Mode of Anti-IBV Activity

Cells and viruses were incubated with 1,8-cineole at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with 1,8-cineole before viral infection, viruses were incubated with 1,8- cineole before infection and cells and viruses were incubated together with 1,8-cineole during adsorption or after penetration of the virus into the host cells. 1,8-cineole was always used at the nontoxic concentration. Cell monolayers were pretreated with 1,8-cineole prior to inoculation with virus by adding 1,8-cineole to the culture medium and incubation for 1h at 37°C. The compound was aspi-rated and cells were washed immediately before the IBV inoculum was added. For pretreatment virus, IBV were incubated in medium containing 1,8-cineole for 1h at room temperature prior to infection of Vero-E6 cells. For analyzing the anti-IBV inhibition during the adsorption period, the same amount of IBV was mixed with the drug and added to the cells immediately. After 1h of adsorption at 37°C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of 1,8-cineole (or ribavirin) against IBV was also tested during the replication period by adding it after adsorption, as typical performed in anti-IBV susceptibility studies. Each assay was run in eight replicates. Ribavirin was used as a positive control.

Statistical Analysis

All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.01 were considered to be significant.

Figure 1: Chemical structure of 1,8-cineole.

CH3

H3C CH3

O

Table IAnti-IBV activity of 1,8-cineole compared with ribavirin.

Compound CC50a (mM) TD0

b (mM)

IBV (Gray strain)

IC50c (mM) SId

1,8-cineole >10.0 3.90 ± 0.22 0.61 ± 0.07 >16.39Ribavirin >1.0 0.78 ± 0.15 0.118 ± 0.02 >8.47

Values in this table represent the mean values (±SD) of three independent experiments (P < 0.01).a,bCytotoxic effect was determined by MTT assay. CC50 was the concentration that showed 50% cytotoxic effects in Vero cells. TD0 was the concentration that showed nontoxic maximum effects in Vero cells. cAntiviral activity was determined by MTT assay. IC50 was the concentration that inhibited 50% of IBV replication in Vero cells.dThe selective index (SI) was calculated as CC50/IC50.

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Docking and Molecular Dynamic Simulations

All the docking and molecular dynamics (MD) simulations were performed with the different modules implemented under InsightII 2005 software package (43) on Linux workstations, using the consistent-valence force-field (CVFF). The structure of 1,8-cineole (Figure 1) was generated with the Builder module. Geometry and partial atomic charges of the substrate were conducted throughout Discover 3.0 module by applying the BFGS algorithm (44) with a convergence criterion of 0.01 kcal mol-1 Å-1. The X-ray crystallography structure of the N- terminal domain of N-protein (PDB code 2GEC) was recovered from the RCSB Protein Data Bank (6). For convenience, it is named as NTD throughout this work. Demonstrated by previous literatures (17-42), the docking and molecular dynamics (MD) simulations were performed to explore and understand the inter-actions between 1,8-cineole and NTD, by the general protocols in the InsightII 2005 software packages (45-46). The MD trajectories were generated using a 1.0-fs time step for a total of 5000 ps, saved at 5.0-ps intervals. The interaction energies of 1,8-cineole with NTD and the respective residues at the NTD active site were calculated by the Docking module (46), over the 1000~5000 ps MD trajectories. More calculated details can be found in Supporting Information or refer to elsewhere (45).

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Figure 2: Antiviral effect of 1,8-cineole (3.9 mM) and ribavirin (0.78 mM) against IBV by incubation at different periods of time during infection. Cells were pretreated with 1,8-cineole or ribavirin prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and 1,8-cineole or ribavirin was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of 3 experiments.

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Figure 3: The total energy of ensemble (Total energies, A) and time-evolution backbone-atom root mean square deviations (RMSD, B) during the molecular dynamic simulation for N terminus of N-protein (NTD) complexed with 1,8-cineole.

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Results and Discussions

Effect of 1,8-cineole Against IBV by MTT Assay

First, the efficacy of 1,8-cineole on IBV replication and cell viability were exam-ined. As shown in Table I, the cytotoxicity of 1,8-cineole on Vero cells were expressed as CC50 and TD0. The CC50 of 1,8-cineole was above 10 mM. And the maximum noncytotoxic concentration (TD0) of 1,8-cineole was determined as 3.90 ± 0.22 mM, which was much higher than that of ribavirin (0.78 ± 0.15 mM) (P < 0.01). These results indicated that 1,8-cineole did not affect the growth of Vero cells. Thus, it seems that the antiviral effects of 1,8-cineole were not due to any cytotoxicity.

Furthermore, 1,8-cineole was found to inhibit IBV with an IC50 of 0.61 mM. Based on the IC50 and CC50 values, the selectivity index (SI) was calculated as >16.39. It is reported that a SI of 4 or more should be appropriate for an antiviral agent (47). This suggests that 1,8-cineole can be judged to have significant anti-IBV activity.

Mode of Anti-IBV Activity by MTT Assay

As shown in Figure 2, 1,8-cineole showed the maximum noncytotoxic antiviral activity when added at a concentration of 3.9 mM during the replication period with inhibition of the viral replication of 82.63% ± 2.11% for IBV. Inhibition of the pretreatment virus phase was 61.68 ± 4.32%, whereas that of the adsorption phase and pretreatment cell phase was only 5.31 ± 3.14% and 12.13 ± 1.40%, respec-tively. Differently from 1,8-cineole, ribavirin only showed antiviral activity at a concentration of 0.78 mM during the replication period with inhibition of the viral

Figure 4: The propeller structure (A), the active site (B) and key residues at the active site (C) of the binding mode of N terminus of N-protein (NTD) with 1, 8-cineole. 1, 8-cine-ole is represented by ball and stick model. N terminus of N-protein (NTD) is in color rib-bon: hydrogen-bonded turns, extended strands and random coils are in blue, yellow and green, respectively. The active site is defined through Binding Site Analysis module, col-ored by electrostatic potentials. Key residues are represented by stick models. The oxygen, nitrogen, carbon, hydrogen in model are col-ored in red, blue, green and white, respec-tively. The important H-bond is labeled in the dashed black line.

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replication of 90.18% ± 4.39%. However, no significant effect on viral replication was detected when ribavirin was used for pretreatment of cells or viruses or when ribavirin was only added during the adsorption phase. These results suggested that the inhibition of IBV by cineole appears to occur moderately before entering the cell but much strongly occur after penetration of the virus into the cell. In addition, biochemical studies indicated that the bioactivity of N protein is essential determi-nant for the replication of IBV virus (6, 8, 10). Hence, we conclude that the func-tion of N protein may be suppressed by 1,8-cineole.

In Silico Inhibition Mechanism of 1,8-cineole

Based on the MTT results above, N terminus of N-protein (NTD) was determined to be the binding location of 1,8-cineole. As the total energies and backbone root-mean-square- deviations (RMSD) in Figure 3 show, the NTD complexed with 1,8-cineole quickly reaches equilibrium and remains rather stable afterwards. Accordingly, the analysis of interactions between the NTD and 1,8-cineole was carried out on the base of the average structure of the 1000~5000 ps trajectories. As shown in Figure 4, the structure of NTD is characterized by twisted antiparallel β-sheet surrounded with several loop regions. The active site that binds to 1,8- cineole is mapped to the loop region on the top of the β-sheet, near the RNA bind-ing site (6), contains a large number of polar (or charged) residues (Figure 4B). The oxygen atom in the core of 1,8-cineole is docked toward residue TyrA92, with the formation of one H-bond (Figure 4C). The length and angel of the H-bond equal to be 1.88 Å and 175.2°, respectively. The three –CH3 groups of 1,8-cineole fit the hydrophobic caves on the NTD active site. Form the spatial analysis, the 1,8-cin-eole holds the residues SerA34, GlnA37, TyrA92, ProA134, PheA137, AspA138, TyrA140 and TrpB155 and hinders the binding of RNA with NTD. The interac-tion energy of 1,8-cineole and NTD is calculated to be at -40.33 kcal mol-1. The vdW interactions play a dominant role during the binding process, contributing to

Figure 5: The multiple sequence alignment of the N terminus of N-protein (NTD) for various IBV strains. The same amino acid residues were represented by dots. The asterisk marks the key amino acid residues in the active sites of NTD (residues Ser34, Gln37, Tyr92, Pro134, Phe137, Asp138, Tyr140 and Trp155). Sequences for IBV-NTD (residues 22 to 160) were obtained from RCSB Protein Data Bank and Swiss-Prot (IBV-G, 2GEC; IBV-B, P69596; IBV-AR, Q64960; IBV-DE, Q9J4B0; IBV-D1, Q9J4A3; IBV-K, P12648; IBV-H1, Q98WJ7; IBV-H5, Q98Y32; IBV-SA, Q8JMI6; IBV-M, Q82616; IBV-VI, Q96598; IBV-V1, Q96605).

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82.0% (-33.07 kcal mol-1). The active site residues that have interaction energies below -1.0 kcal mol-1 were collected in Table II. It was found that 1,8-cineole has strong interactions with residues TyrA92, ProA134, PheA137, AspA138 and TyrA140, the interaction energies (Einter) amount to -9.70, -2.91, -2.05, -3.03 and -4.66 kcal mol-1, respectively. As the five residues are key for RNA bindings (6, 10), it further suggests that the RNA bindings will be interfered with the presence of 1,8-cineole. It commendably explains that the inhibition of 1,8-cineole against IBV occurs strongly after penetration of the virus into the cell. The Figure 5 shows the multiple sequence alignment of NTD (residues 22-160) in various IBV strains. It was found that the key active-site residues are fully or partially conserved. It indicates the 1,8-cineole not only inhibits the N-protein of IBV Gray strain, but also other known strains.

In conclusion, 1,8-cineole possesses anti-IBV properties via embarrassing the bind-ing process between RNA and IBV N-protein. Our results provide the promising information for the potential use of 1,8-cineole in the treatment of IBV infectious disease. Further studies on the anti-IBV activity in vivo are needed to support this point of view.

Supplemental Material

The details of docking and molecular dynamic simulations can be found in supple-mental material. Supplementray material is available at no charge from the authors directly; the supplementary data can also be purchased from Adenine Press for US $50.00.

Acknowledgement

The authors gratefully acknowledge the financial supports by National Natu-ral Science Foundation of China (30770231), Heilongjiang Province Science Foundation for Excellent Youths (JC200704), Agricultural Science and Tech-nology Achievements Transformation Fund Program (2009GB23600514), Key Project of Chinese Ministry of Education (108049), Innovative Program for Importation of International Advanced Agricultural Science and Technology, National Forestry Bureau (2006-4-75), Key Program for Science and Technol-ogy Development of Harbin (2009AA3BS083), Fundamental Research Funds for the Central Universities (DL09EA04), Project for Distinguished Teacher Abroad, Chinese Ministry of Education (MS2010DBLY031) and the Cultivated Funds of Excellent Dissertation of Doctoral Degree Northeast Forestry Univer-sity (grap09).

References

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Table IIThe vdW, electrostatic and total interaction ener-gies (EvdW, Eele and Einter) between 1,8-cineole and the active-site residues of N terminus of N-protein (NTD)a.

Residue Evdw Eele Einter

SerA34 -1.18 -0.12 -1.30 GlnA37 -1.52 -0.31 -1.83 TyrA92 -4.17 -5.53 -9.70 ProA134 -2.79 -0.12 -2.91 PheA137 -1.81 -0.24 -2.05 AspA138 -1.68 -1.35 -3.03 TyrA140 -4.58 -0.08 -4.66 TrpB155 -1.07 -0.06 -1.13

aEnergy units in kcal mol-1.

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Date Received: April 28, 2010

Communicated by the Editor Ramaswamy H. Sarma

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