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

a b

Thomas Efferth

, Nan Wu

a b

, Yujie Fu

a b

, Gang Yang

a b

, Wei Wang

a b

, Yuangang Zu

a b

&

Key Laboratory of Forest Plant Ecology , Ministry of Education, Northeast Forestry

University , Harbin , 150040 , PR China
b

Engineering Research Center of Forest Bio-preparation , Ministry of Education, Northeast

Forestry University , Harbin , 150040 , PR China
c

Department of Pharmaceutical Biology , Institute of Pharmacy and Biochemistry, University

of Mainz , Mainz , 55099 , Germany
Published online: 15 May 2012.

To cite this article: Zhiwei Yang , Nan Wu , Yujie Fu , Gang Yang , Wei Wang , Yuangang Zu & Thomas Efferth (2010) AntiInfectious Bronchitis Virus (IBV) Activity of 1,8-cineole: Effect on Nucleocapsid (N) Protein, Journal of Biomolecular Structure
and 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)

Anti-Infectious Bronchitis Virus (IBV) Activity

of 1,8-cineole: Effect on Nucleocapsid (N) Protein
http://www.jbsdonline.com

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Abstract
In the present study, anti-IBV (infectious bronchitis virus) activity of 1,8-cineole was studied 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 ribavirin (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 entering 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 binding 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.

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 China
2Engineering

Bio-preparation, Ministry of Education,

Northeast Forestry University, Harbin
150040, PR China
3Department

of Pharmaceutical Biology,

Institute of Pharmacy and Biochemistry,

University of Mainz, Mainz 55099,

Introduction

Germany

Infectious bronchitis virus (IBV), the prototype species of the family Coronaviridae, 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 bronchitis (IB), an acute and highly contagious disease of chickens. IB affects respiratory, kidney and oviduct tissues, resulting in respiratory disease, retarded growth
and reduced egg production (1-2). A number of IBV serotypes have been identified 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 sometime 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.

#These

IBV has a single stranded RNA genome, approximately 27 kb in length, of positive

polarity specifies the production of three major structural proteins: the phosphorylated nucleocapsid (N) protein, the membrane (M) glycoprotein and the surface
(S) glycoprotein (spike protein). The N protein is a highly immunogenic phosphoprotein also implicated in viral genome replication and in modulating cell signaling pathways. The N-terminal of N-protein (NTD) serves as a functional unit

Research Center of Forest

two authors contribute equally to

this work.

*Phone:

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

323

324
Yang et al.

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 distinctive 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 antiIBV medicine (6-10).

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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 foliage. 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 0.5C in the
absence of light.
Cell Cultures
Vero-E6 (African green monkey kidney cells) was purchased from Harbin Veterinary Research Institute (Harbin, P. R. China). The cells were grown in monolayer
culture with Dulbeccos modified Eagles medium (DMEM) supplemented with 10%
fetal calf serum (FCS), 100 U/mL penicillin and 100 g/mL streptomycin. The monolayers 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 37C in an atmosphere of 5 % CO2.
Viruses
The IBV Gray strain was purchased from National Control Institute of Veterinatory 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 -80C.
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 various concentrations (10 mM 0.078 mM) of 1,8-cineole for eight replicates and
incubated at 37C 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

325

added to each well, after incubated at 37C for 4 h, remove the supernatants, then
200 L DMSO was added and incubated at 37C 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 doseresponse curves generated from the data.

Anti-Infectious Bronchitis
Virus Activity

Anti-IBV Activity

CH3

Inhibition of virus replication was measured by MTT method. Serial dilution of

the treated virus was adsorbed to the cells for 1 h at 37C. 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 37C, the cultures were measured by MTT method as described above. The concentration of 1,8cineole and ribavirin which inhibited virus numbers by 50% (IC50) was determined
from dose-response curves.

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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 1,8cineole 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 37C. The compound was aspirated 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 antiIBV 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 37C,
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.
Table I
Anti-IBV activity of 1,8-cineole compared with ribavirin.
IBV (Gray strain)
Compound

CC50a (mM)

TD0b (mM)

IC50c (mM)

SId

1,8-cineole
Ribavirin

>10.0
>1.0

3.90 0.22
0.78 0.15

0.61 0.07
0.118 0.02

>16.39
>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. CC was the concentration that showed 50% cytotoxic
50
effects in Vero cells. TD0 was the concentration that showed nontoxic maximum effects in Vero cells.
cAntiviral activity was determined by MTT assay. IC was the concentration that inhibited 50% of IBV
50
replication in Vero cells.
dThe selective index (SI) was calculated as CC /IC .
50
50

H 3C

CH3

Figure 1: Chemical structure

of 1,8-cineole.

326
percent inhibition of virus (%)

100

Yang et al.

90

1,8-Cineole

80

Ribavirin

70
60
50
40
30
20
10
0

pretreatment virus

adsorption

pretreatment cell

replication

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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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 -1. The X-ray crystallography structure of the Nterminal 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 interactions 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).

(A)

(B)
0.8

0.6
1000

RMSD ()

Total energies (kcal mol-1)

1200

800

0.4

0.2
600
0.0
0

1000

2000

3000

MD simulation time (ps)

4000

5000

1000

2000

3000

4000

5000

MD simulation time (ps)

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.

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 examined. 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.

327
Anti-Infectious Bronchitis
Virus Activity

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.

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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%, respectively. 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-cineole is represented by ball and stick model. N
terminus of N-protein (NTD) is in color ribbon: 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, colored by electrostatic potentials. Key residues
are represented by stick models. The oxygen,
nitrogen, carbon, hydrogen in model are colored in red, blue, green and white, respectively. The important H-bond is labeled in the
dashed black line.

328
Yang et al.

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 determinant for the replication of IBV virus (6, 8, 10). Hence, we conclude that the function of N protein may be suppressed by 1,8-cineole.

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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 rootmean-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
-sheet surrounded with several loop regions. The active site that binds to 1,8cineole is mapped to the loop region on the top of the -sheet, near the RNA binding 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 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-cineole holds the residues SerA34, GlnA37, TyrA92, ProA134, PheA137, AspA138,
TyrA140 and TrpB155 and hinders the binding of RNA with NTD. The interaction 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).

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.

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In conclusion, 1,8-cineole possesses anti-IBV properties via embarrassing the binding 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 supplemental 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 Natural Science Foundation of China (30770231), Heilongjiang Province Science
Foundation for Excellent Youths (JC200704), Agricultural Science and Technology 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 Technology 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 University (grap09).
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Anti-Infectious Bronchitis
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Table II
The vdW, electrostatic and total interaction energies (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
GlnA37
TyrA92
ProA134
PheA137
AspA138
TyrA140
TrpB155

-1.18
-1.52
-4.17
-2.79
-1.81
-1.68
-4.58
-1.07

-0.12
-0.31
-5.53
-0.12
-0.24
-1.35
-0.08
-0.06

-1.30
-1.83
-9.70
-2.91
-2.05
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-4.66
-1.13

aEnergy

units in kcal mol-1.

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Date Received: April 28, 2010

Communicated by the Editor Ramaswamy H. Sarma