Download Recombinant core proteins of Japanese encephalitis virus as

Virus Genes
DOI 10.1007/s11262-008-0299-9
Recombinant core proteins of Japanese encephalitis virus
as activators of the innate immune response
Shu-O Chen Æ Shih-Hua Fang Æ Dien-Yun Shih Æ
Tien-Jye Chang Æ Jau-Jin Liu
Received: 11 July 2008 / Accepted: 27 October 2008
Ó Springer Science+Business Media, LLC 2008
Abstract Nitric oxide (NO) has been shown to suppress
Japanese encephalitis virus (JEV) RNA synthesis, viral
protein accumulation, and virus release from infected cells.
In this article, the potential viral structural proteins as the
activators of NO product were studied at the molecular
level. First, the genomic region encoding the JEV structural
proteins was cloned into a prokaryotic expression vector
pET for high-level expression. After purification, these
JEV recombinant proteins were added to macrophages to
examine the productions of NO and pro-inflammatory
mediators. In this study, the recombinant core protein, but
not envelope (E), could trigger NO and pro-inflammatory
mediators (TNF-a, IL-6, and IL-12) productions on macrophages. And their effects were about 85–95% relative to
LPS-stimulated macrophages in a dose-dependent manner.
Meanwhile, the rCore-2D could up regulate promoters of
IL-8 and TNF-a via EGFP expression in reporter plasmid
(IL-8p–EGFP and TNF-ap–EGFP)-transfected cells by
flow cytometric analysis. These results suggest that JEV
core protein could regulate pro-inflammatory mediators
and NO production, and may play a crucial role in the
innate immunity for the host to restrict the initial stage of
JEV infection.
S.-O. Chen T.-J. Chang (&)
Department of Veterinary, College of Veterinary Medicine,
National Chung-Hsing University, Taichung 402, Taiwan
S.-H. Fang
Institute of Athletics, National Taiwan Sport University,
Taichung 404, Taiwan
D.-Y. Shih J.-J. Liu (&)
Department of Microbiology, China Medical University,
Taichung 404, Taiwan
e-mail: [email protected]
Keywords Japanese encephalitis virus Core protein Nitric oxide
Introduction
Japanese encephalitis virus (JEV), a member of the family
Flaviviridae, causes acute encephalitis with a mortality rate
up to one-third of infected patients, and nearly half of the
survivors suffer neurological or mental sequelae in humans
[1, 2]. During JEV infection, there is no established antiviral treatment for JEV, and the recovery from viral
infection mainly depends upon host immunity. The antiJEV effect of nitric oxide (NO) has been suggested to be
one of the important factors of innate immunity in controlling the initial stages of JEV infection [2, 3]. However,
recent reports on Japanese encephalitis (JE) show the elevated levels of cytokines and chemokines in hosts are
associated with a poor outcome during JEV infection [4]
and pro-inflammatory mediators released by activated
microglia induce neuronal death in JE [2].
The mechanisms of JE viral replication have been
extensively studied; nevertheless, little is known about the
molecular mechanisms by which viral components modulate host innate immune responses. The genome of JEV is a
single-stranded positive-sense RNA of approximately
11 kb and contains a single open reading frame with the
potential to encode a large polyprotein. The JEV polyprotein is cleaved by both host and viral proteases to yield
at least 10 distinct products in the order of three structural
proteins (core, M, and E), and seven nonstructural proteins
(NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [5, 6].
The envelope glycoprotein (E), with the presence of a
hypervariable region, appears to play a major role in
inducing protective immunity and determining viral
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Virus Genes
(405 bp) was cut from plasmid pJE-S with restriction
enzymes Nco I and Hind III, and then inserted into the
prokaryotic expression vector pET32a. The other deleted
clones which contained 375 and 209 bp of core gene were
subcloned into pET32a vectors individually by restriction
enzymes (BamH I, Bgl II, or Xho I) digestion and named,
pCore/D and pCore/2D, respectively. The 1,500 bp cDNA
fragment containing full-length E gene of JEV was cloned
into pET32c vector and named pET32/E as described in our
previous study [6]. In this study, JEV recombinant proteins
(rCore, rCore/D, rCore/2D, and rE) expressed by prokaryotic expression pET system are schematically represented in
Fig. 1. Furthermore, the derived cDNA fragments of Core/
2D and E genes were re-subcloned into eukaryotic expression vector pLNCX2 (Clontech) by restriction enzymes, and
named pLN-Core/2D and pLN-E, respectively (Fig. 7a).
The expression of inserted gene in each construct was controlled by of CMVIE promoter.
In this study, EGFP reporter plasmids (IL-2p-EGFP,
IL-8p-EGFP, and TNF-ap-EGFP), each containing promoter (IL-2, IL-8, or TNF-a) linked to EGFP (enhanced
green fluorescent protein), were constructed from chloramphenicol acetyltransferase (CAT) reporter DNA (sIL-2CAT, sIL-8-CAT, and sTNF-a-CAT) which had been built
in our laboratory previously, and the CAT gene was
replaced with EGFP gene derived from pEGFP-N1
(Clontech) (Fig. 1b).
All the inserted sequences of constructed plasmids were
confirmed by automated DNA sequencing.
pathogenicity by defining cellular tropism and triggering
apoptosis in infected cells [6–8]. By contrast, the wellconserved core is a small protein (14 kDa) and possesses
multiple functions involved in the formation of the viral
nucleocapsid as well as affliction of viral RNA replication
[9]. West Nile virus (WNV) is a flavivirus that is genetically related very closely to JEV, its capsid protein causing
neuronal death and the release of neurotoxic factors by
infected astrocytes, coupled with pro-inflammatory gene
induction [10]. As regards JEV, which viral protein plays
the role as an activator of innate immune response and
neurovirulence is not determined yet. In this study, we
constructed and expressed recombinant JEV core and E
proteins to investigate whether JEV core or E proteins can
modulate inflammatory components release by activated
macrophages in the absence of the intact virus.
Materials and methods
Plasmid constructs
Prokaryotic expression system, pET-32 series vectors and
Escherichia coli BL21 (DE3) (Novagen) were used for
expression. For cloning core and E genes of JEV, at first
cDNA of structure genes was generated from RNA genome
of JEV with primers JE1F and JE1R by RT-PCR and cloned
into pGEM-T-Easy, named pJE-S [5, 6]. Second, the cDNA
fragment which encoded full-length of JEV core protein
Fig. 1 a Schematic
representation of JEV
recombinant proteins (rCore,
rCore/D, rCore/2D, and rE)
constructed and expressed by
prokaryotic expression pET
system. The gray box indicates
JEV’s protein and the dot line
represents deleted region. b
Three reporter plasmids (IL-2p–
EGFP, IL-8p–EGFP, and TNFap–EGFP) each containing
promoter (IL-2p, IL-8p, or
TNF-ap) linked to EGFP gene
(A)
Structural genes
5’
C
prM
Nonstructural genes
E
NS1
NS2
NS3
5’CGGAAGATAACCATGGCTA3’
5` CGGAAGATAACCATGGCTA 3`
Nco I
3’
Sph I
5` AAACATCAACCCAATCTGCC 3`
Nco I
prM
Cathepsin L NLS
E
295
137 a.a.
(rCore)
1
1
NS5
JE1R primer
JES1R primer
5’CCAGTGTCAGCATGCACAT
3`
JE1F primer
JES1F
primer
C
NS4
794 a.a.
(rE)
115 a.a.
(rCore/D)
1
27
81
115 a.a.
(rCore/2D)
(B) Promoter-EGFP reporter
IL-2p
IL-8p
TNF-αp
123
IL-2p-EGFP
EGFP
IL-8p-EGFP
EGFP
EGFP
TNF-αp-EGFP
Virus Genes
Protein expression and purification
Cell culture
Growth and induction of E. coli were performed essentially according to the recommendations by Studier et al.
[11]. E. coli transformant cells were induced with 1 mM
isopropyl b-thiogalactoside (IPTG) for 5 h. Then bacterial lysates in 10 mM Tris-Cl, pH 8.0 and 150 mM NaCl
were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained
with Coomassie brilliant blue. Furthermore, the six-histidine residues (His-Tag) at N-terminus of expressed
products could be used for affinity chromatography
purification by cobalt-based immobilized affinity chromatography according to TALONTM Metal Affinity
Resins User Manual (Clontech). The cell extract was
isolated from a 100 ml bacterial culture, and the insoluble fraction was dissolved in lysis buffer (8 M urea,
100 mM NaH2PO4, 10 mM Tris, pH 8.0) and then loaded on an equilibrated Talon resin column (Clontech).
The eluted fraction was rapidly desalted with PBS
exchange by repeated centrifugations at 14,0009g using
protein desalting spin columns (NanosepÒ 10 K Centrifugal Filter Devices). Then, these purified recombinant
proteins were analyzed by 10% SDS-PAGE and further
identified by Western blotting analysis.
Peritoneal exudate macrophages were obtained from mice
by lavage with 10 ml of cold HBSS per mouse at 3 days
after intraperitoneal (i.p.) injection of 2 ml 3% thioglycollate in saline (1.5 ml per mouse, Difco, Detroit, MI)
[12]. Cells were seeded in 96-well cluster plates at a density of 2 9 106 cells/ml and incubated at 37°C in
humidified 5% CO2 to allow macrophages adherence. Two
hours later, the non-adherent cells were removed by
washing with warmed PBS and the remaining cells (90%
macrophages, judged by non-specific esterase stain) were
incubated with medium containing various concentrations
of JEV recombinant proteins. Control cells were grown
under identical conditions but were not exposed to
recombinant proteins or lipopolysaccharide (LPS, E. coli
055: B5).
RPMI-1640 medium, Hank’s balanced salt solution
(HBSS), penicillin, streptomycin, L-glutamine, and fetal
calf serum used above were purchased from Invitrogen.
Western blotting
Western blotting analysis was conducted as previously
described [6]. In brief, proteins were separated by 10%
SDS-PAGE and then transferred to nitrocellulose membranes by electrotransfer (Bio-Rad). The monoclonal antihistidine tag antibody was used as first antibody at 1:1000
dilution, and the HRP-goat anti-mouse IgG conjugate
(Zymed) at 1:1000 dilution was used as secondary antibody. Finally, the DAB substrate (GERBU Biotechnik
GmbH, Gaiberg, Germany) was used for color development to reveal the signal.
Mice
Female BALB/c mice were bought from the Animal Center
of the College of Medicine, National Taiwan University
and maintained in the Animal Center of China Medical
University. The animal room was maintained on a 12-h
light-and-dark cycle with a constant temperature and
humidity. All mice were 8 weeks old, sacrificed under
anesthesia, and used to obtain cells from peritoneal exudates. All procedures adhered to the Guide for the Care and
Use of Laboratory Animals (NRC, USA) and were
approved by the laboratory animals committee of China
Medical University.
Cell viability
Mitochondrial respiration-dependent MTT assay was
employed to determine their cytotoxicity [15]. MTT
(Sigma) was a pale yellow substance that was reduced by
living cells to yield a dark blue formazan product. This
process requires active mitochondria, and even fresh dead
cells do not reduce significant amount of MTT. Recombinant proteins (2 lg/ml) were incubated in a 96-well plate
(2 9 106 cells/ml) for 24 h. MTT in PBS (0.1 mg) was
added into each well and then incubated at 37°C for 4 h.
The MTT formazan (1-(4,5-dimethylthiazol-2-yl)-3,5diphenylformazan) crystals formed due to dye reduction by
viable cells were dissolved using acidified isopropanol
(0.1 M HCl) and mixed well at room temperature. After
20 min, index of cell viability was calculated by measuring
the optical density (OD) of color produced by MTT dye
reduction with a microplate reader (BIO-RAD, model
3550, U.S.A.) at 570 nm (OD570-620). The mean OD
value of the content of four wells was used for assessing
the cell viability expressed as percentage of control.
NO determination
The production of NO was estimated from the accumulation
of nitrite (NO2-), which is the end product of NO metabolism. This was achieved by using Griess reagent (Sigma) in
the medium as described by Green et al. [13]. Cells were
incubated with medium containing 2 lg/ml of recombinant
protein for 24 h. Equal volumes of culture supernatant and
Griess reagent (1:1 mixture of 1% sulfanilamide in 5%
phosphoric acid, and 0.1% a-naphthylethylenediamine
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Virus Genes
dihydrochloride in distilled water) were mixed and incubated for 15 min at room temperature. Then the absorbance
was measured at 540 nm in a spectrophotometer and referred to NaNO2 standard curve to determine the nitrate
concentration in supernatants.
systems (BDIS), San Jose, CA) and Modfit LT (Verity
Software House, Topsham, ME).
Cytokine assay
Construction of JEV expression plasmids
Peritoneal exudate macrophages were treated with various
concentrations (4, 2, 1, 0.5 lg/ml) of rCore/2D and LPS for
48 h, following which the cell supernatants were analyzed
for various secreted cytokines using enzyme-linked
immunosorbent assay (ELISA) as previously established in
our laboratory [12]. Serial dilutions of recombinant mouse
tumor necrosis factor (TNF)-a, interleukin (IL)-6, and IL12 (PharMingen, San Diego, CA) were used as the standard. The sensitivity of ELISA of TNF-a, IL-6, and IL-12
is as low as 15.6 pg/ml. Cell viability was assessed by
trypan blue dye exclusion method and was always greater
than 95%.
In order to obtain a high-level expression of JEV structural
proteins, we cloned core and E genes into a prokaryotic
expression system (pET32 vectors), individually. In this
study, three different deleted clones of core genes (pCore,
pCore/D, and pCore/2D) were constructed and their relative positions of amino acids of expression products (rCore,
rCore/D, and rCore/2D) were delineated as shown in
Fig. 1a. The resultant plasmids were described briefly as
follows: the first plasmid, pCore, encoded 137 amino acids
(a. a.) of full-length JEV core protein; the second plasmid,
pCore/D, lacked hydrophobic sequences (116–137 a. a) at
the C-terminus of core protein; the third plasmid, pCore/
2D, had extra-deleted the hydrophobic region (28–80 a. a.)
in the middle of core protein than pCore/D. The cathepsin
L may be capable of cleaving amino acid residues between
Lys18 and arg19 at the N-terminus of capsid protein [14].
The JEV capsid protein possesses two conserved hydrophobic sequences: one is in the center, an internal sequence
of hydrophobic residues surrounded by charged residues
which contain a nuclear localization signal (NLS) and may
have a role in flavivirus capsid protein dimerization [9, 15];
the other is at the carboxyl-terminus which serves as a
signal sequence of prM in most flaviviruses [16] and
ubiquitin-proteins and is likely to bind specifically at the
region to degrade capsid protein in coordination with Jab1
[17]. The pET32/E which encoded full length of JEV E
protein (rE, 295–794 a. a.) was constructed and is also
shown in Fig. 1a.
Statistics
Experiments were repeated independently for three times.
All experimental data are shown as mean ± SD and statistical analysis was performed using a one-way Analysis
of Variance (ANOVA) followed by Dunnett’s post-hoc
test. In all cases, a probability (P) value of the significant
difference was set at *P \ 0.05; **P \ 0.01.
Flow cytometry
GP2-293 cells were seeded in 12-well dish for 1 day and
then transfected with EGFP reporter plasmid (IL-2p-EGFP,
IL-8p-EGFP and TNF-ap-EGFP) by using Qiagen EffectenceTM (New England BioLab, Beverly, MA) according to
the manufacturer’s instructions (BioLab). Briefly, 0.3 lg
plasmid DNA is added with EC buffer to 75 ll and mixed
with 2.4 ll Enhancer at room temperature for 5 min. Then,
the mixture was added 6 ll EffectenceTM reagent and
800 ll culture medium into cells for 10 min at room
temperature. After 24 h transfection, cells were washed
with 19 PBS and added 2 lg rCore/2D then maintained in
culture medium at 37°C, 5% CO2. Further incubation for
48 h, cells were resuspended in PBS and analyzed by flow
cytometry immediately. For each analysis, 5,000–10,000
gated events were collected from both the EGFP (?) and
EGFP (-) cells subpopulation. The EGFP fluorescence was
directly emitted by 488-nm argon-ion laser, and fluorescence emission was read with a 515/40 band pass filter.
Data analysis was performed using CellQuest software
(custom product; Becton Dickinson Immunocytometry
123
Results
Purification and identification of recombinant core
proteins
These plasmids above were transformed into E. coli, BL21
(DE3) for expression. After 5 h of 1 mM IPTG induction,
the cell lysates were analyzed by 10% SDS-PAGE (Fig. 2).
The results show cell lysates of pCore, pCore/D, and pCore/
2D transformants possess prominent stained bands which
correspond to approximate molecular masses of 34, 31, and
25 kDa, respectively (Fig. 2). It was obviously observed
that these recombinant core products which increased
gradually accompanied by hydrophobic region deletions,
particularly in pCore/2D which retain antigenic regions and
absence of all hydrophobic regions of core protein, have
high-level production in prokaryotic system (Fig. 2, lane 8).
Perhaps, this hydrophobic region of core might have a toxic
Virus Genes
1
2
3
4
5
6
7
8
NO concentration (µM per 10^5 cells)
M
kDa
39
34 kDa
31 kDa
25 kDa
26
19
70
57.84
54.68
60
50
40
30
20.13
20
10
5.12
1.03
1.26
0
cell
LPS
rCore
rCore/D
rCore/2D
rE
Treatment
Fig. 2 Expressed core proteins from E. coli transformants are
analyzed by SDS-PAGE and Coomassie brilliant blue staining. Lanes
1 and 2: pET-32a as vector control; Lanes 3 and 4: pCore; Lanes 5
and 6: pCore/D; Lanes 7 and 8: pCore/2D. Lanes 1, 3, 5, 7: cell
lysates without IPTG induction and lanes 2, 4, 6, 8: cell lysates with
1 mM IPTG induction for 5 h at 37°C. M: protein molecular weight
makers. Arrowheads indicate expressed recombinant core proteins
effect on the host cells. Further processed by affinity column
purification, desalting and PBS exchange, these recombinant core proteins (rCore, rCore/D, and rCore/2D) were
separated with SDS-PAGE (Fig. 3a) and identified by
Western blotting analysis (Fig. 3b). In addition, the rCore/
2D production (25 kDa) also increased with time after IPTG
induction in time course analysis (Fig. 3c).
Effects of core protein on the activation of murine
peritoneal excluded macrophages
For investigating the effects of core protein on macrophages, the peritoneal excluded macrophages were cultured
with the same concentration (2 lg/ml) of LPS, rCore,
rCore/D, rCore/2D, and rE proteins, separately. After 24-h
treatment, the NO level in the culture supernatant was
(A)
Fig. 4 Effects of different JEV recombinant proteins on NO synthesis. Murine peritoneal macrophages were stimulated without or with
2 lg/ml LPS, rCore, rCore/D, rCore/2D, and rE, as indicated.
Supernatants were collected after 24 h, and NO release was determined by the method of Griess. The data represent the mean ± SD of
triplicate cultures
determined by Griess method. The results show NO concentrations released from macrophages by PBS, LPS,
rCore, rCore/D, rCore/2D, and rE proteins stimulation are
1.26 lM, 57.84 lM, 20.13 lM, 5.12 lM, 54.68 lM, and
1.03 lM, respectively (Fig. 4). Especially for rCore/2D
(94.5%), the effect of induced macrophages to secrete a
high level of NO is almost the same as LPS (100%). By
contrast, the effect of rE-triggered NO release on macrophages is as low as the basal level of PBS control (Fig. 4).
In this prokaryotic system, the recombinant JEV protein is
produced with a His-tag on its N-terminus. In order to
examine the interference with His- tag, the purified protein
(His-tag, no insert) derived from pET32a (?) transformant
was used as vector’s protein control, and there is no NOresponse in this test (data not shown).
Macrophages activities by rCore/2D
One of the major players of innate immunity is macrophage, and LPS is a potent activator of macrophages and
(B)
(C)
kDa
kDa
M
1
2
3
4
M
1
2
3
4
M
1
2
3
39
26
26
19
Fig. 3 Purification, Western blotting, and time course analysis of
recombinant core proteins. a Affinity column purification of
recombinant core proteins by SDS-PAGE analysis. Lanes 1–4: pET
TrxA-Tag no insert protein as control, rCore (34 kDa), rCore/D
(31 kDa), and rCore/2D (25 kDa) in order. b Western blotting
analysis of recombinant core proteins with His-Tag monoclonal Ab.
Lanes 1–4: the same as figure A. c Time course analysis of rCore/2D.
Lanes: 1–3, cells with 1 mM IPTG induction for 1, 3, and 5 h,
respectively, at 37°C. M: protein molecular weight makers. The
position of rCore/2D is indicated by arrowhead
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Virus Genes
100
1600
80
70.38
57.84
54.68
60
40
25.79
21.97
20
13.46 12.68
10.45
0
4
2
1
0.5
1200
1006
1000
952
768
600
548
438
400
244
200
4
2
1
0.5
cell
1200
LPS
rCore/2D
1529
1400
1253
1200
1126
1000
842
800
698
600
512
465
372
400
200
0
4
2
1
0.5
cell
Concentration (µg/ml)
may stimulate, through Toll-like receptor (TLR) signaling
for inducing pro-inflammatory cytokine and chemokines
production [18, 19]. In order to estimate the macrophages
activities by rCore/2D, macrophages were treated with
various concentrations (0.5, 1, 2 and 4 lg/ml) of rCore/2D
and LPS separately. Supernatants were collected to determine the release of NO, TNF-a, IL-6, and IL-12 after 24and 48-h cultures. The results reveal that rCore/2D-stimulated macrophages secreted NO as well as proinflammatory cytokines (TNF-a, IL-6 and IL-12) of about
85–95% relative to LPS-stimulated cells in a dose-dependent manner (Fig. 5).
Regulation of cytokine/chemokine promoter
by recombinant JEV proteins
In order to examine whether the recombinant JEV proteins
possess regulatory activity on cytokine/chemokine promoters, we constructed three promoter–EGFP reporter
plasmids (IL-2p–EGFP, IL-8p–EGFP, and TNF-ap–EGFP)
to detect EGFP expression cells by flow cytometric analysis in this study. If promoter was activated by recombinant
JEV protein, the EGFP would be expressed in transfected
cells and detected under continuous high-intensity 488-nm
irradiation. The results showed the rCore/2D and rCore/D
could induce EGFP expression in reporter plasmid-transfected cells (IL-8p–EGFP and TNF-ap–EGFP, but not IL2p–EGFP), and the significant numbers of EGFP-expressing cells were 13.4% (rCore/2D: IL-8p–EGFP), 5.3%
(rCore/D: IL-8p–EGFP), 12.2% (rCore/2D: TNF-ap–
EGFP), and 4.1% (rCore/D: TNF-ap–EGFP), respectively,
The level of IL-12 (pg/m
The level of IL-6 (pg/m
895
800
Concentration (µg/ml)
1846
1800
1600
rCore/2D
1260
0
cell
LPS
1400
Concentration (µg/ml)
2000
123
1438
rCore/2D
The level of TNF-a (pg/m
LPS
79.22
The level of NO (µM)
Fig. 5 Comparison of
macrophage activation between
rCore/2D and LPS. Murine
peritoneal macrophages were
stimulated with various
concentrations of rCore/2D and
LPS as indicated. Supernatants
were collected after 24 h for NO
detection. IL-6, IL-12, and
TNF-a levels were determined
after 48 h. The data represent
the mean ± SD of triplicate
cultures
1000
LPS
1003
rCore/2D
852
800
729
622
600
438
400
358
315 298
200
0
143.4
4
2
1
0.5
cell
Concentration (µg/ml)
with curves of EGFP(?) cells (excited at 488 nm) being
slightly more shifted to the right than the EGFP-negative
population (absorbed at 475 nm) (Fig. 6). As to rE, it does
not induce EGFP expression through up-regulation of
cytokine/chemokine promoters in this experiment (Fig. 6).
In prokaryotic expression system, the recombinant JEV
E protein may not possess properly folding and glycosylation statuses. Therefore, we reconstructed JEV eukaryotic
expression vectors to examine the effect of regulation of
cytokine/chemokine promoter by core and E gene of JEV.
In this study, GP2-293 cells were co-transfected with
eukaryotic vector (pLN-Core/2D and pLN-E) and promoter–reporter plasmid (IL-2p–EGFP, IL-8p–EGFP, and
TNF-ap–EGFP), respectively, to examine the effects of
regulation of cytokine/chemokine promoter by flow cytometric analysis. The results also showed that the core gene
could activate promoters of IL-8 and TNF-a but not IL-2;
by contrast, E gene did not possess the ability to activate
the promoters used in this study (Fig. 7).
Discussion
Japanese encephalitis (JE) is the most common form of
viral encephalitis in Asia, and there is no antiviral treatment [1, 2]. The major players of innate immunity are
macrophages and natural killer cells, which could produce
NO and various cytokines for an efficient host defense
response. NO has been suggested to be an important antiviral factors of innate immunity in controlling the initial
stages of JEV infection and shown to inhibit JEV–RNA
Virus Genes
(A)
IL-2p
TNF-α p
IL-8p
PBS
vector
rE
rCore/D
rCore/2D
EGFP
EGFP
EGFP
(B) 16
14
EGFP-cells (%)
Fig. 6 Regulation of cytokine/
chemokine promoters by JEV
recombinant proteins. a JEV
recombinant proteins (rCore/2D
and rE) were added to GP2-293
cells which had transfected with
promoter–EGFP reporter
plasmids (IL-2p–EGFP, IL-8p–
EGFP, and TNF-ap–EGFP),
respectively, then EGFPexpressing cells were detected
by flow cytometry after 48 h
incubation. PBS added to test
cells was as PBS control, and
the purified protein (Trx-tag no
insert) derived from pET vector
transformants was as vector
control. b The quantitation of
relative percentage of EGFPexpressing cells presented in
panel a. Results are shown as
mean ± SD of triplicate
cultures
13.4
12.2
12
PBS
vector
10
rE
8
rCore/D
6
rCore/2D
5.3
4.1
4
2
15. 16. 1.6 1.5 1.4
1.5 1.7 1.6
1.5 1.6 1.6
0
IL-2p
(A)
pLN-E
Core/2D (209 bp)
CMVIE
(B)
TNF-α p
E (1,500 bp)
CMVIE
pLN-Core/2D
IL-2p
IL-8p
TNF-α p
vector
pLN-E
pLN-Core/2D
EGFP
EGFP
(C) 20
18
17.3
16.1
16
EGFP-cells (%)
Fig. 7 Regulation of cytokine/
chemokine promoters by JEV
eukaryotic plasmids, pLN-Core/
2D or pLN-E. a Schematic
presentation of two constructs
(pLN-E and pLN-Core/2D)
containing the JEV core and E
genes and which are controlled
by CMVIE promoter. b GP2293 cells were co-transfected
with JEV eukaryotic construct
(pLN-Core/2D and pLN-E) as
well as promoter–EGFP
reporter plasmid (IL-2p–EGFP
and IL-8p-EGFP and TNF-apEGFP, respectively). After 48 h
incubation, EGFP-expressing
cells were analyzed by flow
cytometry. Plasmid pLNCX2
(no insert) was used as vector
control. c The quantitation of
relative percentage of EGFPexpressing cells presented in
panel b. Results are shown as
mean ± SD of triplicate
cultures
IL-8p
EGFP
vector
pLN-E
pLN-Core/2D
14
12
10
8
6
4
2
0
1.7
1.6
0.5 0.5 0.7
0.4
0.6
IL-2p
IL-8p
TNF-α p
synthesis, protein accumulation, and virus release in
infected cells [2, 3, 20]. The synthesis of antiviral cytokines and chemokines, not only provides a first line of host
defense against infection by generating an intracellular
environment that restricts virus replication, but also signals
the presence of viral pathogens to the adaptive immune
123
Virus Genes
system [21]. Pro-inflammatory mediators (IL-6, IL-12, and
TNF-a) are typical examples of multifunctional cytokines
involved in the regulation of the immune response,
hematopoiesis, and inflammation [4]. Although innate
immunity can be mobilized to combat invading microorganism during the initial phase of infection, an unregulated
pro-inflammatory response could be detrimental in flavivirus encephalitis [22]. The patients with clinical signs of
brain swelling had significantly higher levels of NO, and
there were correlations between levels of pro-inflammatory
cytokines/chemokines (IL-6, IL-8, TNF-a) and fatal outcome during JE, but whether these are simply a correlate of
severe disease or actually contribute to pathogenesis
remains to be determined [4]. Many previous researches on
neurovirulent flaviviruses suggest that the viral envelope
(E) and capsid (core) proteins play important roles in
neuropathogenesis [2, 10, 23]. In this study, we cloned and
expressed JEV structural genes (core and E) to determine
whether above viral proteins can directly trigger innate
immune response by cytokines release from macrophages.
It has been reported that JEV core protein was detected
in both the cytoplasm and nucleoli of the infected cells, and
that the mutant virus defective in the nuclear localization of
capsid protein exhibited impaired viral growth in mammalian cells and neuroinvasiveness in mice [9]. The
expressed WNV capsid protein, which localized to the
nucleolus, was capable of binding to cellular Jab1 protein
and translocating from nucleus to cytoplasm. The C-terminus of capsid protein would allow the ubiquitination
process to occur in the presence of Jab1 and subsequently
induce the degradation of capsid protein [17]. The aminoterminal region of capsid protein including Jab1-binding
motif is well conserved between JEV and WNV, and it has
been shown that JEV capsid protein can be cleaved of the
amino-terminal 18 amino acids by cathepsin L. And this
processing plays important roles in the viral replication in
mouse neuroblastoma and macrophages and in the pathogenesis of encephalitis [14]. In our study, recombinant core
proteins could be cleaved with cathepsin L at the N-terminal region of the capsid protein in the lysosome during
uptake by macrophages through phagocytosis (Fig. 1) and
escape from Jab1-facilitated degradation. Moreover, rCore/
2D lacking NLS in the middle hydrophobic region, almost
remained in cytoplasm rather than in nucleus (data not
shown). Toll-like receptors (TLRs) have a crucial role in
the detection of microbial infection in mammals. Activation of TLR may be involved in protecting the host from
viruses. TLR-activated transcription factors cooperate
to induce a huge number of effector genes, many of
which have been shown to have roles in anti-viral innate
immunity. Several reports reveal that many viral macromolecules can activate macrophages through TLRs,
leading to induction of pro-inflammatory cytokines such as
123
TNF-a, IL-6, IL-8, IL-12 etc. [24–26]. Our results showed
that recombinant JEV core protein, particularly rCore/2D
but not E protein, could trigger a significant increase the
production of IL -6, IL-8, IL-12, TNF-a, and NO in macrophages. Infection with WNV, the major structural protein
E blocks the production of antiviral and pro-inflammatory
cytokines triggering by TLR signaling, and impairs the
innate immune response [27]. Recently, van Marle et al.
reported that WNV core protein plays an important role in
induction of neuroinflammatory genes by activation of glial
cells and leads to neuronal injury and death [10]. These
data show that JEV structural protein (core and E) may
have similar effects as WNV in innate immune response.
Because of JEV replication, products will accumulate in
the cytosol during replication, and the endosomal TLR in
phagocytic cells can detect viral products by phagocytosis
of infected cell or debris from lysed cells. Our previous
study has shown that the transient-expressed JEV E protein
induced apoptosis in Vero cells [6–8] and that the core
protein could cause cells to undergo necrosis (data not
shown). In this study, the recombinant capsid protein may
play as a ligand of TLR and trigger the pro-inflammatory
mediators release in the absence of viral infection or cause
necrotic cells to yield endogenous host-derived products
such as heat shock proteins, and initiate inflammation and
immune response, in contrast to apoptotic cell death, which
does not trigger inflammation. Moreover, the pathway(s)
responsible for activation of macrophages that are dependent and/or independent of TLR signaling by JEV core
protein needs further investigation.
In summary, this is the first study to report that the
recombinant JEV core protein, but not envelope protein,
could trigger the production of pro-inflammatory mediators
on macrophages in the absence of the intact virus. The E
protein appears to play a dominant role in generation of
neutralizing antibodies and induction of adaptive immune
response [28], whereas the core protein initiates the innate
immune response and provides the first line of host defense
in JEV infection. Further understanding of the role played
by viral proteins during JEV infection may lead to new
treatment of JEV-induced immune-mediated encephalitis.
Acknowledgments We thank Professor Min-Liang Wong (Department of Veterinary Medicine, National Chung-Hsing University) for
critical reading of our manuscript, and Dr. Chia-Hsien Chang for
expert technical assistance. This study was supported by CMU95-084
and CMU94-104 of China Medical University, Taiching, Taiwan.
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