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Research Paper
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Acta Biochim Biophys Sin 2005,37:607-612 |
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doi:10.1111/j.1745-7270.2005.00082.x |
Activation of NF-kB by the Full-length Nucleocapsid Protein of the
SARS Coronavirus
Qing-Jiao LIAO#, Lin-Bai YE*, Khalid Amine TIMANI#, Ying-Chun ZENG,
Ying-Long SHE, Li YE, and Zheng-Hui WU
State Key Laboratory of Virology,
Received:
March 8, 2005
Accepted:
May 19, 2005
# These authors contributed equally to
this work
*Corresponding
author: Tel, 86-27-68752372; Fax, 86-27-68764763; E-mail, [email protected]
Abstract The
severe acute respiratory syndrome coronavirus (SARS-CoV) is the major causative
agent for the worldwide outbreak of SARS in 2003. The mechanism by which
SARS-CoV causes atypical pneumonia remains unclear. The nuclear factor kappa B
(NF-kB) is a key
transcription factor that activates numerous genes involved in cellular immune
response and inflammation. Many studies have shown that NF-kB plays an important role in the pathogenesis of
lung diseases. In this study, we investigated the possible regulatory
interaction between the SARS-CoV nucleocapsid (N) protein and NF-kB by luciferase activity assay. Our results showed
that the SARS-CoV N protein can significantly activate NF-kB only in Vero E6 cells, which are susceptible to
SARS-CoV infection, but not in Vero or HeLa cells. This suggests that NF-kB activation is cell-specific. Furthermore, NF-kB activation in Vero E6 cells expressing the N
protein is dose-dependent. Further experiments showed that there is more than
one function domain in the N protein responsible for NF-kB activation. Our data indicated the possible role
of the N protein in the pathogenesis of SARS.
Key words
severe acute respiratory syndrome coronavirus (SARS-CoV); nucleocapsid
protein; NF-kB
Severe acute respiratory
syndrome (SARS) is a newly emerging infectious disease that has spread to many
countries. The causative agent of SARS has been identified as a novel
coronavirus, namely, the SARS-associated coronavirus [1,2]. The SARS-CoV is an
enveloped, positive-sense RNA virus with a genome comprising about 30,000
nucleotides predicted to contain 1315 open reading frames (ORFs). A sequence
comparison with corresponding ORFs of other known coronaviruses has revealed a
pattern of gene organization similar to typical coronaviruses [3,4]. The high
viral virulence resulting in a significant mortality rate of infected patients
has created widespread scientific interest in understanding the mechanisms of
pathogenicity of this virus.
The SARS-CoV
nucleocapsid (N) protein (NP) is a 46 kDa structural protein and shares little
homology with other members of the coronavirus family. Besides its nucleocapsid
assembly during the viral life cycle, the N protein has also been reported to
activate the activator protein 1 (AP1) signal transduction pathway and induce
apoptosis in COS-1 cells in the absence of growth factors [5,6].
The nuclear factor kB (NF-kB) belongs to
a highly conserved Rel-related protein family, which includes RelA (p65),
RelB, c-Rel, NF-kB1 (p105/p50)
and NF-kB2
(p100/p52). The p50/p65 heterodimer, commonly referred to NF-kB, is the most abundant and ubiquitous. NF-kB is the key transcription factor that activates
many genes involved in cellular immune response and inflammation, such as
interferon-b, tumor
necrosis factor (TNF)-a, interleukin
(IL)-2, IL-6 and IL-8 [7].
It has been reported
that NF-kB plays an
important role in the pathogenesis of many lung diseases [8]. The clinical
symptom of SARS patients is atypical pneumonia characterized by progressive
respiratory failure leading to lung fibrosis and the formation of cysts [9].
In order to understand
the role of NF-kB in SARS-CoV
infection, we studied the regulatory interaction between the SARS-CoV N
protein and NF-kB. Our
results showed that the full-length N protein can significantly increase NF-kB activity only in the Vero E6 cells and this
activation is dose-dependent.
Materials and Methods
Plasmid construction
The recombinant plasmids
with different deletion mutations of the N protein gene were constructed by
inserting the corresponding DNA fragments into the eukaryotic expression vector
pcDNA3 under the immediate early CMV promoter. The DNA fragments were
amplified by PCR from pGEMT-NP [10] using the primers in Table 1.
Primers
Cell culture and transfection
Vero, HeLa and Vero E6
cells obtained from China Centre for Type Culture Collection (Wuhan, China)
were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, USA)
containing 10% fetal bovine serum (Hyclone, Logan, USA) at 37 ºC, with 5% CO2. The DNA transfection
experiment was performed using LipofectamineTM 2000 (Gibco) according to the
manufacturer's instructions.
Western blot analysis
The total cell proteins
were separated by 15% SDS-polyacrylamide gel electrophoresis (PAGE) and
transferred to a polyvinylidene difluoride membrane 24 h after transfection.
The blots were first blocked with 5% non-fat milk in Tris buffer saline (TBS)
containing 0.1% Tween-20, and then probed with the first antibodies, rabbit
anti-N protein poly antibodies or anti-actin antibody (sc-1616; Santa Cruz
Biotechnology, Santa Cruz, USA), for 1 h at 37 ºC. After extensive washing,
secondary antibodies conjugated with horseradish peroxidase (HRP) were applied
onto the blots for at least 1 h at 37 ºC. The blots were washed 5 times with
TBS containing 0.1% Tween-20. Reagents for enhanced chemiluminescence were
applied to the blots and the light signals were detected by X-ray film.
Luciferase activity
assay
The pNF-kB-Luc vector (Stratagene,
Statistical analysis
Data were expressed as
mean+/- SD. The activation of NF-kB was
considered to be statistically significant if the relative luciferase activity
showed an increase higher than 2 folds.
Results
Identification of SARS N
protein and its mutants
The expressions of the N
protein in three cell lines and N protein mutants in the Vero E6 cell line were
measured by Western blotting. As shown in Fig. 1, the N protein was
expressed with the correct molecular weight of 46 kDa in HeLa, Vero and Vero E6
cells [Fig. 1(A)], and N protein mutants were expressed with
corresponding molecular weights (N protein, 46 kDa; N1-225, 25 kDa;
N226-422, 21 kDa; N355-422, 10 kDa;
and N226-300, 9 kDa) in Vero E6 cells [Fig. 1(B)].
The expression levels of different N protein mutants were a little different at
the same transfection concentrations because they interacted differently with
the antibodies (anti-N protein rabbit serum). This phenomenon was also observed
when we performed an indirect-immunofluorescence assay for the localization of
N protein mutants (data not shown).
Activation of NF-kB in Vero E6 cells by N protein
The effect of the N
protein on NF-kB activity in
HeLa, Vero and Vero E6 cells was investigated by the luciferase activity assay.
In Vero E6 cells, the expression of the N protein resulted in a significant
increase in NF-kB activity
compared with the control (Fig. 2). With the increase in the
concentration of pcNP, the NF-kB activity
was 2-fold to 8-fold higher than that of the control [Fig. 3(A)],
clearly showing that activation of NF-kB by the
SARS-CoV N protein is dose-dependent. In contrast, the increase in NF-kB activity in HeLa cells was not obvious, being
only about 2-fold higher than that of the control (Fig. 2). In Vero
cells transfected with pcNP, there was almost no change in the NF-kB activity compared with the control (<2 folds,
Fig. 2). The expression of the N protein in Vero E6 cells transfected
with different concentrations of pcNP plasmids was also detected by Western
blotting. The result clearly showed that the expression level of the N protein
is linearly related to the amount of pcNP plasmid transfected [Fig. 3(B)].
On the basis of these results, we concluded that the N protein can activate NF-kB only in Vero E6 cells and that this activation
is dose-dependent.
Function domain of the N
protein for NF-kB activation
To determine the
function domain of the N protein for NF-kB activation, we constructed four N protein mutants [Fig.
4(A)]. First, we examined two mutants, N1-225 and N226-422. N1-225 contains the
N-terminal part of the N protein with an SR-rich region which may be
responsible for the N protein phosphorylation. N226-422 contains
the C-terminal fragment of the N protein. As indicated in Fig. 4(B),
N226-422 exhibited a higher increase in NF-kB activity. This suggests that the function
domain might be located in the C-terminal of the N protein, so we examined two other
mutants, N226-300 containing the
middle part of the N protein and N355422 containing the C-terminal of the N
protein. We found it very strange that these two mutants both had an increase
in NF-kB activity [Fig.
4(B)]. It seems that all the N protein mutants can activate NF-kB, but the increase in activity in every mutant is
lower than that of the full-length N protein. It has been reported that the N
protein can be cleaved into several small fragments in virus-infected cells by
caspases [11]. NF-kB activation
may be crucial for virus replication or proliferation; to minimize the loss of
the N protein through degradation caused by the host factor, there are several
function domains in the N protein responsible for NF-kB activation and the activation caused by the N protein
is the synergistic effect of all the function domains.
Discussion
In this study, we have
shown that the SARS-CoV N protein significantly activates NF-kB in Vero E6 cells, but not in HeLa or Vero cells,
and that this activation is dose-dependent. This is in accordance with the
results of a previous study, which showed that the SARS-CoV N protein can not
activate NF-kB in both
Vero and Huh-7 cells [5]. This suggests that the activation of NF-kB by the N protein is cell-specific. The Vero E6
cell line is highly susceptible to virus infection. There must be some specific
cellular factors which support SARS-CoV proliferation and participate in the
activation of NF-kB by the N
protein in Vero E6 cells. Further experiments have to be conducted to find
these factors.
In addition, we also
found that the full-length N protein had the highest NF-kB activity. This suggests that there is more than
one function domain in the N protein responsible for NF-kB activation and that the activation caused by the
N protein is a result of the synergistic effect of all the function domains.
Furthermore, the localization of these mutants is different. The full-length N
protein and N355-422 are located mainly
in the cytoplasmid, while N1-225, N226-300 and N226-422 are located in both
the cytoplasmid and nucleolus, and the latter two can also be found in the
nucleoli (data not shown). The mechanism by which NF-kB is activated by these mutants is unclear. Some mutants
may bind directly with the kB binding
site in the promoter of the target gene or form a complex with other nuclear
factors and then bind with the kB binding
site. Other mutants may activate NF-kB through
signal pathways.
Three nuclear
localization signal (NLS) motifs in the N protein were identified using PSORT
II [Fig. 4(A)]. We found that the NF-kB activation caused by the N protein is slightly related
to the NLS motifs. The more NLS motifs the mutant has, the higher the NF-kB activity that can be detected. The full-length N
protein has three NLS motifs and has the highest NF-kB activity, while N1225 and N355422 have only one NLS
motif and have the lowest NF-kB activity.
NLS is a basic amino acid-rich sequence and can be recognized by proteins of
the importin superfamily that mediate transport across the nuclear envelope
[12]. But it is unclear as to how NLS affects NF-kB activation; the basic characteristic of NLS may play an
important role.
NF-kB is a critical regulator of the immediate early
pathogen response, playing an important role in promoting inflammation and
regulating cell proliferation and survival [13]. NF-kB is highly activated at sites of inflammation in diverse
diseases and it induces the transcription of pro-inflammatory cytokines (e.g.
IL-1b, TNF-a and IL-6), chemokines (e.g. IL-8) and adhesion
molecules, including intercellular adhesion molecule 1 (ICAM-1), matrix
metalloproteinases (MMPs), cyclooxygenase 2 (COX2) and inducible nitric oxide
synthase (iNOS) [14]. Because of the multiple functions of NF-kB, many viruses, including several human
pathogens, such as the human immunodeficiency virus (HIV)-1, human T-cell
leukemia virus (HTLV)-1, herpes simplex virus (HSV)-1, hepatitis C virus (HCV)
and Epstein-Barr virus, have evolved different strategies to modulate the activity
of NF-kB [1519].
Some of them modulate the NF-kB activity
through the binding of the viral particles to the NF-kB receptor, while others modulate the activity through
viral proteins. The activation of NF-kB may be a
strategy used by viruses to control the host cells in order to facilitate the
early release of virus progeny or help the emerging virus evade the host immune
system.
NF-kB has been shown to regulate the production of
acute inflammatory mediators in a variety of cells and animal models developed
to elucidate the pathobiology of lung diseases, including acute respiratory
distress syndrome (ARDS), systemic inflammatory response syndrome (SIRS),
asthma, respiratory viral infections, occupational and environmental lung
disease and cystic fibrosis [8]. A high IL-8 expression resulting from NF-kB activation is always observed in cell or animal
models of these lung diseases [2022]. In addition, there are some emerging
clinical data related to NF-kB activation
in the pathogenesis of ARDS, SIRS and asthma. In ARDS and SIRS, NF-kB activation in alveolar macrophages and other
types of lung cells very likely modulates neutrophilic alveolitis and lung
injury [23]. In asthma, NF-kB activation
in airway epithelial cells and other types of cells may affect initiation or
maintenance of the inflammatory phenotype that characterizes the disease [24].
SARS is characterized by
a persistent fever and respiratory symptoms with lung consolidation,
lymphopenia and respiratory failure in life-threatening cases [9]. SARS
sequelae, such as transendothelial migration of polymorphonuclear cells into
the lung tissues, multiple organ dysfunction and ARDS, have been postulated to
be associated with cytokine and chemokine dysregulation [25]. High IL-8 and
IL-2 levels have been observed in SARS patients [26]. This indicates that NF-kB might also play an important role during SARS
infection.
Although the basic
function of the N protein of a virus is to bind with the genome and form a
virus core, many viruses N proteins, such as the HCV core protein and hepatitis
B virus (HBV) X protein, have shown a regulatory effect on NF-kB activation [18,27]. In this article, the
regulatory effect of the SARS-CoV N protein on NF-kB was studied. Our results show that the full-length N
protein activates NF-kB activity
only in Vero E6 cells and that the activation is dose-dependent. This indicates
that the SARS-CoV N protein may be involved in the pathogenesis of SARS and
this finding can be used in the development of therapeutics for the treatment
of SARS.
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