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Research Express@NCKU - Articles Digest
Research Express@NCKU
Volume 9 Issue 2 - June 5, 2009
[ http://research.ncku.edu.tw/re/articles/e/20090605/4.html ]
How to defense virus infection in fish: grouper
Mx confers resistance to nodavirus and
interacts with coat protein
Young-Mao Chena, Yung-Lin Sua, Pei-Shiuan Shiea, Shao-Ling Huanga,
Huey-Lang Yanga, b, c, and Tzong-Yueh Chena, b, c,*
aInstitute
of Biotechnology, College of Bioscience and Biotechnology
bResearch
Center of Ocean Environment and Technology
cAgriculture
Biotechnology Research Center, National Cheng Kung University, Tainan,
70101, Taiwan
[email protected]
Develepmental and Comparative Immunology 32:825-836, 2008
I
n recent years, with the rapidly developing aquaculture industry, viral
diseases have affected severely many highly valued species such as grouper
causing heavy economic losses in Taiwan and SE Asia. Until now, one
bottleneck of the grouper farming industry is the control of fish disease,
especially the larva stage of grouper that have hazarded by the virus disease
NNV (nerves necrosis virus), these diseases on grouper larva cause over
99.7% fatality rate. But we know that develop the grouper vaccines or
increase immunity could be prevent the diseases.
Fish interferons (IFNs) are the main cytokines induced in the innate
immune response directed against viral infection. Gene products regulated
by IFNs are the major effectors of IFN-mediated biological actions; antiviral
products among the IFN-stimulated genes include dsRNA-activated protein kinase (PKR), guanylate
binding protein (GBP), Mx proteins, and 2′-5′ -oligoadenylate synthetase (OAS). These IFN-inducible
antiviral proteins inhibit viral replication at the levels of penetration, uncoating, mRNA synthesis, protein
synthesis, and assembly. One of IFN-inducible antiviral proteins, Mx proteins are members of the
superfamily of dynamin-like GTPases involved in intracellular membrane remodeling and intracellular
trafficking, and whose primary function is the propensity to self-assemble into defined structures capable of
binding activity. They seem to act as force-generating molecules capable of self-assembly into rings and
stacks of interconnected rings in solution. Mx proteins have been used as molecular markers for IFN
production and virus infection in mammals and fish. In addition, antiviral pathways of Mx-mediated
inhibition of viruses have been identified, in which one of the pathways involving the intrinsic structure of
fish Mx species contributes to the intracellular localization of Mx proteins, with a differing antiviral
spectrum of Mx proteins. The viral protein partner in the Mx-virus interaction correlates with their
specificity of antiviral activity. Various cellular factors that are dependent on the cell type in which the
factor is expressed might account for the contrasting results of antiviral activity. Grouper Mx (gMx) proteins
can inhibit nodavirus propagation. However, molecular mechanism of this antiviral function is unknown,
and the viral target is still poorly characterized.
The available evidence suggests that triggering of IFN synthesis and induction of gMx protein production
may be important in the interaction of nodavirus proteins with the innate immune system. This is expected,
as gMx is induced exclusively by polyinosinic polycytidylic acid (poly [I:C]), accumulates in the cytoplasm of
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cells, and interferes with viral antigens in fish system. Grouper cells that overexpress Mx are highly
resistant to viral infection. However, the role and the antiviral mechanisms of gMx protein in host cell
defenses against nodavirus infection in grouper cells remains unclear.
Our team focuses at the host-virus interaction study over six years. Piscine nodavirus, a member of the
Betanodavirdae family, is the causative agent of viral nervous necrosis (fish encephalitis) that results in high
mortality rates in hatchery-reared larvae and juveniles of marine fishes in Taiwan, Japan, Australia, and
Europe. Betanodaviruses are neuropathogenic and inflicts conspicuous damage characterized by
vacuolation and degeneration of neurons throughout the central nervous system. This piscine nodavirus is a
non-enveloped, icosahedral capsid with a genome comprised of two positive-sense single-stranded RNA
strands: 3.1 kb RNA1 and 1.4 kb RNA2. RNA1 encodes an RNA-dependent RNA polymerase (RdRp), while
RNA2 encodes coat protein. In addition, a subgenomic RNA3 transcribed from the 3’ end of RNA1 encodes
B2 protein. We found nodavirus coat protein which is multifunction, multidomain protein that is the focus
of intense study as an effector for numerous viral functions and the induction of molecular processes.
Nodavirus coat protein influences viral self-assembly and budding during the latter stages of viral
replication. Coat protein contains the signal required for nucleolar localization; through virus infection into
the cells, coat protein was demonstrated to spread within the nucleolus and cytoplasm. Correspondingly,
the transport machinery of the host assists virus replication. Grouper nervous necrosis virus coat protein
contains three domains: (i) basic amino acids residues in the N-terminal region comprising residues 1-50,
which may be involved in the protein-RNA interaction necessary for encapsidation, (ii) N-terminal domain
residues 83-216 that form a β-sandwich that provides structural scaffolding, and (iii) residues 217 to the Cterminus, which likely form a surface-protruding domain involved in specific biological processes such as
host cell recognition. However, while coat proteins are postulated to move from the endoplasmic reticulum
through the nuclear pore complex to the nucleolus, little is actually known about the transport process.
Several functions of Mx protein may contribute to the link between reduced viral yields and protein
association. Fish interferon-induced proteins are a downstream effector molecule of the diverse biological
actions of interferons and are thought to mediate many regulatory functions toward antiviral responses. In
the previous study, we have been cloned the full nucleotide and amino acid sequences of an Epinephelus
coioides Mx cDNA and promoter from nodavirus-infected grouper. The grouper Mx gene has been shown to
be inducible in vivo by injection of live fish with fish pathogen nodavirus. The expression of Mx mRNA
from healthy grouper by RT-PCR showed that the Mx mRNA was constitutively expressed at low levels in
the eye, gills and heart, and minimally expressed in blood, brain, kidney, spleen, muscle, liver and
intestine. The gMx gene transcript began to increase 6 h after nodavirus injection and peaked at 72 h in the
brain. The levels of expression of gMx from brain of a nerves necrosis virus (NNV)-infected grouper over a
96 hours time course. However, the molecular mechanisms of gMx for antiviral function in grouper are less
studied and unclear, these problems will be solved.
Presently, we examined the inducibility and
transcriptional activity of the gMx gene, the cells of
GF-1 treated with dsRNA poly[I:C] or NNV infection
(Fig.1). Therefore, we found that gMx effectively
prevented nodavirus infection in cultured grouper
hepatocytes (GLa), a cell line able to nodavirus
infection and supportive of nodavirus replication.
Because incubation of nodavirus with the stable cell
lines that express gMx-GFP, could be prevented the
infection of nodavirus to the cells, and incubation of
nodavirus with GLa cell lines showed no inhibitory
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effect on nodavirus infection (Fig.2). These results
show that the promoter activity of gMx protein could
be inducible by poly[I:C] and may have the antivirus
activity. In this study, we explored the possibility
that grouper Mx carried out its physiological function
by participating in or interfering with translocation
events. Based on the results supported the Mx
protein may interact with the proteins in NNV.
Specifically, we investigated the hypothesis that
grouper Mx-related induction expression is involved
in coat protein binding and perturbance of its
intracellular localization. The N-terminus of coat
protein contains a functional nucleolus localization
signal (NLS) that is required for many of its activities
including viral RNA replication, package signal,
Fig. 1. Immunological subcellular
localization of grouper Mx proteins in
nodavirus infected or poly[I:C] induced Mx
expressing grouper cells. Grouper Mx proteins
were inducible after treatment with either
nodavirus (MOI=0.1) or poly[I:C] (0.1 mg mL-1) for
24 h. Cells were fixed, stained with polyclonal
rabbit anti-grouper Mx and Texas-Red 594 labeled
translocation, and virion assembly. We used the Farsheep anti-rabbit immunoglobulins.
western blotting to identify NNV proteins which
associated with gMx protein. From Fig. 3, we found that gMx may form a complex with gMx and the coat
protein of NNV in vitro, and the coimmunoprecipitation result also confirmed the association of gMx and
the coat protein of NNV in vivo. Then, from GST pull down assay, we identified and characterized to find
only the effector domain of gMx protein interacted with coat protein. On the other hand, we deleted the Nterminal region of coat protein which all lost the binding activity for gMx. Therefore, we demonstrated that
the antiviral activity of gMx protein was due to the interaction of gMx with nodavirus coat protein. From
these results, we would like to confirm that Mx protein could be directly interacted with the coat protein for
their antivirus function. We also report that gMx interacts with nodavirus proteins, suggesting that the
presence of gMx leads to the repression of viral gene expression. As human Mx retains the ability to binds to
viral components, this observation offers supports for a fish model to study Mx-mediated repression of gene
expression. These results indicate that gMx plays an important role in depleting nodavirus activity by
preventing coat proteins translocate to nucleous (Fig.4).
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Fig. 2. Nodavirus activity of the mx-expressing clones. (A) Cells were seeded in a 96-well plate
until subconfluent growth developed before stimulation with 0.1–10 μg mL-1 poly [I:C] directly added
to culture medium for 24 h before challenge with nodavirus. Cell viability was determined by MTT assay.
(B) Cells grown in 96-well plates were lipofectin-transfected with 1–2 μg mL-1 of poly [I:C] for 24 h
before challenge with nodavirus. Cell viability was determined four days later using the MTT assay. Two
independent experiments were performed in triplicate. (C) Stable Mx-expressing clones were obtained
via transfection with the pcDNA3.1mx-gfp and pcDNA3.1mx-his6 constructs and subsequent neomycin
selection. The expression of Mx protein fused to GFP, and V5/His epitope-tagged grouper Mx proteins
was determined in GF-1 cells. Western blots were performed to detect expression of the Mx-GFP and MxHis6 proteins. The asterisk to the right of the figure indicates an unidentified cross-reacting protein, and
the positions of molecular mass markers are indicated to the left. (D) Following infection with
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nodavirus, the Mx-expressing clone (pcDNA3.1mx-gfp), permanently Mx-expressing clone
(pcDNA3.1mx-his6), and untreated cells (control) were seeded in 96-well plates until subconfluence and
cell viability was determined using the MTT assay. Two independent experiments were performed in
triplicate. (E) Growth rates of Mx-expressing cell lines. Equal numbers of cells from each line were
seeded onto 24-well plates. Cell were trypsinized and counted from three wells for each line every day
after plating. (F) Viability of Mx-expressing cells after nodavirus infection. At each time point, viability
was determined by the trypan blue exclusion method. GF-1 cells were used as a control (squares) for
viability in cells treated with nodavirus. The stable clones of expressing Mx were GF-1 cells containing
the grouper mx-gfp expression plasmid are indicated by the triangles and mx-his6 expression plasmid
are denoted by circles. In panels E and F, the data is representative of one of three independent
experiments (mean ± SEM).
Fig. 3. Interaction between nodavirus coat
protein and grouper Mx protein.
Fig. 4. The potential anti-nodavirus
mechanism of grouper Mx.
Over the past years, piscine nodavirus has devastated the grouper (Epinephelus spp.) culture industry in
Taiwan and other Asian countries and so a better understanding of fish natural defense mechanisms against
such pathogens is needed. We hope that knowledge of grouper Mx protein will contribute to the
understanding of the antivirus molecular mechanism and gene function which associated interferon
signaling molecules in grouper. Furthermore, this study will be provided to understanding the molecular
mechanism and a novel direction which increases the grouper to protective ability against nodavirus in
aquacultural industry.
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