- Email: [email protected]
Antiviral role of grouper STING against iridovirus infection
Accepted Manuscript Antiviral role of grouper STING against iridovirus infection Youhua Huang, Zhengliang Ouyang, Wei Wang, Yepin Yu, Pengfei Li, Sheng Zhou, Shina Wei, Jingguang Wei, Xiaohong Huang, Ph D., Professor, Qiwei Qin, Ph D., Professor PII:
S1050-4648(15)30139-X
DOI:
10.1016/j.fsi.2015.09.014
Reference:
YFSIM 3602
To appear in:
Fish and Shellfish Immunology
Received Date: 9 August 2015 Revised Date:
3 September 2015
Accepted Date: 5 September 2015
Please cite this article as: Huang Y, Ouyang Z, Wang W, Yu Y, Li P, Zhou S, Wei S, Wei J, Huang X, Qin Q, Antiviral role of grouper STING against iridovirus infection, Fish and Shellfish Immunology (2015), doi: 10.1016/j.fsi.2015.09.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Antiviral role of grouper STING against iridovirus infection
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Youhua Huang#, Zhengliang Ouyang#, Wei Wang, Yepin Yu, Pengfei Li, Sheng Zhou,
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Shina Wei, Jingguang Wei, Xiaohong Huang**, Qiwei Qin*
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Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea
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Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road,
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Guangzhou 510301, China
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*Corresponding author:
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Qiwei Qin, Ph D., Professor,
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Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea
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Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road,
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Guangzhou 510301, China.
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Phone & Fax: +86-20-89023638, E-mail: [email protected]
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Xiaohong Huang, Ph D., Professor,
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Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea
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Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road,
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Guangzhou 510301, China.
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Phone & Fax: +86-20-89023197, E-mail: [email protected]
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#These authors contributes equally to this work.
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Abstract Stimulator of interferon genes (STING, also known as MITA, ERIS, MPYS or
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TMEM173) has been identified as a central component in the innate immune response
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to cytosolic DNA and RNA derived from different pathogens. However, the detailed
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role of STING during fish iridovirus infection still remained largely unknown. Here,
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the STING homolog from grouper Epinephelus coioides (EcSTING) was cloned and
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its effects on IFN response and antiviral activity were investigated. The full-length
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EcSTING cDNA was composed of 1590 bp and encoded a polypeptide of 409 amino
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acids with 80% identity to STING homolog from large yellow croaker. Amino acid
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alignment analysis indicated that EcSTING contained 4 predicated transmembrane
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motifs (TMs) in the N terminal, and a C-terminal domain (CTD) which consisted of a
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dimerization domain (DD), c-di-GMP-binding domain (CBD) and a C-terminal tail
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(CTT). Expression profile analysis revealed that EcSTING was abundant in gill,
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spleen, brain, skin, and liver. Upon different stimuli in vivo, the EcSTING transcript
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was dramatically up-regulated after challenging with Singapore grouper iridovirus
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(SGIV), lipopolysaccharide (LPS) and polyinosin-polycytidylic acid (poly I:C).
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Reporter gene assay showed that EcSTING activated ISRE, zebrafish type I IFN and
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type III IFN promoter in vitro. Mutant analysis showed that IFN promoter activity
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was mostly mediated by the phosphorylation sites at serine residue S379 and S387.
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Moreover, EcSTING induced type I and III IFN promoter activity could be impaired
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by overexpression of EcIRF3-DN or EcIRF7-DN, suggesting that EcSTING mediated
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IFN response in IRF3/IRF7 dependent manner. In addition, the cytopathic effect (CPE)
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ACCEPTED MANUSCRIPT progression of SGIV infection and viral protein synthesis was significantly inhibited
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by overexpression of EcSTING, and the inhibitory effect was abolished in serine
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residue S379 and S387 mutant transfected cells. Together, our results demonstrated
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that EcSTING might be an important regulator of grouper innate immune response
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against iridovirus infection.
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Key words: STING; Grouper; Interferon; SGIV; Viral replication
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1. Introduction Increased reports demonstrated that stimulator of interferon genes (STING, also
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known as MITA, ERIS, MPYS or TMEM173) functioned as a central and
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multifaceted mediator in innate immune response and attracted much attention in
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recent years [1-3]. As the cytosolic DNA sensor, STING initiates a cascade of events,
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including moving from the ER to the Golgi, activating the cytosolic kinases,
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TANK-binding kinase 1 (TBK1) and IκB kinase (IKK) which phosphorylate the IFN
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regulatory factor 3 (IRF3) and transcription factors nuclear factor κB (NF-κB) [4-6].
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These phosphorylated transcription factors induce type I IFN and pro-inflammatory
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cytokine production, and then trigger the host immune response. Meanwhile, STING
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could be triggered to recruit STAT6 to the endoplasmic reticulum, leading to STAT6
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phosphorylation on Ser (407) by TBK1 and Tyr (641), independent of JAKs [7]. In
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addition to its established role as a signaling adaptor in the IFN response to cytosolic
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DNA, STING also functions as a direct pattern recognition receptor (PRR) for cyclic
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dinucleotides [5], such as cyclic diguanylate monophosphate (c-di-GMP) and cyclic
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diadenylate monophosphate (c-di-AMP) [8]. Although the roles of STING in
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mammalian innate immune responses were detailed explored, few work focused on
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fish until now [9-11]. Recent studies indicated that overexpression of crucian carp
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STING induced zebrafish IFN1 and IFN3 promoter activation dependent on IRF3 or
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IRF7 [9], and silencing of zebrafish STING significantly inhibited DNA
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virus-induced antiviral responses, and the inhibitory effect was determined by a
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conserved serine residue (S373) [11].
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ACCEPTED MANUSCRIPT Groupers, Epinephelus spp. are widely cultured in China and Southeast Asian
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countries. However, the emergence of iridoviral pathogens caused heavy economic
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losses in grouper aquaculture [12, 13]. Singapore grouper iridovirus (SGIV) was
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isolated from diseased groupers and caused more than 90% mortality in grouper and
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sea bass [14]. As a large DNA virus, SGIV infection in vitro induced a novel type of
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programmed cell death-paraptosis [15], and MAPK signaling pathway was
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demonstrated to be involved in SGIV infection [16]. In addition, our previous studies
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demonstrated that SGIV infection could regulate the expression of a certain interferon
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stimulated or induced genes, suggested that interferon stimulated genes might exert
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crucial roles during SGIV infection [17]. Up to now, only two fish STING
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orthologues have been cloned from freshwater fish, including crucian carp and
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zebrafish [9, 11, 18]. However, little work has been performed on the role of STING
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from marine fish in response to virus infection.
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In the present study, we cloned an STING homolog from grouper and evaluated
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the roles of EcSTING during SGIV infection. Our data will provide new insight into
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the function of fish STING during DNA virus infection.
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2. Material and methods
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2.1 Fish, cell and virus.
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Orange-spotted groupers, E. coioides, about 50 g in body weight, were purchased
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from a fish farm in Hainan province, China. Fishes were kept in a laboratory
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recirculating seawater system at 25°C for two weeks before use. Grouper spleen cells
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(GS) were maintained in Leibovitz’s L-15 medium supplemented with 10% fetal 5
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bovine serum (FBS) (Gibco, USA) at 25°C [19]. Singapore grouper iridovirus (SGIV)
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was propagated in GS cells and the virus stocks were stored at 80°C until used.
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2.2. Cloning of EcSTING and bioinformatic analysis Based on the EST sequences of EcSTING from grouper spleen transcriptome
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annotation [17], the full length of the EcSTING cDNA was amplified with a SMART
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RACE cDNA amplification kit as described previously [20]. To explore the structural
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characteristics of EcSTING, the obtained nucleotide sequence was edited and
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analyzed using the WWW BLAST server. The transmembrane domain prediction was
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carried out using TMHMM program. The potential serine phosphorylation sites were
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predicted using NetPhos program. Multiple amino acid sequences alignment was
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carried out using ClustalX 1.83 and edited with GeneDoc software.
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2.3. Expression profiles of EcSTING in healthy and challenged grouper
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To detect the tissue distribution pattern of EcSTING in healthy orange-spotted
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grouper, total RNA was extracted from head kidney, heart, liver, spleen, intestine,
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muscle, brain, skin, gill, stomach and kidney, respectively, using the SV Total RNA
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Isolation Kit (Promega) according to manufacturer’s instructions. Expression of
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EcSTING in different tissues was determined by quantitative real-time PCR
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(qRT-PCR) using primers shown in Table 1.
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To determine the expression profiles of EcSTING upon challenge, groupers were
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injected with PBS, SGIV, poly I:C and LPS as described previously [20]. At indicated
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time points, the spleen of different groups (n>3) were collected for RNA extraction
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and qRT-PCR analysis. 6
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2.4. Plasmid construction To study the roles of EcSTING in vitro, the full length of EcSTING cDNA was
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PCR amplified and subcloned into the vector pEGFP-N3 or pcDNA3.1-HA. All the
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primers were listed in Table 1. The constructed plasmids pEGFP-EcSTING and
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HA-EcSTING were subsequently confirmed by DNA sequencing. To further identify
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the potential roles of putative phosphorylation residues in EcSTING, we generated 6
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EcSTING mutants in which serine (S) residues were mutated to alanine (A) either
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individually, or two adjacent serines in combination. All the mutants were cloned into
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pcDNA3.1-HA vector and confirmed by DNA sequencing.
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2.5. Cell transfection
Cell transfection was performed using Lipofectamine 2000 reagent (Invitrogen)
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as described previously [20]. In brief, GS cells were grown in 24-well plates, and
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incubated with the mixture of Lipofectamine 2000 and plasmids for 6 h. After
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replacing the fresh medium, cells were cultured at 25°C for further analysis.
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To generate stable cells overexpressing EcSTING or its mutants, the recombinant
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plasmids were transfected into GS cells and then cells were incubated with medium
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containing 800 ng/ml G418 (Gibco). After propagation under selective pressure for 4
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weeks, cells stably expressing EcSTING or its mutants were obtained for further
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analysis. Cells transfected with pcDNA3.1-HA was chosen as control.
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2.6. Subcellular localization
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To elucidate the subcellular localization of EcSTING in vitro, pEGFP-N3 and
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pEGFP-EcSTING were transfected into GS as described above. At 24 h post 7
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transfection, cells were incubated with ER-tracker and Hochest 33342 for observation
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under fluorescence microscopy.
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2.7. Reporter gene assay To evaluate the effects of wild type EcSTING and mutants on the promoter
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activity of ISRE and zebrafish IFN, luciferase activity assays were carried out as
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described previously [21]. In first set of experiment, GS cells were cultured in 24-well
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plates, and then cotransfected with 0.4 µg ISRE-Luc, DrIFN1-Luc or DrIFN3-Luc and
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0.4 µg EcSTING or its mutants using Lipofectamine 2000 as described above. A total
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of 0.05 µg pRL-TK was included to normalize the expression level. For the second set
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of
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EcIRF3/EcIRF3-DN/EcIRF7/EcIRF7-DN/empty vector and 0.4 µg DrIFN1-Luc or
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DrIFN3-Luc, respectively. At 48 h posttransfection, the transfected cells were
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harvested and lysed according to the Dual-Luciferase Reporter Assay System
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(Promega). Luciferase activities were measured using a Victor X5 Multilabel plate
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reader (PerkinElmer). The results were representative of three independent
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experiments, and each independent experiment was performed in triplicate. Luciferase
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activities were expressed as the fold relative to the empty vector transfected cells.
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2.8. Quantitative real-time PCR (qRT-PCR) analysis
cells
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experiment,
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The expression of target genes, including EcSTING, grouper Mx-I, ISG15,
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viperin and β-actin (internal control) were detected by qRT-PCR using the SYBR
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Green realtime PCR Kit (Toyobo) according to the manufacturers’ instructions. In
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brief, after reverse transcription, qRT-PCR was carried out in a Roche 480 Real Time 8
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Detection System (Roche, German) as described previously [20]. The relative
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expression level of virus genes was analyzed using typical Ct method (2
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[22]. The data were calculated as the folds based on the expression level of targeted
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genes normalized to β-actin at the indicated time points. The Data were represented as
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mean ± SD, and the statistic analysis were performed using SPSS software.
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2.9. Immune fluorescence microscopy
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method)
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immune fluorescence was carried out using SGIV VP19 antibody as described
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previously [23]. In brief, cells were seeded into 24-wells plate and then transfected
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with plasmids pEGFP-N3 or pEGFP-EcSTING. At 24 h post transfection, cells were
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infected with SGIV for another 24 h and fixed with 4% paraformaldehyde. After
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blocking with 2% bovine serum albumin (BSA), cells were incubated with anti-VP19
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(1:100), followed by Rhodamine-conjugated goat anti-mouse antibodies (Pierce).
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Finally, samples were stained with 1 µg/ml 6-diamidino-2-pheny-lindole (DAPI), and
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observed under fluorescence microscopy (Leica, Germany).
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2.10. Virus titer assay
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To investigate the effect of wild type EcSTING, S379 mutant or S387 mutant
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overexpression on SGIV production, virus titer assay was carried out as described
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previously [16]. Briefly, the stable cell lines were cultured in 24-well plates separately
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and infected with SGIV at multiplicity of infection (MOI) of 1. At 48 h p.i.,
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virus-infected cell lysates were harvested, and viral titers were determined by 50%
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tissue culture infectious does (TCID50) assay (Reed & Muench, 1938). 9
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3.1. Results
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3.1. Sequence characterization of EcSTING Using the RACE method, the full length cDNA of EcSTING which was
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composed of 1590 bp was obtained in this study. It contained a 1227 bp open reading
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frame (ORF) encoding a 409-aa protein (Fig. 1). Blastp analysis showed that
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EcSTING shared 80%, 76%, 46%, and 46% homology with the known STING
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sequences of large yellow croaker (Larimichthys crocea), Oreochromis niloticus,
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zebrafish and Carassius auratus, respectively. Amino acid alignment showed that
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EcSTING contained four predicted transmembrane motifs (TMs) in the N terminal
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and a C-terminal domain (CTD) which consisted of a dimerization domain (DD),
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c-di-GMP-binding domain (CBD) and a C-terminal tail (CTT). In addition, the critical
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serine residues S358 and S366 in human STING which were essential for
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TBK1-mediated phosphorylation of STING and STING-dependent IRF3 activation
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were also conserved in EcSTING and other fishes (Fig. 2).
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3.2. Tissue distribution and expression pattern of EcSTING
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The expression of EcSTING mRNA in various tissues was determined by
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qRT-PCR. As shown in Fig. 3, a constitutively expression of EcSTING was obviously
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observed in most examined tissues, including gill, spleen, brain, skin, liver, muscle,
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heart, kidney, head kidney, intestine. The predominant expression was detected in the
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gill, spleen, brain, skin and liver (Fig. 3A).
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To explore the expression of EcSTING after challenged with different stimulates,
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the transcript of EcSTING was determined at the indicated time after challenge with 10
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EcSTING was increased up to 32- and 165-fold in LPS and poly I:C challenged fish
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spleen at 6 h post injection compared with 0 h post injection. Similar results were
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observed in SGIV infected groupers, and the expression of EcSTING was
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significantly increased up to 62-fold at 12 h post injection. With the infection time
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EcSTING expression gradually decreased, and its expression decreased to the basic
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level at 96 h post-injection.
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3.3. The subcellular localization of EcSTING
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STING contains transmembrane motifs in the N-terminus, which predominantly
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anchors itself in the endoplasmic reticulum (ER) or the outer membrane of
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mitochondria [1, 24]. To explore the subcellular localization of EcSTING in vitro,
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pEGFP-N3 or pEGFP-EcSTING was transfected into GS cells and then stained with
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ER-tracker. As show in Fig. 4, the green fluorescence omitted from pEGFP-EcSTING
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transfected cells showed three forms with the transfection time increased, including
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evenly distribution, punctuates and bright aggregates. All the green fluorescence was
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overlapped with the red fluorescence from ER tracker red. Differently, in pEGFP-N3
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transfected cells, the green fluorescence was distributed throughout the cells. Thus,
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EcSTING was proposed to encode an ER-localized protein.
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3.4. Ectopic expression of EcSTING induced ISRE and IFN promoter activity
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Due to the structural similarity between EcSTING and mammalian STING, we
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firstly examined the effect of EcSTING overexpression on ISRE and IFN promoter
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activity in grouper cells. As shown in Fig. 5, overexpression of EcSTING could 11
ACCEPTED MANUSCRIPT induce a strong activation of ISRE-Luc by up to 27-fold compared to the control
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vector pcDNA3.1-HA. In addition, the promoter activity of zebrafish type I IFN
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(DrIFN1-Luc) and type III IFN (DrIFN3-Luc) were significantly increased in
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EcSTING transfected cells compared to pcDNA3.1-HA transfected cells. In detail, the
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luciferase activity of DrIFN1-Luc and DrIFN3-Luc was increased up to 11-folds and
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6-folds in EcSTING transfected cells, respectively. This result indicated that
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EcSTING was capable of activating fish IFN response in vitro. has
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phophorylation-dependent manner. Using NetPhos program, we predicted the putative
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phophorylation sites of EcSTING. The 6 EcSTING mutants in which one or two
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potential phosphorylation serine residues were mutated to alanines were constructed.
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The potential roles of these serine residues in controlling EcSTING function were
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examined using luciferase reporter assays. As shown in Fig. 6, mutation of S379 and
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S387 to alanine significantly reduced the activation of ISRE promoter, while the
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mutation of S73, S143 and S148, S195 and S200, S248, S277 and S281 to alanine
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displayed similar effects to that of EcSTING wild type. The results suggested that
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S379 and S387 might be important for EcSTING-mediate IFN signaling. To further
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determine which phophorylation sites were crucial in IFN response induced by
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EcSTING, the S379 or S387 mutants were constructed, respectively. Interestingly, the
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ability of activate ISRE-Luc, zebrafish DrIFN1-Luc and DrIFN3-Luc promoter were
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both significantly decreased in S379 and S387 mutant transfected cells, in compared
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to wild type EcSTING transfected cells. Especially in S387 mutant transfected cells,
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the activity of all these three promoters were almost completely abolished like control
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vector transfected cells. Taken together, the results indicated that S379 and S387
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exerted crucial roles in EcSTING induced IFN immune response. In addition, we also evaluated the effects of EcSTING and S379 or S387 mutants
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on the expression of IFN related genes, including ISG15, MX-I and viperin. As shown
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in Fig. 7, the transcripts of all examined genes were significantly induced by wild type
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EcSTING, compared to the control vector. Differently, ability of inducing IFN related
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genes expression were obviously inhibited in S379 and S387 mutant transfected cells,
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and inhibitory effect evoked by S387 mutant was stronger than by S379 mutant.
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3.5. EcSTING activated IFN immune response via IRF3 and IRF7 The previous studies showed that EcIRF3 and EcIRF7 could induce the
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promoters activity of DrIFN1-Luc or DrIFN3-Luc [25, 26]. Our results also indicated
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that overexpression of EcIRF3-DN and EcIRF7-DN could inhibit the activation of
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IFN1 and IFN3 promoter induced by EcIRF3 and EcIRF7, respectively (Fig. 8). In
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order to further understand whether IRF3 or IRF7 mediated the activating fish IFN
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response induced by EcSTING in vitro, we cotransfected EcSTING construct,
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EcIRF3-DN (or EcIRF7-DN) and DrIFN1-Luc (or DrIFN3-Luc) in GS cells,
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respectively. As shown in Fig. 8, EcSTING-induced IFN promoter activities were
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significantly impaired in GS cells when cotransfected with either EcIRF3-DN or
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EcIRF7-DN. However, the inhibitory effect of EcIRF7-DN was weaker than that of
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EcIRF7-DN. Thus, our results indicated that EcSTING activated IFN response via
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both IRF3 and IRF7, but IRF3 exert more crucial roles.
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3.6. Overexpression of EcSTING inhibited SGIV replication To determine the roles of EcSTING during iridovirus infection, we examined the
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viral protein synthesis in SGIV infected EcSTING expressing cells. As shown in Fig.
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9, severe cytopathic effect (CPE) was observed in SGIV infected control cells at 48 h
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p.i., and no obvious CPE was observed in infected EcSTING-transfected cells.
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Moreover, EcSTING overexpression obviously inhibited the synthesis of viral
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structural protein VP19 in compared to control cells. The virus production was also
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evaluated using cells stably expressed EcSTING and pcDNA3.1-HA. Consistently,
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EcSTING overexpression evoked the reduction of virus titer from 7.78-Log
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TCID50/ml to 6.83 -Log TCID50/ml. In contrast, in S379 or S387 mutants expressing
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cells, the inhibitory effect on virus titer were not detected. Together, the EcSTING
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exerted important antiviral roles during iridovirus infection, and the antiviral effects
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were determined by the critical residues S379 or S387 at the phosphorylation sites
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(Fig. 9).
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4. Discussion
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Increased reports have demonstrated that STING is a crucial component in the
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innate immune response to cytosolic DNA and RNA derived from different pathogens,
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including viruses, bacteria and parasites [5, 27, 28]. During the co-evolution,
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pathogens, such as gammaherpesviruses, could block cGAS-STING-mediated
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antiviral immunity by encoding inhibitors, and modulate this pathway for viral
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transmission in the human population [29]. To our knowledge, only several reports
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focused on the roles of STING in fresh water fish [18, 9, 11]. To better understanding
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the function of STING in teleost, a STING gene from marine fish, grouper, was
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identified and characterized in this study. Bioinformatics analysis showed that EcSTING contained 4 predicted TM
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domains in the N terminal. TM domains of human STING are not only essential for
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its localization and dimerization [30], but also play important roles during its
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interaction with MAVS to active IRF3 and induce IFNs [24]. The amino acid
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sequences in the regions of former 4 TM domains were highly divergent between fish
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and mammals, whether these TM domains exert different roles remained unclear. In
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our study, EcSTING expression could be detected in all tissues tested, and a high
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level expression was found in gill, brain, skin, pleen, liver. Upon challenge with poly
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(I:C), LPS and SGIV, EcSTING expression could be differently regulated at various
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stages of chanllenge, suggested that EcSTING might mainly play vital roles in host
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immune response against different pathogens.
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Studies from mammals indicated that overexpression of STING led to strong
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induction of IFN immune response [1, 24, 30]. After phosphorylation, the adaptor
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proteins STING could activate the downstream protein kinase TBK1, which
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phosphorylates the transcription factor IRF3, and then drives type I IFN production
18
[31]. In this study, the ectopic expression of EcSTING in vitro induced the activation
19
of ISRE and IFN promoter activity. The expressions of a certain interferon induced
20
genes, including ISG15, MX and viperin were also significantly increased in
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EcSTING overexpressing cells, suggested that EcSTING also mediated IFN immune
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response like mammals. In addition, we also found that IFN promoter activated by
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EcSTING overexpression could be significantly inhibited by IRF3-DN or IRF7-DN
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co-transfection, suggesated that EcSTING activated IFN signaling dependent on
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functional IRF3 or IRF7. In response to different stimulation, STING functioned in a phosphorylation
5
dependent manner [3]. In human STING, mutation of S280, S358, or S366 to alanine
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affected the TBK1-mediated phosphorylation of STING. Further studies indicated that
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S358 in STING was an important target residue phosphorylated by TBK1 and also
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contributeed to the aggregation of STING, while conserved residue S366 was
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identified as a key amino acid required for STING-dependent IRF3 activation and
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subsequent IFN induction [31, 32]. Our results showed that serine residues S379 and
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S387 were critical for EcSTING induced IFN promoter activation. Moreover, the
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ability of inducing IFN related gene expression was significantly decreased after in
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serine residues S379 and S387 mutant transfected cells. We speculated that S379 and
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S387 in EcSTING might exert similar roles like S358 and S366 from human STING.
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It has been reported that overexpression of mammalian STING decreases
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intracellular viral load during Japanese encephalitis virus infection [33]. Similarly,
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ectopic expression of crucian carp and grass carp STING in vitro significantly inhibits
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virus replication through activating IRF3/7-dependent IFN response [9, 10]. In
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addition, overexpression of zebrafish STING induced a strong anti-viral immunity
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against both RNA and DNA viruses [18, 11]. In the present study, the effects of
21
EcSTING expression on iridovirus replication were also investigated. Our results
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showed that the ectopic expression of EcSTING delayed the CPE progression induced
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Moreover, the antiviral activity was dependent on the critical residue S379 and S387.
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In zebrafish, a conserved serine residue (S373) was essential for the action of zSTING
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which could mediate DNA virus-induced antiviral responses [11]. Although STING
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from zebrafish and grouper shared only 46% identity, the amino acid alignment
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showed that serine residue at DrSTING (S373) and EcSTING (S387) was conserved
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as well as human STING (S366). Whether S379 and S387 exert more regulatory roles
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during the antiviral actions of EcSTING needed further investigation.
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In summary, we characterized a functional STING homolog from marine fish,
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which contained evolutionarily conserved domains like mammals. The transcription
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of EcSTING was induced in vivo upon different stimuli, including poly (I:C) and
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SGIV and LPS. Overexpression of EcSTING in vitro induced type I and III IFN
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promoter activity which was mediated by IRF3 or IRF7. Furthermore, SGIV
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replication was significantly inhibited by EcSTING overexpression in GS cells.
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Together, our results shed new lights on understanding the roles of STING in fish
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immune response against iridovirus infection.
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Acknowledgment
This work was supported by grants from the National Basic Research Program of
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China (973) (2012CB114404; 2012CB114402), the National High Technology
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Development Program of China (863) (2014AA093507), the National Natural Science
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Foundation of China (31101936, 31172437, 31172445, 31372566), and China
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Postdoctoral Science Foundation (2011M501348). 17
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ACCEPTED MANUSCRIPT Figure legends
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Fig.1. The nucleotide and deduced amino acid sequences of EcSTING.
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Fig.2. Multiple sequence alignment of STINGs. The tansmembranes domains (TMs),
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c-di-GMP-binding domain, and C-terminal tail were labeled above the sequences.
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The STING dimerization domain (DD) was indicated with rectangles, and the
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conserved serine residues (human S358 and S366) were indicated with asterisks.
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Fig.3. The expression pattern of EcSITNG in healthy and challenged grouper. (A) The
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relative mRNA level of EcSTING in different tissues. Temporal expression
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analysis of EcSTING mRNA in fish spleen after challenge with different
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stimulates. At the indicated time points, the transcript of EcSTING were detected
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after challenge with poly I:C (B) , LPS (C) and SGIV (D), respectively. *, p<
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0.05.
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Fig.4 Subcellular localization of EcSTING in grouper cells. GS cells were transfected with pEGFP-N3 and pEGFP-EcSTING, and then stained with ER-tracker red. Fig. 5 The effects of EcSTING on ISRE and IFN promoter activity in grouper cells.
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Fig. 6 The S379 and S387 residues were crucial for action of EcSTING. (A) The
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effect of 6 sets of EcSTING mutants on ISRE promoter activity induced by EcSTING. The ISRE(B), IFN1(C) or IFN3(D) promoter activity induced by EcSTING was regulated by S379 and S387. *, p< 0.05.
Fig. 7 The effects of EcSTING and S379 or S387 mutants on the expression of IFN related genes, including ISG15 (A), MXI(B) and Viperin (C). *, p< 0.05. Fig. 8 EcSTING activated IFN response via both IRF3 and IRF7. (A) The roles of 23
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EcIRF3-DN and EcIRF7-DN on IFN promoter activity induced by EcIRF3 or
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EcIRF7. (B) EcSTING induced IFN promoter activity was abolished by
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EcIRF3-DN and EcIRF7-DN. *, p< 0.05. Fig.9 Overexpression of EcSTING decreased SGIV production. (A) Overexpression
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of EcSTING delayed the CPE progression of SGIV infection. (B) Protein
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synthesis of VP19 was significantly inhibited in EcSTING expressing cells. (C)
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EcSTING overexpression decreased SGIV production, and the S379 and S387
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residue were essential for antiviral function.
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Table 1 Primers used in this study Nucleotide sequence (5’ 3’)
5’EcSTING-R1 5’EcSTING-R2 3’EcSTING-R1 3’EcSTING-R2 PcDNA3.1-HA-EcSTING-F PcDNA3.1-HA-EcSTING-R RT- EcSTING-F RT- EcSTING-R EcSTING-S73A-F EcSTING-S73A-R EcSTING-S143+148A-F EcSTING-S143+148A-R EcSTING-S195+200A-F EcSTING-S195+200A-R EcSTING-S248A-F EcSTING-S248A-R EcSTING-S277+281A-F EcSTING-S277+281A-R EcSTING-S379+387A-F EcSTING-S379+387A-R EcISG15-RT-F EcISG15-RT-R EcMXI-RT-F EcMXI-RT-R EcViperin-RT-F EcViperin-RT-R
TGCTGACCTGATGGGTGGCACGAA GCACTTGGCGGGGCACTTGATGTA TCTATGAACCAGGTGCCAATGAGC CGGAGGCGAGTCTTCAAGCACAGC CAGGATCCACGCCACCATGGAGTGCCTCCAAGATC GCATCCTCGAGTATTCTTCCTTGATAATGGT GGAGGCATCGTGTTTCTGTCTC CGGCCTCGGTACCTTGTACTT GATGCTTTACCATGCAAGTACAAGGTACC GGTACCTTGTACTTGCATGGTAAAGCAT GTCCTGCGGAGGTGGAGGTGGCAGAC CTGCCACCTCCACCTCCGCAGGACC AGATCCGCCTGGGGTCGTGGCGCCA TGGCGCCACGACCCCAGGCGGATCT GCGAGTCTTCAAGCACGCAGTCTAC TCTGTAGACTGCGTGCTTGAAGACT TGTACAGCATGGCCCAGGAGAGCGCAGCT AGCTGCGCTCTCCTGGGCCATGCTGTA TGGCCAGGGAGCCTACACTCATGTTCGCCATG ATGGCGAACATGAGTGTAGGCTCCCTGGCCA CCTATGACATCAAAGCTGACGAGAC GTGCTGTTGGCAGTGACGTTGTAGT CGAAAGTACCGTGGACGAGAA TGTTTGATCTGCTCCTTGACCAT GGATGAAGACATGACGGAGAACA ATCCAGGATCAGGTAGGAGTTCC
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Gene name
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SGIV
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LPS
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Ploy I:C
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pEGFP-EcSTING
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pEGFP-EcSTING
pEGFP-N3
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EGFP ER-tracker
Figure 4
Merge
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ISG15
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Viperin
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EGFP
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ACCEPTED MANUSCRIPT Highlights: EcSTING shared 80% identity with large yellow croaker.
2.
EcSTING transcript was differently regulated upon different challenge.
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EcSTING encoded an ER-localized protein.
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EcSTING mediated IFN response through IRF3/IRF7 dependent manner.
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Overexpression of EcSTING inhibited SGIV replication.
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S1050-4648(15)30139-X
DOI:
10.1016/j.fsi.2015.09.014
Reference:
YFSIM 3602
To appear in:
Fish and Shellfish Immunology
Received Date: 9 August 2015 Revised Date:
3 September 2015
Accepted Date: 5 September 2015
Please cite this article as: Huang Y, Ouyang Z, Wang W, Yu Y, Li P, Zhou S, Wei S, Wei J, Huang X, Qin Q, Antiviral role of grouper STING against iridovirus infection, Fish and Shellfish Immunology (2015), doi: 10.1016/j.fsi.2015.09.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Antiviral role of grouper STING against iridovirus infection
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Youhua Huang#, Zhengliang Ouyang#, Wei Wang, Yepin Yu, Pengfei Li, Sheng Zhou,
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Shina Wei, Jingguang Wei, Xiaohong Huang**, Qiwei Qin*
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Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea
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Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road,
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Guangzhou 510301, China
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*Corresponding author:
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Qiwei Qin, Ph D., Professor,
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Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea
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Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road,
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Guangzhou 510301, China.
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Phone & Fax: +86-20-89023638, E-mail: [email protected]
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Xiaohong Huang, Ph D., Professor,
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Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea
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Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road,
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Guangzhou 510301, China.
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Phone & Fax: +86-20-89023197, E-mail: [email protected]
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#These authors contributes equally to this work.
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Abstract Stimulator of interferon genes (STING, also known as MITA, ERIS, MPYS or
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TMEM173) has been identified as a central component in the innate immune response
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to cytosolic DNA and RNA derived from different pathogens. However, the detailed
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role of STING during fish iridovirus infection still remained largely unknown. Here,
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the STING homolog from grouper Epinephelus coioides (EcSTING) was cloned and
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its effects on IFN response and antiviral activity were investigated. The full-length
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EcSTING cDNA was composed of 1590 bp and encoded a polypeptide of 409 amino
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acids with 80% identity to STING homolog from large yellow croaker. Amino acid
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alignment analysis indicated that EcSTING contained 4 predicated transmembrane
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motifs (TMs) in the N terminal, and a C-terminal domain (CTD) which consisted of a
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dimerization domain (DD), c-di-GMP-binding domain (CBD) and a C-terminal tail
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(CTT). Expression profile analysis revealed that EcSTING was abundant in gill,
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spleen, brain, skin, and liver. Upon different stimuli in vivo, the EcSTING transcript
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was dramatically up-regulated after challenging with Singapore grouper iridovirus
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(SGIV), lipopolysaccharide (LPS) and polyinosin-polycytidylic acid (poly I:C).
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Reporter gene assay showed that EcSTING activated ISRE, zebrafish type I IFN and
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type III IFN promoter in vitro. Mutant analysis showed that IFN promoter activity
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was mostly mediated by the phosphorylation sites at serine residue S379 and S387.
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Moreover, EcSTING induced type I and III IFN promoter activity could be impaired
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by overexpression of EcIRF3-DN or EcIRF7-DN, suggesting that EcSTING mediated
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IFN response in IRF3/IRF7 dependent manner. In addition, the cytopathic effect (CPE)
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ACCEPTED MANUSCRIPT progression of SGIV infection and viral protein synthesis was significantly inhibited
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by overexpression of EcSTING, and the inhibitory effect was abolished in serine
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residue S379 and S387 mutant transfected cells. Together, our results demonstrated
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that EcSTING might be an important regulator of grouper innate immune response
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against iridovirus infection.
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Key words: STING; Grouper; Interferon; SGIV; Viral replication
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1. Introduction Increased reports demonstrated that stimulator of interferon genes (STING, also
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known as MITA, ERIS, MPYS or TMEM173) functioned as a central and
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multifaceted mediator in innate immune response and attracted much attention in
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recent years [1-3]. As the cytosolic DNA sensor, STING initiates a cascade of events,
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including moving from the ER to the Golgi, activating the cytosolic kinases,
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TANK-binding kinase 1 (TBK1) and IκB kinase (IKK) which phosphorylate the IFN
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regulatory factor 3 (IRF3) and transcription factors nuclear factor κB (NF-κB) [4-6].
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These phosphorylated transcription factors induce type I IFN and pro-inflammatory
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cytokine production, and then trigger the host immune response. Meanwhile, STING
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could be triggered to recruit STAT6 to the endoplasmic reticulum, leading to STAT6
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phosphorylation on Ser (407) by TBK1 and Tyr (641), independent of JAKs [7]. In
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addition to its established role as a signaling adaptor in the IFN response to cytosolic
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DNA, STING also functions as a direct pattern recognition receptor (PRR) for cyclic
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dinucleotides [5], such as cyclic diguanylate monophosphate (c-di-GMP) and cyclic
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diadenylate monophosphate (c-di-AMP) [8]. Although the roles of STING in
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mammalian innate immune responses were detailed explored, few work focused on
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fish until now [9-11]. Recent studies indicated that overexpression of crucian carp
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STING induced zebrafish IFN1 and IFN3 promoter activation dependent on IRF3 or
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IRF7 [9], and silencing of zebrafish STING significantly inhibited DNA
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virus-induced antiviral responses, and the inhibitory effect was determined by a
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conserved serine residue (S373) [11].
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ACCEPTED MANUSCRIPT Groupers, Epinephelus spp. are widely cultured in China and Southeast Asian
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countries. However, the emergence of iridoviral pathogens caused heavy economic
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losses in grouper aquaculture [12, 13]. Singapore grouper iridovirus (SGIV) was
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isolated from diseased groupers and caused more than 90% mortality in grouper and
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sea bass [14]. As a large DNA virus, SGIV infection in vitro induced a novel type of
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programmed cell death-paraptosis [15], and MAPK signaling pathway was
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demonstrated to be involved in SGIV infection [16]. In addition, our previous studies
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demonstrated that SGIV infection could regulate the expression of a certain interferon
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stimulated or induced genes, suggested that interferon stimulated genes might exert
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crucial roles during SGIV infection [17]. Up to now, only two fish STING
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orthologues have been cloned from freshwater fish, including crucian carp and
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zebrafish [9, 11, 18]. However, little work has been performed on the role of STING
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from marine fish in response to virus infection.
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In the present study, we cloned an STING homolog from grouper and evaluated
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the roles of EcSTING during SGIV infection. Our data will provide new insight into
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the function of fish STING during DNA virus infection.
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2. Material and methods
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2.1 Fish, cell and virus.
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Orange-spotted groupers, E. coioides, about 50 g in body weight, were purchased
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from a fish farm in Hainan province, China. Fishes were kept in a laboratory
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recirculating seawater system at 25°C for two weeks before use. Grouper spleen cells
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(GS) were maintained in Leibovitz’s L-15 medium supplemented with 10% fetal 5
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bovine serum (FBS) (Gibco, USA) at 25°C [19]. Singapore grouper iridovirus (SGIV)
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was propagated in GS cells and the virus stocks were stored at 80°C until used.
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2.2. Cloning of EcSTING and bioinformatic analysis Based on the EST sequences of EcSTING from grouper spleen transcriptome
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annotation [17], the full length of the EcSTING cDNA was amplified with a SMART
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RACE cDNA amplification kit as described previously [20]. To explore the structural
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characteristics of EcSTING, the obtained nucleotide sequence was edited and
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analyzed using the WWW BLAST server. The transmembrane domain prediction was
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carried out using TMHMM program. The potential serine phosphorylation sites were
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predicted using NetPhos program. Multiple amino acid sequences alignment was
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carried out using ClustalX 1.83 and edited with GeneDoc software.
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2.3. Expression profiles of EcSTING in healthy and challenged grouper
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To detect the tissue distribution pattern of EcSTING in healthy orange-spotted
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grouper, total RNA was extracted from head kidney, heart, liver, spleen, intestine,
15
muscle, brain, skin, gill, stomach and kidney, respectively, using the SV Total RNA
16
Isolation Kit (Promega) according to manufacturer’s instructions. Expression of
17
EcSTING in different tissues was determined by quantitative real-time PCR
18
(qRT-PCR) using primers shown in Table 1.
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20
injected with PBS, SGIV, poly I:C and LPS as described previously [20]. At indicated
21
time points, the spleen of different groups (n>3) were collected for RNA extraction
22
and qRT-PCR analysis. 6
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2.4. Plasmid construction To study the roles of EcSTING in vitro, the full length of EcSTING cDNA was
3
PCR amplified and subcloned into the vector pEGFP-N3 or pcDNA3.1-HA. All the
4
primers were listed in Table 1. The constructed plasmids pEGFP-EcSTING and
5
HA-EcSTING were subsequently confirmed by DNA sequencing. To further identify
6
the potential roles of putative phosphorylation residues in EcSTING, we generated 6
7
EcSTING mutants in which serine (S) residues were mutated to alanine (A) either
8
individually, or two adjacent serines in combination. All the mutants were cloned into
9
pcDNA3.1-HA vector and confirmed by DNA sequencing.
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2.5. Cell transfection
Cell transfection was performed using Lipofectamine 2000 reagent (Invitrogen)
12
as described previously [20]. In brief, GS cells were grown in 24-well plates, and
13
incubated with the mixture of Lipofectamine 2000 and plasmids for 6 h. After
14
replacing the fresh medium, cells were cultured at 25°C for further analysis.
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To generate stable cells overexpressing EcSTING or its mutants, the recombinant
16
plasmids were transfected into GS cells and then cells were incubated with medium
17
containing 800 ng/ml G418 (Gibco). After propagation under selective pressure for 4
18
weeks, cells stably expressing EcSTING or its mutants were obtained for further
19
analysis. Cells transfected with pcDNA3.1-HA was chosen as control.
20
2.6. Subcellular localization
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To elucidate the subcellular localization of EcSTING in vitro, pEGFP-N3 and
22
pEGFP-EcSTING were transfected into GS as described above. At 24 h post 7
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transfection, cells were incubated with ER-tracker and Hochest 33342 for observation
2
under fluorescence microscopy.
3
2.7. Reporter gene assay To evaluate the effects of wild type EcSTING and mutants on the promoter
5
activity of ISRE and zebrafish IFN, luciferase activity assays were carried out as
6
described previously [21]. In first set of experiment, GS cells were cultured in 24-well
7
plates, and then cotransfected with 0.4 µg ISRE-Luc, DrIFN1-Luc or DrIFN3-Luc and
8
0.4 µg EcSTING or its mutants using Lipofectamine 2000 as described above. A total
9
of 0.05 µg pRL-TK was included to normalize the expression level. For the second set
10
of
11
EcIRF3/EcIRF3-DN/EcIRF7/EcIRF7-DN/empty vector and 0.4 µg DrIFN1-Luc or
12
DrIFN3-Luc, respectively. At 48 h posttransfection, the transfected cells were
13
harvested and lysed according to the Dual-Luciferase Reporter Assay System
14
(Promega). Luciferase activities were measured using a Victor X5 Multilabel plate
15
reader (PerkinElmer). The results were representative of three independent
16
experiments, and each independent experiment was performed in triplicate. Luciferase
17
activities were expressed as the fold relative to the empty vector transfected cells.
18
2.8. Quantitative real-time PCR (qRT-PCR) analysis
cells
were
cotransfected
with
0.4
µg
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The expression of target genes, including EcSTING, grouper Mx-I, ISG15,
20
viperin and β-actin (internal control) were detected by qRT-PCR using the SYBR
21
Green realtime PCR Kit (Toyobo) according to the manufacturers’ instructions. In
22
brief, after reverse transcription, qRT-PCR was carried out in a Roche 480 Real Time 8
ACCEPTED MANUSCRIPT 1
Detection System (Roche, German) as described previously [20]. The relative
2
expression level of virus genes was analyzed using typical Ct method (2
3
[22]. The data were calculated as the folds based on the expression level of targeted
4
genes normalized to β-actin at the indicated time points. The Data were represented as
5
mean ± SD, and the statistic analysis were performed using SPSS software.
6
2.9. Immune fluorescence microscopy
ct
method)
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To examine the effect of EcSTING overexpression on viral protein synthesis,
8
immune fluorescence was carried out using SGIV VP19 antibody as described
9
previously [23]. In brief, cells were seeded into 24-wells plate and then transfected
10
with plasmids pEGFP-N3 or pEGFP-EcSTING. At 24 h post transfection, cells were
11
infected with SGIV for another 24 h and fixed with 4% paraformaldehyde. After
12
blocking with 2% bovine serum albumin (BSA), cells were incubated with anti-VP19
13
(1:100), followed by Rhodamine-conjugated goat anti-mouse antibodies (Pierce).
14
Finally, samples were stained with 1 µg/ml 6-diamidino-2-pheny-lindole (DAPI), and
15
observed under fluorescence microscopy (Leica, Germany).
16
2.10. Virus titer assay
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To investigate the effect of wild type EcSTING, S379 mutant or S387 mutant
18
overexpression on SGIV production, virus titer assay was carried out as described
19
previously [16]. Briefly, the stable cell lines were cultured in 24-well plates separately
20
and infected with SGIV at multiplicity of infection (MOI) of 1. At 48 h p.i.,
21
virus-infected cell lysates were harvested, and viral titers were determined by 50%
22
tissue culture infectious does (TCID50) assay (Reed & Muench, 1938). 9
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3.1. Results
2
3.1. Sequence characterization of EcSTING Using the RACE method, the full length cDNA of EcSTING which was
4
composed of 1590 bp was obtained in this study. It contained a 1227 bp open reading
5
frame (ORF) encoding a 409-aa protein (Fig. 1). Blastp analysis showed that
6
EcSTING shared 80%, 76%, 46%, and 46% homology with the known STING
7
sequences of large yellow croaker (Larimichthys crocea), Oreochromis niloticus,
8
zebrafish and Carassius auratus, respectively. Amino acid alignment showed that
9
EcSTING contained four predicted transmembrane motifs (TMs) in the N terminal
10
and a C-terminal domain (CTD) which consisted of a dimerization domain (DD),
11
c-di-GMP-binding domain (CBD) and a C-terminal tail (CTT). In addition, the critical
12
serine residues S358 and S366 in human STING which were essential for
13
TBK1-mediated phosphorylation of STING and STING-dependent IRF3 activation
14
were also conserved in EcSTING and other fishes (Fig. 2).
15
3.2. Tissue distribution and expression pattern of EcSTING
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The expression of EcSTING mRNA in various tissues was determined by
17
qRT-PCR. As shown in Fig. 3, a constitutively expression of EcSTING was obviously
18
observed in most examined tissues, including gill, spleen, brain, skin, liver, muscle,
19
heart, kidney, head kidney, intestine. The predominant expression was detected in the
20
gill, spleen, brain, skin and liver (Fig. 3A).
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To explore the expression of EcSTING after challenged with different stimulates,
22
the transcript of EcSTING was determined at the indicated time after challenge with 10
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2
EcSTING was increased up to 32- and 165-fold in LPS and poly I:C challenged fish
3
spleen at 6 h post injection compared with 0 h post injection. Similar results were
4
observed in SGIV infected groupers, and the expression of EcSTING was
5
significantly increased up to 62-fold at 12 h post injection. With the infection time
6
EcSTING expression gradually decreased, and its expression decreased to the basic
7
level at 96 h post-injection.
8
3.3. The subcellular localization of EcSTING
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STING contains transmembrane motifs in the N-terminus, which predominantly
10
anchors itself in the endoplasmic reticulum (ER) or the outer membrane of
11
mitochondria [1, 24]. To explore the subcellular localization of EcSTING in vitro,
12
pEGFP-N3 or pEGFP-EcSTING was transfected into GS cells and then stained with
13
ER-tracker. As show in Fig. 4, the green fluorescence omitted from pEGFP-EcSTING
14
transfected cells showed three forms with the transfection time increased, including
15
evenly distribution, punctuates and bright aggregates. All the green fluorescence was
16
overlapped with the red fluorescence from ER tracker red. Differently, in pEGFP-N3
17
transfected cells, the green fluorescence was distributed throughout the cells. Thus,
18
EcSTING was proposed to encode an ER-localized protein.
19
3.4. Ectopic expression of EcSTING induced ISRE and IFN promoter activity
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Due to the structural similarity between EcSTING and mammalian STING, we
21
firstly examined the effect of EcSTING overexpression on ISRE and IFN promoter
22
activity in grouper cells. As shown in Fig. 5, overexpression of EcSTING could 11
ACCEPTED MANUSCRIPT induce a strong activation of ISRE-Luc by up to 27-fold compared to the control
2
vector pcDNA3.1-HA. In addition, the promoter activity of zebrafish type I IFN
3
(DrIFN1-Luc) and type III IFN (DrIFN3-Luc) were significantly increased in
4
EcSTING transfected cells compared to pcDNA3.1-HA transfected cells. In detail, the
5
luciferase activity of DrIFN1-Luc and DrIFN3-Luc was increased up to 11-folds and
6
6-folds in EcSTING transfected cells, respectively. This result indicated that
7
EcSTING was capable of activating fish IFN response in vitro. has
been
reported
that
STING
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with
IRF3
in
a
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phophorylation-dependent manner. Using NetPhos program, we predicted the putative
10
phophorylation sites of EcSTING. The 6 EcSTING mutants in which one or two
11
potential phosphorylation serine residues were mutated to alanines were constructed.
12
The potential roles of these serine residues in controlling EcSTING function were
13
examined using luciferase reporter assays. As shown in Fig. 6, mutation of S379 and
14
S387 to alanine significantly reduced the activation of ISRE promoter, while the
15
mutation of S73, S143 and S148, S195 and S200, S248, S277 and S281 to alanine
16
displayed similar effects to that of EcSTING wild type. The results suggested that
17
S379 and S387 might be important for EcSTING-mediate IFN signaling. To further
18
determine which phophorylation sites were crucial in IFN response induced by
19
EcSTING, the S379 or S387 mutants were constructed, respectively. Interestingly, the
20
ability of activate ISRE-Luc, zebrafish DrIFN1-Luc and DrIFN3-Luc promoter were
21
both significantly decreased in S379 and S387 mutant transfected cells, in compared
22
to wild type EcSTING transfected cells. Especially in S387 mutant transfected cells,
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the activity of all these three promoters were almost completely abolished like control
2
vector transfected cells. Taken together, the results indicated that S379 and S387
3
exerted crucial roles in EcSTING induced IFN immune response. In addition, we also evaluated the effects of EcSTING and S379 or S387 mutants
5
on the expression of IFN related genes, including ISG15, MX-I and viperin. As shown
6
in Fig. 7, the transcripts of all examined genes were significantly induced by wild type
7
EcSTING, compared to the control vector. Differently, ability of inducing IFN related
8
genes expression were obviously inhibited in S379 and S387 mutant transfected cells,
9
and inhibitory effect evoked by S387 mutant was stronger than by S379 mutant.
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3.5. EcSTING activated IFN immune response via IRF3 and IRF7 The previous studies showed that EcIRF3 and EcIRF7 could induce the
12
promoters activity of DrIFN1-Luc or DrIFN3-Luc [25, 26]. Our results also indicated
13
that overexpression of EcIRF3-DN and EcIRF7-DN could inhibit the activation of
14
IFN1 and IFN3 promoter induced by EcIRF3 and EcIRF7, respectively (Fig. 8). In
15
order to further understand whether IRF3 or IRF7 mediated the activating fish IFN
16
response induced by EcSTING in vitro, we cotransfected EcSTING construct,
17
EcIRF3-DN (or EcIRF7-DN) and DrIFN1-Luc (or DrIFN3-Luc) in GS cells,
18
respectively. As shown in Fig. 8, EcSTING-induced IFN promoter activities were
19
significantly impaired in GS cells when cotransfected with either EcIRF3-DN or
20
EcIRF7-DN. However, the inhibitory effect of EcIRF7-DN was weaker than that of
21
EcIRF7-DN. Thus, our results indicated that EcSTING activated IFN response via
22
both IRF3 and IRF7, but IRF3 exert more crucial roles.
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3.6. Overexpression of EcSTING inhibited SGIV replication To determine the roles of EcSTING during iridovirus infection, we examined the
3
viral protein synthesis in SGIV infected EcSTING expressing cells. As shown in Fig.
4
9, severe cytopathic effect (CPE) was observed in SGIV infected control cells at 48 h
5
p.i., and no obvious CPE was observed in infected EcSTING-transfected cells.
6
Moreover, EcSTING overexpression obviously inhibited the synthesis of viral
7
structural protein VP19 in compared to control cells. The virus production was also
8
evaluated using cells stably expressed EcSTING and pcDNA3.1-HA. Consistently,
9
EcSTING overexpression evoked the reduction of virus titer from 7.78-Log
10
TCID50/ml to 6.83 -Log TCID50/ml. In contrast, in S379 or S387 mutants expressing
11
cells, the inhibitory effect on virus titer were not detected. Together, the EcSTING
12
exerted important antiviral roles during iridovirus infection, and the antiviral effects
13
were determined by the critical residues S379 or S387 at the phosphorylation sites
14
(Fig. 9).
15
4. Discussion
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Increased reports have demonstrated that STING is a crucial component in the
17
innate immune response to cytosolic DNA and RNA derived from different pathogens,
18
including viruses, bacteria and parasites [5, 27, 28]. During the co-evolution,
19
pathogens, such as gammaherpesviruses, could block cGAS-STING-mediated
20
antiviral immunity by encoding inhibitors, and modulate this pathway for viral
21
transmission in the human population [29]. To our knowledge, only several reports
22
focused on the roles of STING in fresh water fish [18, 9, 11]. To better understanding
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the function of STING in teleost, a STING gene from marine fish, grouper, was
2
identified and characterized in this study. Bioinformatics analysis showed that EcSTING contained 4 predicted TM
4
domains in the N terminal. TM domains of human STING are not only essential for
5
its localization and dimerization [30], but also play important roles during its
6
interaction with MAVS to active IRF3 and induce IFNs [24]. The amino acid
7
sequences in the regions of former 4 TM domains were highly divergent between fish
8
and mammals, whether these TM domains exert different roles remained unclear. In
9
our study, EcSTING expression could be detected in all tissues tested, and a high
10
level expression was found in gill, brain, skin, pleen, liver. Upon challenge with poly
11
(I:C), LPS and SGIV, EcSTING expression could be differently regulated at various
12
stages of chanllenge, suggested that EcSTING might mainly play vital roles in host
13
immune response against different pathogens.
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Studies from mammals indicated that overexpression of STING led to strong
15
induction of IFN immune response [1, 24, 30]. After phosphorylation, the adaptor
16
proteins STING could activate the downstream protein kinase TBK1, which
17
phosphorylates the transcription factor IRF3, and then drives type I IFN production
18
[31]. In this study, the ectopic expression of EcSTING in vitro induced the activation
19
of ISRE and IFN promoter activity. The expressions of a certain interferon induced
20
genes, including ISG15, MX and viperin were also significantly increased in
21
EcSTING overexpressing cells, suggested that EcSTING also mediated IFN immune
22
response like mammals. In addition, we also found that IFN promoter activated by
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EcSTING overexpression could be significantly inhibited by IRF3-DN or IRF7-DN
2
co-transfection, suggesated that EcSTING activated IFN signaling dependent on
3
functional IRF3 or IRF7. In response to different stimulation, STING functioned in a phosphorylation
5
dependent manner [3]. In human STING, mutation of S280, S358, or S366 to alanine
6
affected the TBK1-mediated phosphorylation of STING. Further studies indicated that
7
S358 in STING was an important target residue phosphorylated by TBK1 and also
8
contributeed to the aggregation of STING, while conserved residue S366 was
9
identified as a key amino acid required for STING-dependent IRF3 activation and
10
subsequent IFN induction [31, 32]. Our results showed that serine residues S379 and
11
S387 were critical for EcSTING induced IFN promoter activation. Moreover, the
12
ability of inducing IFN related gene expression was significantly decreased after in
13
serine residues S379 and S387 mutant transfected cells. We speculated that S379 and
14
S387 in EcSTING might exert similar roles like S358 and S366 from human STING.
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It has been reported that overexpression of mammalian STING decreases
16
intracellular viral load during Japanese encephalitis virus infection [33]. Similarly,
17
ectopic expression of crucian carp and grass carp STING in vitro significantly inhibits
18
virus replication through activating IRF3/7-dependent IFN response [9, 10]. In
19
addition, overexpression of zebrafish STING induced a strong anti-viral immunity
20
against both RNA and DNA viruses [18, 11]. In the present study, the effects of
21
EcSTING expression on iridovirus replication were also investigated. Our results
22
showed that the ectopic expression of EcSTING delayed the CPE progression induced
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2
Moreover, the antiviral activity was dependent on the critical residue S379 and S387.
3
In zebrafish, a conserved serine residue (S373) was essential for the action of zSTING
4
which could mediate DNA virus-induced antiviral responses [11]. Although STING
5
from zebrafish and grouper shared only 46% identity, the amino acid alignment
6
showed that serine residue at DrSTING (S373) and EcSTING (S387) was conserved
7
as well as human STING (S366). Whether S379 and S387 exert more regulatory roles
8
during the antiviral actions of EcSTING needed further investigation.
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In summary, we characterized a functional STING homolog from marine fish,
10
which contained evolutionarily conserved domains like mammals. The transcription
11
of EcSTING was induced in vivo upon different stimuli, including poly (I:C) and
12
SGIV and LPS. Overexpression of EcSTING in vitro induced type I and III IFN
13
promoter activity which was mediated by IRF3 or IRF7. Furthermore, SGIV
14
replication was significantly inhibited by EcSTING overexpression in GS cells.
15
Together, our results shed new lights on understanding the roles of STING in fish
16
immune response against iridovirus infection.
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Acknowledgment
This work was supported by grants from the National Basic Research Program of
20
China (973) (2012CB114404; 2012CB114402), the National High Technology
21
Development Program of China (863) (2014AA093507), the National Natural Science
22
Foundation of China (31101936, 31172437, 31172445, 31372566), and China
23
Postdoctoral Science Foundation (2011M501348). 17
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Stallings CL, Virgin HW, Cox JS. The Cytosolic Sensor cGAS Detects 21
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Mycobacterium tuberculosis DNA to Induce Type I Interferons and Activate
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Autophagy. Cell Host Microbe 2015; 17(6): 811-819. [28] Majumdar T, Chattopadhyay S, Ozhegov E, Dhar J, Goswami R, Sen GC, Barik
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Toxoplasma gondii. PLoS Pathog 2015; 11(3): e1004779.
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signaling through dimerization. Proc Natl Acad Sci USA 2009; 106: 8653-8658.
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PPM1A regulates antiviral signaling by antagonizing TBK1-mediated STING
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[33] Nazmi A, Mukhopadhyay R, Dutta K, Basu A. STING mediates neuronal innate
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immune response following Japanese encephalitis virus infection. Sci Rep 2012;
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2: 347.
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ACCEPTED MANUSCRIPT Figure legends
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Fig.1. The nucleotide and deduced amino acid sequences of EcSTING.
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Fig.2. Multiple sequence alignment of STINGs. The tansmembranes domains (TMs),
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c-di-GMP-binding domain, and C-terminal tail were labeled above the sequences.
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The STING dimerization domain (DD) was indicated with rectangles, and the
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conserved serine residues (human S358 and S366) were indicated with asterisks.
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Fig.3. The expression pattern of EcSITNG in healthy and challenged grouper. (A) The
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relative mRNA level of EcSTING in different tissues. Temporal expression
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analysis of EcSTING mRNA in fish spleen after challenge with different
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stimulates. At the indicated time points, the transcript of EcSTING were detected
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after challenge with poly I:C (B) , LPS (C) and SGIV (D), respectively. *, p<
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Fig.4 Subcellular localization of EcSTING in grouper cells. GS cells were transfected with pEGFP-N3 and pEGFP-EcSTING, and then stained with ER-tracker red. Fig. 5 The effects of EcSTING on ISRE and IFN promoter activity in grouper cells.
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Fig. 6 The S379 and S387 residues were crucial for action of EcSTING. (A) The
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effect of 6 sets of EcSTING mutants on ISRE promoter activity induced by EcSTING. The ISRE(B), IFN1(C) or IFN3(D) promoter activity induced by EcSTING was regulated by S379 and S387. *, p< 0.05.
Fig. 7 The effects of EcSTING and S379 or S387 mutants on the expression of IFN related genes, including ISG15 (A), MXI(B) and Viperin (C). *, p< 0.05. Fig. 8 EcSTING activated IFN response via both IRF3 and IRF7. (A) The roles of 23
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EcIRF3-DN and EcIRF7-DN on IFN promoter activity induced by EcIRF3 or
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EcIRF7. (B) EcSTING induced IFN promoter activity was abolished by
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EcIRF3-DN and EcIRF7-DN. *, p< 0.05. Fig.9 Overexpression of EcSTING decreased SGIV production. (A) Overexpression
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of EcSTING delayed the CPE progression of SGIV infection. (B) Protein
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synthesis of VP19 was significantly inhibited in EcSTING expressing cells. (C)
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EcSTING overexpression decreased SGIV production, and the S379 and S387
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residue were essential for antiviral function.
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Table 1 Primers used in this study Nucleotide sequence (5’ 3’)
5’EcSTING-R1 5’EcSTING-R2 3’EcSTING-R1 3’EcSTING-R2 PcDNA3.1-HA-EcSTING-F PcDNA3.1-HA-EcSTING-R RT- EcSTING-F RT- EcSTING-R EcSTING-S73A-F EcSTING-S73A-R EcSTING-S143+148A-F EcSTING-S143+148A-R EcSTING-S195+200A-F EcSTING-S195+200A-R EcSTING-S248A-F EcSTING-S248A-R EcSTING-S277+281A-F EcSTING-S277+281A-R EcSTING-S379+387A-F EcSTING-S379+387A-R EcISG15-RT-F EcISG15-RT-R EcMXI-RT-F EcMXI-RT-R EcViperin-RT-F EcViperin-RT-R
TGCTGACCTGATGGGTGGCACGAA GCACTTGGCGGGGCACTTGATGTA TCTATGAACCAGGTGCCAATGAGC CGGAGGCGAGTCTTCAAGCACAGC CAGGATCCACGCCACCATGGAGTGCCTCCAAGATC GCATCCTCGAGTATTCTTCCTTGATAATGGT GGAGGCATCGTGTTTCTGTCTC CGGCCTCGGTACCTTGTACTT GATGCTTTACCATGCAAGTACAAGGTACC GGTACCTTGTACTTGCATGGTAAAGCAT GTCCTGCGGAGGTGGAGGTGGCAGAC CTGCCACCTCCACCTCCGCAGGACC AGATCCGCCTGGGGTCGTGGCGCCA TGGCGCCACGACCCCAGGCGGATCT GCGAGTCTTCAAGCACGCAGTCTAC TCTGTAGACTGCGTGCTTGAAGACT TGTACAGCATGGCCCAGGAGAGCGCAGCT AGCTGCGCTCTCCTGGGCCATGCTGTA TGGCCAGGGAGCCTACACTCATGTTCGCCATG ATGGCGAACATGAGTGTAGGCTCCCTGGCCA CCTATGACATCAAAGCTGACGAGAC GTGCTGTTGGCAGTGACGTTGTAGT CGAAAGTACCGTGGACGAGAA TGTTTGATCTGCTCCTTGACCAT GGATGAAGACATGACGGAGAACA ATCCAGGATCAGGTAGGAGTTCC
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Viperin
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ACCEPTED MANUSCRIPT Highlights: EcSTING shared 80% identity with large yellow croaker.
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EcSTING transcript was differently regulated upon different challenge.
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EcSTING encoded an ER-localized protein.
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EcSTING mediated IFN response through IRF3/IRF7 dependent manner.
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Overexpression of EcSTING inhibited SGIV replication.
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