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Molecular and functional characterization of tilapia DDX41 in IFN regulation
Journal Pre-proof Molecular and functional characterization of tilapia DDX41 in IFN regulation Zhen Gan, Jun Cheng, Jing Hou, Hongli Xia, Wenjie Chen, Liqun Xia, Pin Nie, Yishan Lu PII:
S1050-4648(20)30117-0
DOI:
https://doi.org/10.1016/j.fsi.2020.02.031
Reference:
YFSIM 6830
To appear in:
Fish and Shellfish Immunology
Received Date: 1 December 2019 Revised Date:
30 January 2020
Accepted Date: 16 February 2020
Please cite this article as: Gan Z, Cheng J, Hou J, Xia H, Chen W, Xia L, Nie P, Lu Y, Molecular and functional characterization of tilapia DDX41 in IFN regulation, Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/j.fsi.2020.02.031. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Ltd.
1
Molecular and functional characterization of tilapia DDX41 in IFN regulation
2 a, b, c, d, e, 1
, Jun Cheng a, c, d, 1, Jing Hou a, b, c, d, e, Hongli Xia
3
Zhen Gan
4
Chen a, b, c, d, e, Liqun Xia a, c, d, Pin Nie b, f, *, Yishan Lu a, c, d, **
a, c, d
, Wenjie
5 6
a
7
b
8
Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
9
c
College of Fishery, Guangdong Ocean University, Zhanjiang 524025, China
10
d
Guangdong Provincial Engineering Research Center for Aquatic Animal Health
11
Assessment, and Shenzhen Public Service Platform for Evaluation of Marine
12
Economic Animal Seedings, Shenzhen 518120, China
13
e
14
Shenzhen 518120, China
15
f
16
Qingdao, Shandong Province, 266109, China
Shenzhen Institute of Guangdong Ocean University, Shenzhen 518120, China State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of
Shenzhen Dapeng New District Science and Technology Innovation Service Center,
School of Marine Science and Engineering, Qingdao Agricultural University,
17 18
*
19
7 of South Donghu Road, Wuchang District, Wuhan, Hubei Province, 430072, China.
20
E-mail addresses: [email protected]; [email protected] (P. Nie).
Corresponding author. Institute of Hydrobiology, Chinese Academy of Sciences, No.
21 22
**
23
Binhai 2nd Road, Dapeng New District, Shenzhen, Guangdong Province, 518120,
24
China.
25
E-mail address: [email protected] (Y. Lu).
Corresponding author. Shenzhen Institute of Guangdong Ocean University, No. 3 of
26 27 28
1
Indicating the two first authors.
1
ABSTRACT
2
DEAD-box helicase 41 (DDX41) is a key cytosolic DNA sensor playing critical
3
roles in the regulation of type I IFN responses, and their functions have been
4
well-characterized in mammals. However, little information is available regarding the
5
function of fish DDX41. In this study, a DDX41 gene, named On-DDX41, was
6
identified in Nile tilapia, Oreochromis niloticus. The predicted protein of On-DDX41
7
contains several structural features known in DDX41, including conserved DEADc
8
and HELICc domains, and a conserved sequence “Asp-Glu-Ala-Asp (D-E-A-D)”.
9
On-DDX41 gene was constitutively expressed in all tissues examined, with the
10
highest expression level observed in liver and muscle, and was inducible after
11
poly(I:C) stimulation. Moreover, the overexpression of On-DDX41 can elicit a strong
12
activation of both zebrafish IFN1 and IFN3 promoter in fish cells treated with
13
poly(dA:dT). The present study thus contributes to a better understanding of the
14
functional properties of DDX41 in fish.
15 16
Keywords: Oreochromis niloticus; DDX41; cytosolic DNA sensors; type I interferon;
17
promoter activity.
18 19 20
21
1. Introduction
22
Type I interferons (IFNs) are a subset of pleiotropic cytokines playing central
23
roles in antiviral immune defense [1, 2]. From an evolutionary point of view, it is
24
generally accepted that type I IFNs are ubiquitously present in all classes of jawed
25
vertebrates [3-6]. However, large divergence may exist in the composition of type I
26
IFNs from different classes of vertebrates. Specifically, jawed fish may harbor most
27
primitive type I IFNs, which are encoded by intron-containing genes consisting of 5
28
exons and 4 introns [3, 7-9], whereas type I IFN genes in amniotes are intronless [6,
29
10]. Interestingly, recent studies have revealed that intronless and intron-containing
30
type I IFN genes coexist in amphibians, and the difference in genomic organisations
31
of vertebrate type I IFNs may be resulted from three retroposition events occurred in
32
the clawed frog Xenopus and the Tibetan frog Nanorana parkeri, and amniotes,
33
respectively [4, 5].
34
In spite of considerate divergence exist among type I IFN repertoire in different
35
vertebrates, the main regulatory mechanisms of type I IFN responses appear to
36
conserve from fish to mammals [3]. It is believed that three major families of pattern
37
recognition receptors (PRRs) are involved in the regulation of type I IFN production
38
[11-13]. The first class of PRRs is retinoic acid-inducible gene I (RIG-I)-like
39
receptors (RLRs), which consists of three members, including RIG-I, melanoma
40
differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology
41
2 (LGP2) [11]. The second category of viral nucleic acid sensors is some members
42
from toll-like receptors (TLRs), such as TLR3, TLR7, TLR8, and TLR9 [12]. The
43
third class of PRRs is a series of cytosolic DNA sensors, in which DEAD-box
44
helicase 41 (DDX41) is a critical regulator of type I IFN responses [14].
45
Structurally, as a member of the DExD/H-box helicases superfamily, DDX41 is
46
characterized by two conserved RecA-like domains, including DEADc and HELICc
47
domains, and the former contains a strictly conserved sequence “Asp-Glu-Ala-Asp
48
(D-E-A-D)” [15-18]. Upon detecting viral double-stranded DNAs (dsDNAs) or
49
bacterial cyclic dinucleotides (CDNs), DDX41 can interact with the stimulator of IFN
50
genes (STING) to activate TANK-binding kinase 1 (TBK1). These activated TBK1
51
phosphorylates interferon regulatory factor 3/7 (IRF3/7), eventually elicit the
52
production of type I IFNs and interferon-stimulated genes (ISGs) [14, 19]. Although
53
functional properties and signalling pathways of DDX41 have been well-characterized
54
in mammals, little information is available to date regarding fish DDX41. DDX41 has
55
only been cloned from a few species of teleosts, including zebrafish (Danio rerio),
56
Japanese flounder (Paralichthys olivaceus), and orange spotted grouper (Epinephelus
57
coioides) [20-24], and the studies about functional properties of fish DDX41 is rather
58
limited.
59
Tilapia (Oreochromis spp.) is the second farmed fish species worldwide, serving
60
as an important protein source in many countries [25-28]. In recent years, a series of
61
emerging viral diseases caused by tilapia lake virus (TiLV) and tilapia larvae
62
encephalitis virus (TLEV) have been severe, posing a potential threat to the global
63
tilapia industry [29-31]. To prevent the outbreak of above viral diseases has become a
64
major task for the sustainable development of tilapia industry, and the understanding
65
of their immune system, particularly their antiviral immune system, may provide clues
66
for the development of strategies on the prevention of such diseases. In this study, a
67
DDX41 gene (named On-DDX41) was cloned from Nile tilapia, O. niloticus, and its
68
expression profiles and functional properties were investigated. The present results
69
thus contribute to better understanding of the mechanism of antiviral immune
70
responses in tilapia.
71 72
2. Materials and methods
73
2.1. Experimental animals and cells
74
Samples of tilapia (average weight of 100 ± 10 g) were obtained from a
75
commercial
farm
in
Guangzhou,
Guangdong
province,
China.
Prior
to
76
experimentation, fish were acclimated in fiber-reinforced plastic tanks (1000 L each)
77
with a stocking rate of 4 g L−1 under 28 ± 2 °C for 4 weeks [32, 33]. All animal
78
protocols were performed following the Guide for the Care and Use of Laboratory
79
Animals of the Guangdong Ocean University and approved by the Shenzhen Institute
80
of Guangdong Ocean University. Epithelioma papulosum cyprini (EPC) cells were
81
cultured in medium 199 (Life Technologies) supplemented with 10% FBS (Life
82
Technologies), 100 U/ml penicillin, and 100 µg/ml streptomycin at 26 °C with 5%
83
CO2.
84 85
2.2. Cloning of DDX41 gene in tilapia
86
The putative open reading frame (ORF) of the DDX41 gene, named On-DDX41,
87
were predicted by in silico analysis. By using Trizol Reagent (Invitrogen), total RNA
88
from various tissues including spleen, kidney, and liver of healthy tilapia was
89
extracted and mixed. For first-strand cDNA synthesis, above mixed total RNA was
90
treated with DNase I to remove genomic DNA contamination, before being reverse
91
transcribed using RevertAid™ First Strand cDNA Synthesis Kit (Thermo Fisher
92
Scientific). The first-strand cDNA was served as template to amplify ORF sequence of
93
On-DDX41 by PCR using specific primers designed according to predicted data. All
94
PCR products were ligated into the pMD18-T vector (TaKaRa) and transformed into
95
competent Escherichia coli cells. Then the positive clones were sequenced by Sangon
96
Biotech (Shanghai, China). The sequences of all PCR primers used in this study are
97
summarized in Supplementary Table 1.
98 99
2.3. Sequence analysis of DDX41 gene in tilapia
100
The protein sequences were deduced from the nucleic acid sequences using the
101
program on the Expasy Web site (http://ca.expasy.org/tools), and multiple sequence
102
alignments for amino acids were generated using the Clustal X program. Homology
103
between sequences was calculated using the Megalign program within the DNASTAR
104
package, and location of domains was predicted using the InterProScan program
105
(http://www.ebi.ac.uk/Tools/pfa/iprscan/). Phylogenetic tree for On-DDX41 was
106
constructed using MEGA5 package with neighbor-joining (NJ) algorithm, with 1000
107
time repeat of bootstrap analysis. Chromosomal locations of DDX41 genes of tilapia,
108
zebrafish (Danio rerio), chicken (Gallus gallus), mouse (Mus musculus), and human
109
(Homo
110
(https://www.ncbi.nlm.nih.gov/projects/sviewer/). All sequences used in the analysis
sapiens)
were
obtained
from
the
Sequence
Viewer
111
were listed in Supplementary Table 2.
112 113
2.4. Quantitative analysis of DDX41 mRNA expression in tilapia
114
For analyzing the expression of DDX41 gene in healthy tilapia, organs/tissues
115
were collected from three animals to extract total RNA with Trizol. For analyzing the
116
expression
117
intraperitoneally with polyinosinic:polycytidylic acid [poly(I:C); Sigma; 10 µg/g body
118
weight] and were divided into four groups, each with three individuals. 12 healthy
119
individuals as control were injected intraperitoneally with phosphate buffered saline
120
(PBS) and were also divided into four groups. At 3, 6, 12 and 24 hours post-injection
121
(hpi), three animals in each group were scarified for extracting RNA with Trizol from
122
organs/tissues. Total RNA from organs/tissues of different individuals was used
123
separately for the synthesis of the first strand cDNA.
after
stimulation,
12
healthy
individuals
were
each
injected
124
Specific primers (Supplementary Table 1) were used to amplify DDX41 and
125
β-actin fragments by PCR, and the PCR products were sequenced as described above.
126
The copy number was calculated following a method described previously [34, 35].
127
To establish a standard curve, sequenced plasmid DNA of positive clones was
128
extracted with Plasmid Mini Kit (Omega Bio-tek) and was diluted in serial 10-fold
129
dilution, ranging from 10−6 to 10−2, before being quantified with PCR using CFX96
130
Real-Time PCR Detection System (BioRad). A final volume of 20 µl PCR reaction
131
system contained 1 µl cDNA template, 1 µl of each primers, 7 µl sterile water, and 10
132
µl iQTM SYBR® Green Supermix (BioRad), with the PCR protocol as the followings:
133
one cycle of 95 °C for 3 min, followed by 45 cycles of 95 °C for 10 s, 57 °C for 20 s
134
and 72 °C for 40 s. Each sample was run in triplicate, and the gene expression for
135
each sample was normalized against β-actin. Data analysis was performed using the
136
2−∆∆Ct method [36], and fold changes were calculated relative to control group.
137 138
2.5. Examination of type I IFN promoter activation by DDX41 from tilapia
139
For determining the type I IFN promoter activation by tilapia DDX41, luciferase
140
activity assay was transfected into EPC cells as previously reported with minor
141
modification [37]. Briefly, the entire ORF of On-DDX41 was amplified and
142
subcloned into pcDNA3.1/myc-His (-) A vector (Invitrogen; called pcDNA3.1 in this
143
research), which was named as pcDNA3.1-On-DDX41. The EPC cells seeded in
144
24-well plates (2 × 105 cells/well) at were co-transfected with 100 ng IFN1pro-Luc or
145
IFN3pro-Luc, two zebrafish type I IFN promoter-driving luciferase plasmid [37],
146
together with 200 ng pcDNA3.1-On-DDX41 or empty pcDNA3.1 (as vector control),
147
and 10 ng pRL-TK (Promega), following the protocol of Lipofectamine 2000
148
Transfection Reagent (Invitrogen), with the stimulation of poly(dA:dT) (1 µg/ml,
149
Sigma) or not. 24 hours post-transfection, the cells were harvested and lysed using
150
Dual-Luciferase Reporter System (Promega), with luciferase activity measured on a
151
Junior LB9509 luminometer (Berthold, Germany). Data were normalized to the
152
Renilla Luciferase activity, and fold changes were calculated relative to control group
153
transfected with empty vector pcDNA3.1.
154 155
2.6. Statistical analysis
156
Statistical analyses were based on three repeated experiments, with one-way
157
ANOVA carried out in SPSS 17.0. Data were presented as mean ± standard error, and
158
statistical significance was defined as P < 0.05.
159 160
3. Results
161
3.1. Coning and sequence analysis of DDX41 gene in tilapia
162
By searching the tilapia genome, a DDX41 gene, named On-DDX41, was
163
identified on chromosome LG2 (NCBI Reference Sequence: NC_031966.2). To test
164
whether predicted DDX41 gene are expressed in vivo at transcription level, the ORF
165
sequence of DDX41 gene was cloned from tilapia, with the following accession
166
number in GenBank: MN729497. The ORF of On-DDX41 is 1845 bp in size, which
167
encodes a protein of 614 amino acids. On-DDX41 share very high identity, being
168
85.8–86.2%, 86.8–87.3%, 85.2–86.8%, and 93.8–97.1%, with DDX41 in mammals,
169
birds, reptiles and other fish, respectively (Supplementary Figure 1), and several
170
important structural characteristics known in DDX41 are conserved in predicted
171
protein of On-DDX41, including two conserved RecA-like domains, DEADc and
172
HELICc domains, and a strictly conserved sequence “Asp-Glu-Ala-Asp (D-E-A-D)”
173
(Fig. 1).
174
To understand the phylogenetic relationship between On-DDX41 and DDX41 in
175
other vertebrates, a NJ tree was constructed using protein sequences. In general, all
176
the DDX41 in tetrapods were clustered into a major clade at the top of the
177
phylogenetic tree, and On-DDX41 and DDX41 in other fish were grouped together to
178
form a separate clade with the support of high bootstrap value (Fig. 2). In addition, the
179
analysis of the genes flanking DDX41 revealed that On-DDX41 was closely linked to
180
docking protein 3 (DOK3) gene, which was similar to zebrafish, chicken, mouse, and
181
human DDX41 (Fig. 3).
182 183
3.2. Expression pattern of DDX41 gene in tilapia
184
In healthy tilapia, the mRNA of DDX41 was detected in all organs/tissues
185
examined, with the highest level of DDX41 observed in liver (Fig. 4A). Following
186
poly(I:C) stimulation, the transcript level of DDX41 was significantly up-regulated in
187
spleen at 6 h post-stimulation, in head kidney at 12 h post-stimulation, and in liver at
188
3 h and 6 h post-stimulation, respectively (Fig. 4B).
189 190
3.3. Luciferase assay on the activation of IFN promoter by tilapia DDX41
191
To determine whether tilapia DDX41 can induce the activation of IFN, the
192
constructed expression plasmid, pcDNA3.1-On-DDX41 was co-transfected with IFN
193
reporter plasmid in EPC cells for luciferase assay. As shown in Figure 5, the
194
overexpression of On-DDX41 provoked a strong activation of both IFN1 and IFN3
195
promoter in EPC cells treated with poly(dA:dT), by up to 18-fold and 39-fold against
196
the vector control, respectively (the fifth and sixth columns).
197 198
4. Discussion
199
DDX41 is a key cytosolic DNA sensor involved in the regulation of type I IFN
200
responses, and their functions have been well-characterized in mammals [14].
201
However, little information is available to date regarding the functions of fish DDX41.
202
In the present study, a DDX41 gene named as On-DDX41, was identified in tilapia.
203
The predicted protein of On-DDX41 possesses several structural features known in
204
DDX41, including DEADc and HELICc domains, and a conserved “D-E-A-D”
205
sequence [15]. In addition, phylogenetic analysis showed that On-DDX41 and
206
DDX41 in other fish were grouped together to form a separate clade in phylogenetic
207
tree, and syntenic analysis revealed that the linkage of DDX41 with DOK3 was
208
conserved throughout jawed vertebrate evolution. These results indicate that
209
On-DDX41 is indeed the ortholog of mammalian DDX41, and DDX41 is structurally
210
conserved from fish to mammals.
211
Previous studies have shown that fish DDX41 can be induced by the stimulation
212
of dsDNA virus infection or synthetic dsDNA analogue, and the induced expression of
213
type I IFNs and ISGs following the stimulation of dsDNA virus infection or synthetic
214
dsDNA analogue was significantly reduced by the knockdown of DDX41 in fish [20,
215
23], reflecting the importance of fish DDX41 in the viral dsDNA-induced type I IFN
216
responses. Interestingly, our data revealed that On-DDX41 is inducible in tilapia in
217
response to the stimulation of poly(I:C), a synthetic dsRNA analogue, which has been
218
proven to be recognized by RLRs in fish [38], implying that there may exist a
219
cross-talk between fish cytosolic DNA sensors and RLRs in the regulation of type I
220
IFN-mediated antiviral response.
221
According to conserved cysteine residue pattern, fish type I IFNs can be divided
222
into two main groups, group I and group II IFNs [3]. It is generally accepted that
223
considerable difference may exist in the receptor usage, expression pattern, and
224
functional properties of group I and II type I IFNs, as exemplified by the comparison
225
of zebrafish IFN1 (group I IFN) and IFN3 (group II IFN). Zebrafish IFN1 induces a
226
delayed but higher level of ISGs through a receptor complex composed of CRFB1 and
227
CRFB5, whereas IFN3 provokes a rapid and transient expression of ISGs through a
228
distinct receptor complex consisted of CRFB2 and CRFB5 [39-41]. Intriguingly, it is
229
revealed that both zebrafish IFN1 and IFN3 promoter can be markedly activated by
230
the overexpression of On-DDX41 in fish cells treated with poly(dA:dT), with a higher
231
level of IFN3 promoter activation, indicating that tilapia DDX41 may play essential
232
roles in viral dsDNA-induced type I IFN responses, and the distinct expression pattern
233
of fish group I and II type I IFNs during viral infection may partly result from the
234
regulation of DDX41.
235
In conclusion, a DDX41 gene, named On-DDX41, was identified in Nile tilapia,
236
O. niloticus. On-DDX41 gene was constitutively expressed in all tissues examined,
237
with the highest expression level observed in liver and muscle, and was inducible
238
after poly(I:C) stimulation. In addition, On-DDX41 has been proven to induce a
239
strong activation of both zebrafish IFN1 and IFN3 promoter in fish cells treated with
240
poly(dA:dT). The present study thus contributes to a better understanding of the
241
functional evolution of DDX41 in fish.
242 243
Acknowledgments
244
We thank all the laboratory members for their critical reviews and comments on
245
this manuscript. This work was financially supported by grants (No. 31320103913)
246
from the National Natural Science Foundation of China (NSFC), and also by China
247
Agriculture Research System (CARS-46), and by a special talent programme “One
248
Thing One Decision (Yishi Yiyi)”, and by the Shenzhen Science and Technology
249
Project (JCYJ20180306173022502).
250 251 252 253
Disclosures The authors have no financial conflicts of interest.
254
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recognizes the bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response, Nat Immunol. 13 (2012) 1155-1161. 20. R. Ge, Y. Zhou, R. Peng, R. Wang, M. Li, Y. Zhang, et al., Conservation of the STING-Mediated Cytosolic DNA Sensing Pathway in Zebrafish, J Virol. 89 (2015) 7696-7706. 21. N.T. Quynh, J. Hikima, Y.R. Kim, F.F. Fagutao, M.S. Kim, T. Aoki, et al., The cytosolic sensor, DDX41, activates antiviral and inflammatory immunity in response to stimulation with double-stranded DNA adherent cells of the olive flounder, Paralichthys olivaceus, Fish Shellfish Immunol. 44 (2015) 576-583. 22. J.M.S. Lazarte, Y.R. Kim, J.S. Lee, S.P. Im, S.W. Kim, J.W. Jung, et al., Enhancement of glycoprotein-based DNA vaccine for viral hemorrhagic septicemia virus (VHSV) via addition of the molecular adjuvant, DDX41, Fish Shellfish Immunol. 62 (2017) 356-365. 23. J.X. Ma, J.Y. Li, D.D. Fan, W. Feng, A.F. Lin, L.X. Xiang, et al., Identification of DEAD-Box RNA Helicase DDX41 as a Trafficking Protein That Involves in Multiple Innate Immune Signaling Pathways in a Zebrafish Model, Front Immunol. 9 (2018) 1327. 24. J. Liu, Y. Huang, X. Huang, C. Li, S.W. Ni, Y. Yu, et al., Grouper DDX41 exerts antiviral activity against fish iridovirus and nodavirus infection, Fish Shellfish Immunol. 91 (2019) 40-49. 25. M. Wang, M. Lu, Tilapia polyculture: a global review, Aquac Res. 47 (2016) 2363-2374. 26. A.M. Haygood, R. Jha, Strategies to modulate the intestinal microbiota of Tilapia (Oreochromis sp.) in aquaculture: a review, Rev Aquac. 10 (2018) 320-333. 27. Z. Gan, S. Chen, J. Hou, H. Huo, X. Zhang, B. Ruan, et al., Molecular and functional characterization of peptidoglycan-recognition protein SC2 (PGRP-SC2) from Nile tilapia (Oreochromis niloticus) involved in the immune response to Streptococcus agalactiae, Fish Shellfish Immunol. 54 (2016) 1-10. 28. Z. Gan, B. Wang, J. Tang, Y. Lu, J. Jian, Z. Wu, et al., Molecular characterization and expression of CD2 in Nile tilapia (Oreochromis niloticus) in response to Streptococcus agalactiae stimulus, Fish Shellfish Immunol. 50 (2016) 101-108. 29. M.D. Jansen, H.T. Dong, C.V. Mohan, Tilapia lake virus: a threat to the global tilapia industry?, Rev Aquac. (2018), https://doi.org/10.1111/raq.12254. 30. M. Shlapobersky, M.S. Sinyakov, M. Katzenellenbogen, R. Sarid, J. Don, R.R. Avtalion, Viral encephalitis of tilapia larvae: primary characterization of a novel herpes-like virus, Virology. 399 (2010) 239-247. 31. J. Keawcharoen, S. Techangamsuwan, A. Ponpornpisit, E.D. Lombardini, T. Patchimasiri, N. Pirarat, Genetic characterization of a betanodavirus isolated from a clinical disease outbreak in farm-raised tilapia Oreochromis niloticus (L.) in Thailand, J Fish Dis. 38 (2015) 49-54. 32. Z. Gan, B. Wang, Y. Lu, S. Cai, J. Cai, J. Jian, et al., Molecular characterization and expression of CD2BP2 in Nile tilapia (Oreochromis niloticus) in response to Streptococcus agalactiae stimulus, Gene. 548 (2014) 126-133. 33. Z. Gan, B. Wang, W. Zhou, Y. Lu, W. Zhu, J. Tang, et al., Molecular and functional characterization of CD59 from Nile tilapia (Oreochromis niloticus) involved in the immune response to Streptococcus agalactiae, Fish Shellfish Immunol. 44 (2015) 50-59. 34. Z.A. Laghari, S.N. Chen, L. Li, B. Huang, Z. Gan, Y. Zhou, et al., Functional, signalling and transcriptional differences of three distinct type I IFNs in a perciform fish, the mandarin fish Siniperca chuatsi, Dev Comp Immunol. 84 (2018) 94-108. 35. J. Hou, Z. Gan, S.N. Chen, P. Nie, Molecular and functional characterization of a short-type
342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362
peptidoglycan recognition protein, PGRP-S in the amphibian Xenopus laevis, Dev Comp Immunol. 98 (2019) 13-19. 36. K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆Ct method, Methods. 25 (2001) 402-408. 37. P.F. Zou, M.X. Chang, N.N. Xue, X.Q. Liu, J.H. Li, J.P. Fu, et al., Melanoma differentiation-associated gene 5 in zebrafish provoking higher interferon-promoter activity through signalling enhancing of its shorter splicing variant, Immunology. 141 (2014) 192-202. 38. M. Chang, B. Collet, P. Nie, K. Lester, S. Campbell, C.J. Secombes, et al., Expression and functional characterization of the RIG-I-like receptors MDA5 and LGP2 in Rainbow trout (Oncorhynchus mykiss), J Virol. 85 (2011) 8403-8412. 39. J.P. Levraud, P. Boudinot, I. Colin, A. Benmansour, N. Peyrieras, P. Herbomel, et al., Identification of the zebrafish IFN receptor: implications for the origin of the vertebrate IFN system, J Immunol. 178 (2007) 4385-4394. 40. D. Aggad, M. Mazel, P. Boudinot, K.E. Mogensen, O.J. Hamming, R. Hartmann, et al., The two groups of zebrafish virus-induced interferons signal via distinct receptors with specific and shared chains, J Immunol. 183 (2009) 3924-3931. 41. A. Lopez-Munoz, F.J. Roca, J. Meseguer, V. Mulero, New insights into the evolution of IFNs: zebrafish group II IFNs induce a rapid and transient expression of IFN-dependent genes and display powerful antiviral activities, J Immunol. 182 (2009) 3440-3449.
363
Figure Legends
364 365
Fig. 1. Multiple alignments of deduced DDX41 protein sequence in tilapia. Identical amino acids
366
are indicated with “*”, and amino acids with high and low similarity are indicated with “:” and “.”,
367
respectively. The DEADc and HELICc domains are highlighted in solid lines above the alignment,
368
and a strictly conserved sequence “Asp-Glu-Ala-Asp (D-E-A-D)” are in bold. The amino acid
369
sequences from different species are named accordingly and abbreviated as: human (H. sapiens, as
370
Hs), mouse (M. musculus, as Mm), zebrafish (D. rerio, as Dr), and Japanese flounder (P. olivaceus,
371
as Po).
372 373
Fig. 2. Phylogenetic tree of tilapia DDX41. The tree was constructed by using neighbor-joining
374
(NJ) method, and numbers at each branch in the NJ tree indicated the percentage bootstrap values
375
on 1000 replicates. The amino acid sequences from different species are named accordingly and
376
abbreviated as: human (H. sapiens, as Hs), mouse (M. musculus, as Mm), pig (Sus scrofa, as Ss),
377
cattle (Bos taurus, as Bt), chicken (G. gallus, as Gg), mallard (Anas platyrhynchos, as Ap),
378
band-tailed pigeon (Patagioenas fasciata, as Pf), green anole lizard (Anolis carolinensis, as Ac),
379
green sea turtle (Chelonia mydas, as Cm), Australian saltwater crocodile (Crocodylus porosus, as
380
Cp), zebrafish (D. rerio, as Dr), and Japanese flounder (P. olivaceus, as Po). Sequences used for
381
phylogenetic tree construction are given in Supplementary Table 2.
382 383
Fig. 3. Gene synteny analyses of DDX41 in vertebrates. DDX41 are marked using red, and
384
PRELID1, MXD3, and FAM193B are illustrated in dark, common and light blue, respectively;
385
DOK3 and PDLIM7 appear as dark and light purple, respectively, and neurogenin-2,
386
uncharacterized protein LOC112841964, UNC5A, and ATOH1B are dyed using white. All genes
387
are indicated with arrow symbols pointing to the transcription direction. Abbreviations for genes
388
used in this figure: PRELID1, PRELI domain containing 1; MXD3, MAX dimerization protein 3;
389
FAM193B, family with sequence similarity 193 member B; DOK3, docking protein 3; PDLIM7,
390
PDZ and LIM domain 7; UNC5A, unc-5 netrin receptor A; ATOH1B, atonal bHLH transcription
391
factor 1B.
392
393
Fig. 4. The expression of DDX41 gene in different organs/tissues of healthy tilapia (A) and tilapia
394
treated with poly(I:C) (B). Gene expression was measured by quantitative RT-PCR, and the
395
expression data were normalized against the level of β-actin. Injection of PBS served as control,
396
and fold changes were calculated relative to control group. Data were expressed as mean ± SE,
397
with * indicating p < 0.05.
398 399
Fig. 5. Luciferase assay on the induction of fish IFN promoter activity by On-DDX41. Detection
400
of luciferase activity in EPC cells which were transfected with IFNpro-Luc, 1 pRL-TK together
401
with pcDNA3.1-On-DDX41 or empty pcDNA3.1, with the stimulation of poly(dA:dT) or not.
402
Data were normalized to the Renilla luciferase activity, and fold changes were calculated relative
403
to control group transfected with empty vector pcDNA3.1. Data were expressed as mean ± SE,
404
with * indicating p < 0.05.
405 406 407
1
2 3 4 5
Figure 1
1
2 3 4 5 6
Figure 2
7 8
9 10 11
Figure 3
1
12 13
14 15 16
Figure 4
2
17 18
19 20 21
Figure 5
22
3
Highlights A DDX41 gene was identified and characterized in tilapia. Structurally, DDX41 is highly conserved from fish to mammals. Tilapia DDX41 was ubiquitously expressed in all tested tissues and up-regulated after poly(I:C) stimulation. Tilapia DDX41 elicits a strong activation of fish IFN promoter with the stimulation of poly(dA:dT).
S1050-4648(20)30117-0
DOI:
https://doi.org/10.1016/j.fsi.2020.02.031
Reference:
YFSIM 6830
To appear in:
Fish and Shellfish Immunology
Received Date: 1 December 2019 Revised Date:
30 January 2020
Accepted Date: 16 February 2020
Please cite this article as: Gan Z, Cheng J, Hou J, Xia H, Chen W, Xia L, Nie P, Lu Y, Molecular and functional characterization of tilapia DDX41 in IFN regulation, Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/j.fsi.2020.02.031. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Ltd.
1
Molecular and functional characterization of tilapia DDX41 in IFN regulation
2 a, b, c, d, e, 1
, Jun Cheng a, c, d, 1, Jing Hou a, b, c, d, e, Hongli Xia
3
Zhen Gan
4
Chen a, b, c, d, e, Liqun Xia a, c, d, Pin Nie b, f, *, Yishan Lu a, c, d, **
a, c, d
, Wenjie
5 6
a
7
b
8
Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
9
c
College of Fishery, Guangdong Ocean University, Zhanjiang 524025, China
10
d
Guangdong Provincial Engineering Research Center for Aquatic Animal Health
11
Assessment, and Shenzhen Public Service Platform for Evaluation of Marine
12
Economic Animal Seedings, Shenzhen 518120, China
13
e
14
Shenzhen 518120, China
15
f
16
Qingdao, Shandong Province, 266109, China
Shenzhen Institute of Guangdong Ocean University, Shenzhen 518120, China State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of
Shenzhen Dapeng New District Science and Technology Innovation Service Center,
School of Marine Science and Engineering, Qingdao Agricultural University,
17 18
*
19
7 of South Donghu Road, Wuchang District, Wuhan, Hubei Province, 430072, China.
20
E-mail addresses: [email protected]; [email protected] (P. Nie).
Corresponding author. Institute of Hydrobiology, Chinese Academy of Sciences, No.
21 22
**
23
Binhai 2nd Road, Dapeng New District, Shenzhen, Guangdong Province, 518120,
24
China.
25
E-mail address: [email protected] (Y. Lu).
Corresponding author. Shenzhen Institute of Guangdong Ocean University, No. 3 of
26 27 28
1
Indicating the two first authors.
1
ABSTRACT
2
DEAD-box helicase 41 (DDX41) is a key cytosolic DNA sensor playing critical
3
roles in the regulation of type I IFN responses, and their functions have been
4
well-characterized in mammals. However, little information is available regarding the
5
function of fish DDX41. In this study, a DDX41 gene, named On-DDX41, was
6
identified in Nile tilapia, Oreochromis niloticus. The predicted protein of On-DDX41
7
contains several structural features known in DDX41, including conserved DEADc
8
and HELICc domains, and a conserved sequence “Asp-Glu-Ala-Asp (D-E-A-D)”.
9
On-DDX41 gene was constitutively expressed in all tissues examined, with the
10
highest expression level observed in liver and muscle, and was inducible after
11
poly(I:C) stimulation. Moreover, the overexpression of On-DDX41 can elicit a strong
12
activation of both zebrafish IFN1 and IFN3 promoter in fish cells treated with
13
poly(dA:dT). The present study thus contributes to a better understanding of the
14
functional properties of DDX41 in fish.
15 16
Keywords: Oreochromis niloticus; DDX41; cytosolic DNA sensors; type I interferon;
17
promoter activity.
18 19 20
21
1. Introduction
22
Type I interferons (IFNs) are a subset of pleiotropic cytokines playing central
23
roles in antiviral immune defense [1, 2]. From an evolutionary point of view, it is
24
generally accepted that type I IFNs are ubiquitously present in all classes of jawed
25
vertebrates [3-6]. However, large divergence may exist in the composition of type I
26
IFNs from different classes of vertebrates. Specifically, jawed fish may harbor most
27
primitive type I IFNs, which are encoded by intron-containing genes consisting of 5
28
exons and 4 introns [3, 7-9], whereas type I IFN genes in amniotes are intronless [6,
29
10]. Interestingly, recent studies have revealed that intronless and intron-containing
30
type I IFN genes coexist in amphibians, and the difference in genomic organisations
31
of vertebrate type I IFNs may be resulted from three retroposition events occurred in
32
the clawed frog Xenopus and the Tibetan frog Nanorana parkeri, and amniotes,
33
respectively [4, 5].
34
In spite of considerate divergence exist among type I IFN repertoire in different
35
vertebrates, the main regulatory mechanisms of type I IFN responses appear to
36
conserve from fish to mammals [3]. It is believed that three major families of pattern
37
recognition receptors (PRRs) are involved in the regulation of type I IFN production
38
[11-13]. The first class of PRRs is retinoic acid-inducible gene I (RIG-I)-like
39
receptors (RLRs), which consists of three members, including RIG-I, melanoma
40
differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology
41
2 (LGP2) [11]. The second category of viral nucleic acid sensors is some members
42
from toll-like receptors (TLRs), such as TLR3, TLR7, TLR8, and TLR9 [12]. The
43
third class of PRRs is a series of cytosolic DNA sensors, in which DEAD-box
44
helicase 41 (DDX41) is a critical regulator of type I IFN responses [14].
45
Structurally, as a member of the DExD/H-box helicases superfamily, DDX41 is
46
characterized by two conserved RecA-like domains, including DEADc and HELICc
47
domains, and the former contains a strictly conserved sequence “Asp-Glu-Ala-Asp
48
(D-E-A-D)” [15-18]. Upon detecting viral double-stranded DNAs (dsDNAs) or
49
bacterial cyclic dinucleotides (CDNs), DDX41 can interact with the stimulator of IFN
50
genes (STING) to activate TANK-binding kinase 1 (TBK1). These activated TBK1
51
phosphorylates interferon regulatory factor 3/7 (IRF3/7), eventually elicit the
52
production of type I IFNs and interferon-stimulated genes (ISGs) [14, 19]. Although
53
functional properties and signalling pathways of DDX41 have been well-characterized
54
in mammals, little information is available to date regarding fish DDX41. DDX41 has
55
only been cloned from a few species of teleosts, including zebrafish (Danio rerio),
56
Japanese flounder (Paralichthys olivaceus), and orange spotted grouper (Epinephelus
57
coioides) [20-24], and the studies about functional properties of fish DDX41 is rather
58
limited.
59
Tilapia (Oreochromis spp.) is the second farmed fish species worldwide, serving
60
as an important protein source in many countries [25-28]. In recent years, a series of
61
emerging viral diseases caused by tilapia lake virus (TiLV) and tilapia larvae
62
encephalitis virus (TLEV) have been severe, posing a potential threat to the global
63
tilapia industry [29-31]. To prevent the outbreak of above viral diseases has become a
64
major task for the sustainable development of tilapia industry, and the understanding
65
of their immune system, particularly their antiviral immune system, may provide clues
66
for the development of strategies on the prevention of such diseases. In this study, a
67
DDX41 gene (named On-DDX41) was cloned from Nile tilapia, O. niloticus, and its
68
expression profiles and functional properties were investigated. The present results
69
thus contribute to better understanding of the mechanism of antiviral immune
70
responses in tilapia.
71 72
2. Materials and methods
73
2.1. Experimental animals and cells
74
Samples of tilapia (average weight of 100 ± 10 g) were obtained from a
75
commercial
farm
in
Guangzhou,
Guangdong
province,
China.
Prior
to
76
experimentation, fish were acclimated in fiber-reinforced plastic tanks (1000 L each)
77
with a stocking rate of 4 g L−1 under 28 ± 2 °C for 4 weeks [32, 33]. All animal
78
protocols were performed following the Guide for the Care and Use of Laboratory
79
Animals of the Guangdong Ocean University and approved by the Shenzhen Institute
80
of Guangdong Ocean University. Epithelioma papulosum cyprini (EPC) cells were
81
cultured in medium 199 (Life Technologies) supplemented with 10% FBS (Life
82
Technologies), 100 U/ml penicillin, and 100 µg/ml streptomycin at 26 °C with 5%
83
CO2.
84 85
2.2. Cloning of DDX41 gene in tilapia
86
The putative open reading frame (ORF) of the DDX41 gene, named On-DDX41,
87
were predicted by in silico analysis. By using Trizol Reagent (Invitrogen), total RNA
88
from various tissues including spleen, kidney, and liver of healthy tilapia was
89
extracted and mixed. For first-strand cDNA synthesis, above mixed total RNA was
90
treated with DNase I to remove genomic DNA contamination, before being reverse
91
transcribed using RevertAid™ First Strand cDNA Synthesis Kit (Thermo Fisher
92
Scientific). The first-strand cDNA was served as template to amplify ORF sequence of
93
On-DDX41 by PCR using specific primers designed according to predicted data. All
94
PCR products were ligated into the pMD18-T vector (TaKaRa) and transformed into
95
competent Escherichia coli cells. Then the positive clones were sequenced by Sangon
96
Biotech (Shanghai, China). The sequences of all PCR primers used in this study are
97
summarized in Supplementary Table 1.
98 99
2.3. Sequence analysis of DDX41 gene in tilapia
100
The protein sequences were deduced from the nucleic acid sequences using the
101
program on the Expasy Web site (http://ca.expasy.org/tools), and multiple sequence
102
alignments for amino acids were generated using the Clustal X program. Homology
103
between sequences was calculated using the Megalign program within the DNASTAR
104
package, and location of domains was predicted using the InterProScan program
105
(http://www.ebi.ac.uk/Tools/pfa/iprscan/). Phylogenetic tree for On-DDX41 was
106
constructed using MEGA5 package with neighbor-joining (NJ) algorithm, with 1000
107
time repeat of bootstrap analysis. Chromosomal locations of DDX41 genes of tilapia,
108
zebrafish (Danio rerio), chicken (Gallus gallus), mouse (Mus musculus), and human
109
(Homo
110
(https://www.ncbi.nlm.nih.gov/projects/sviewer/). All sequences used in the analysis
sapiens)
were
obtained
from
the
Sequence
Viewer
111
were listed in Supplementary Table 2.
112 113
2.4. Quantitative analysis of DDX41 mRNA expression in tilapia
114
For analyzing the expression of DDX41 gene in healthy tilapia, organs/tissues
115
were collected from three animals to extract total RNA with Trizol. For analyzing the
116
expression
117
intraperitoneally with polyinosinic:polycytidylic acid [poly(I:C); Sigma; 10 µg/g body
118
weight] and were divided into four groups, each with three individuals. 12 healthy
119
individuals as control were injected intraperitoneally with phosphate buffered saline
120
(PBS) and were also divided into four groups. At 3, 6, 12 and 24 hours post-injection
121
(hpi), three animals in each group were scarified for extracting RNA with Trizol from
122
organs/tissues. Total RNA from organs/tissues of different individuals was used
123
separately for the synthesis of the first strand cDNA.
after
stimulation,
12
healthy
individuals
were
each
injected
124
Specific primers (Supplementary Table 1) were used to amplify DDX41 and
125
β-actin fragments by PCR, and the PCR products were sequenced as described above.
126
The copy number was calculated following a method described previously [34, 35].
127
To establish a standard curve, sequenced plasmid DNA of positive clones was
128
extracted with Plasmid Mini Kit (Omega Bio-tek) and was diluted in serial 10-fold
129
dilution, ranging from 10−6 to 10−2, before being quantified with PCR using CFX96
130
Real-Time PCR Detection System (BioRad). A final volume of 20 µl PCR reaction
131
system contained 1 µl cDNA template, 1 µl of each primers, 7 µl sterile water, and 10
132
µl iQTM SYBR® Green Supermix (BioRad), with the PCR protocol as the followings:
133
one cycle of 95 °C for 3 min, followed by 45 cycles of 95 °C for 10 s, 57 °C for 20 s
134
and 72 °C for 40 s. Each sample was run in triplicate, and the gene expression for
135
each sample was normalized against β-actin. Data analysis was performed using the
136
2−∆∆Ct method [36], and fold changes were calculated relative to control group.
137 138
2.5. Examination of type I IFN promoter activation by DDX41 from tilapia
139
For determining the type I IFN promoter activation by tilapia DDX41, luciferase
140
activity assay was transfected into EPC cells as previously reported with minor
141
modification [37]. Briefly, the entire ORF of On-DDX41 was amplified and
142
subcloned into pcDNA3.1/myc-His (-) A vector (Invitrogen; called pcDNA3.1 in this
143
research), which was named as pcDNA3.1-On-DDX41. The EPC cells seeded in
144
24-well plates (2 × 105 cells/well) at were co-transfected with 100 ng IFN1pro-Luc or
145
IFN3pro-Luc, two zebrafish type I IFN promoter-driving luciferase plasmid [37],
146
together with 200 ng pcDNA3.1-On-DDX41 or empty pcDNA3.1 (as vector control),
147
and 10 ng pRL-TK (Promega), following the protocol of Lipofectamine 2000
148
Transfection Reagent (Invitrogen), with the stimulation of poly(dA:dT) (1 µg/ml,
149
Sigma) or not. 24 hours post-transfection, the cells were harvested and lysed using
150
Dual-Luciferase Reporter System (Promega), with luciferase activity measured on a
151
Junior LB9509 luminometer (Berthold, Germany). Data were normalized to the
152
Renilla Luciferase activity, and fold changes were calculated relative to control group
153
transfected with empty vector pcDNA3.1.
154 155
2.6. Statistical analysis
156
Statistical analyses were based on three repeated experiments, with one-way
157
ANOVA carried out in SPSS 17.0. Data were presented as mean ± standard error, and
158
statistical significance was defined as P < 0.05.
159 160
3. Results
161
3.1. Coning and sequence analysis of DDX41 gene in tilapia
162
By searching the tilapia genome, a DDX41 gene, named On-DDX41, was
163
identified on chromosome LG2 (NCBI Reference Sequence: NC_031966.2). To test
164
whether predicted DDX41 gene are expressed in vivo at transcription level, the ORF
165
sequence of DDX41 gene was cloned from tilapia, with the following accession
166
number in GenBank: MN729497. The ORF of On-DDX41 is 1845 bp in size, which
167
encodes a protein of 614 amino acids. On-DDX41 share very high identity, being
168
85.8–86.2%, 86.8–87.3%, 85.2–86.8%, and 93.8–97.1%, with DDX41 in mammals,
169
birds, reptiles and other fish, respectively (Supplementary Figure 1), and several
170
important structural characteristics known in DDX41 are conserved in predicted
171
protein of On-DDX41, including two conserved RecA-like domains, DEADc and
172
HELICc domains, and a strictly conserved sequence “Asp-Glu-Ala-Asp (D-E-A-D)”
173
(Fig. 1).
174
To understand the phylogenetic relationship between On-DDX41 and DDX41 in
175
other vertebrates, a NJ tree was constructed using protein sequences. In general, all
176
the DDX41 in tetrapods were clustered into a major clade at the top of the
177
phylogenetic tree, and On-DDX41 and DDX41 in other fish were grouped together to
178
form a separate clade with the support of high bootstrap value (Fig. 2). In addition, the
179
analysis of the genes flanking DDX41 revealed that On-DDX41 was closely linked to
180
docking protein 3 (DOK3) gene, which was similar to zebrafish, chicken, mouse, and
181
human DDX41 (Fig. 3).
182 183
3.2. Expression pattern of DDX41 gene in tilapia
184
In healthy tilapia, the mRNA of DDX41 was detected in all organs/tissues
185
examined, with the highest level of DDX41 observed in liver (Fig. 4A). Following
186
poly(I:C) stimulation, the transcript level of DDX41 was significantly up-regulated in
187
spleen at 6 h post-stimulation, in head kidney at 12 h post-stimulation, and in liver at
188
3 h and 6 h post-stimulation, respectively (Fig. 4B).
189 190
3.3. Luciferase assay on the activation of IFN promoter by tilapia DDX41
191
To determine whether tilapia DDX41 can induce the activation of IFN, the
192
constructed expression plasmid, pcDNA3.1-On-DDX41 was co-transfected with IFN
193
reporter plasmid in EPC cells for luciferase assay. As shown in Figure 5, the
194
overexpression of On-DDX41 provoked a strong activation of both IFN1 and IFN3
195
promoter in EPC cells treated with poly(dA:dT), by up to 18-fold and 39-fold against
196
the vector control, respectively (the fifth and sixth columns).
197 198
4. Discussion
199
DDX41 is a key cytosolic DNA sensor involved in the regulation of type I IFN
200
responses, and their functions have been well-characterized in mammals [14].
201
However, little information is available to date regarding the functions of fish DDX41.
202
In the present study, a DDX41 gene named as On-DDX41, was identified in tilapia.
203
The predicted protein of On-DDX41 possesses several structural features known in
204
DDX41, including DEADc and HELICc domains, and a conserved “D-E-A-D”
205
sequence [15]. In addition, phylogenetic analysis showed that On-DDX41 and
206
DDX41 in other fish were grouped together to form a separate clade in phylogenetic
207
tree, and syntenic analysis revealed that the linkage of DDX41 with DOK3 was
208
conserved throughout jawed vertebrate evolution. These results indicate that
209
On-DDX41 is indeed the ortholog of mammalian DDX41, and DDX41 is structurally
210
conserved from fish to mammals.
211
Previous studies have shown that fish DDX41 can be induced by the stimulation
212
of dsDNA virus infection or synthetic dsDNA analogue, and the induced expression of
213
type I IFNs and ISGs following the stimulation of dsDNA virus infection or synthetic
214
dsDNA analogue was significantly reduced by the knockdown of DDX41 in fish [20,
215
23], reflecting the importance of fish DDX41 in the viral dsDNA-induced type I IFN
216
responses. Interestingly, our data revealed that On-DDX41 is inducible in tilapia in
217
response to the stimulation of poly(I:C), a synthetic dsRNA analogue, which has been
218
proven to be recognized by RLRs in fish [38], implying that there may exist a
219
cross-talk between fish cytosolic DNA sensors and RLRs in the regulation of type I
220
IFN-mediated antiviral response.
221
According to conserved cysteine residue pattern, fish type I IFNs can be divided
222
into two main groups, group I and group II IFNs [3]. It is generally accepted that
223
considerable difference may exist in the receptor usage, expression pattern, and
224
functional properties of group I and II type I IFNs, as exemplified by the comparison
225
of zebrafish IFN1 (group I IFN) and IFN3 (group II IFN). Zebrafish IFN1 induces a
226
delayed but higher level of ISGs through a receptor complex composed of CRFB1 and
227
CRFB5, whereas IFN3 provokes a rapid and transient expression of ISGs through a
228
distinct receptor complex consisted of CRFB2 and CRFB5 [39-41]. Intriguingly, it is
229
revealed that both zebrafish IFN1 and IFN3 promoter can be markedly activated by
230
the overexpression of On-DDX41 in fish cells treated with poly(dA:dT), with a higher
231
level of IFN3 promoter activation, indicating that tilapia DDX41 may play essential
232
roles in viral dsDNA-induced type I IFN responses, and the distinct expression pattern
233
of fish group I and II type I IFNs during viral infection may partly result from the
234
regulation of DDX41.
235
In conclusion, a DDX41 gene, named On-DDX41, was identified in Nile tilapia,
236
O. niloticus. On-DDX41 gene was constitutively expressed in all tissues examined,
237
with the highest expression level observed in liver and muscle, and was inducible
238
after poly(I:C) stimulation. In addition, On-DDX41 has been proven to induce a
239
strong activation of both zebrafish IFN1 and IFN3 promoter in fish cells treated with
240
poly(dA:dT). The present study thus contributes to a better understanding of the
241
functional evolution of DDX41 in fish.
242 243
Acknowledgments
244
We thank all the laboratory members for their critical reviews and comments on
245
this manuscript. This work was financially supported by grants (No. 31320103913)
246
from the National Natural Science Foundation of China (NSFC), and also by China
247
Agriculture Research System (CARS-46), and by a special talent programme “One
248
Thing One Decision (Yishi Yiyi)”, and by the Shenzhen Science and Technology
249
Project (JCYJ20180306173022502).
250 251 252 253
Disclosures The authors have no financial conflicts of interest.
254
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363
Figure Legends
364 365
Fig. 1. Multiple alignments of deduced DDX41 protein sequence in tilapia. Identical amino acids
366
are indicated with “*”, and amino acids with high and low similarity are indicated with “:” and “.”,
367
respectively. The DEADc and HELICc domains are highlighted in solid lines above the alignment,
368
and a strictly conserved sequence “Asp-Glu-Ala-Asp (D-E-A-D)” are in bold. The amino acid
369
sequences from different species are named accordingly and abbreviated as: human (H. sapiens, as
370
Hs), mouse (M. musculus, as Mm), zebrafish (D. rerio, as Dr), and Japanese flounder (P. olivaceus,
371
as Po).
372 373
Fig. 2. Phylogenetic tree of tilapia DDX41. The tree was constructed by using neighbor-joining
374
(NJ) method, and numbers at each branch in the NJ tree indicated the percentage bootstrap values
375
on 1000 replicates. The amino acid sequences from different species are named accordingly and
376
abbreviated as: human (H. sapiens, as Hs), mouse (M. musculus, as Mm), pig (Sus scrofa, as Ss),
377
cattle (Bos taurus, as Bt), chicken (G. gallus, as Gg), mallard (Anas platyrhynchos, as Ap),
378
band-tailed pigeon (Patagioenas fasciata, as Pf), green anole lizard (Anolis carolinensis, as Ac),
379
green sea turtle (Chelonia mydas, as Cm), Australian saltwater crocodile (Crocodylus porosus, as
380
Cp), zebrafish (D. rerio, as Dr), and Japanese flounder (P. olivaceus, as Po). Sequences used for
381
phylogenetic tree construction are given in Supplementary Table 2.
382 383
Fig. 3. Gene synteny analyses of DDX41 in vertebrates. DDX41 are marked using red, and
384
PRELID1, MXD3, and FAM193B are illustrated in dark, common and light blue, respectively;
385
DOK3 and PDLIM7 appear as dark and light purple, respectively, and neurogenin-2,
386
uncharacterized protein LOC112841964, UNC5A, and ATOH1B are dyed using white. All genes
387
are indicated with arrow symbols pointing to the transcription direction. Abbreviations for genes
388
used in this figure: PRELID1, PRELI domain containing 1; MXD3, MAX dimerization protein 3;
389
FAM193B, family with sequence similarity 193 member B; DOK3, docking protein 3; PDLIM7,
390
PDZ and LIM domain 7; UNC5A, unc-5 netrin receptor A; ATOH1B, atonal bHLH transcription
391
factor 1B.
392
393
Fig. 4. The expression of DDX41 gene in different organs/tissues of healthy tilapia (A) and tilapia
394
treated with poly(I:C) (B). Gene expression was measured by quantitative RT-PCR, and the
395
expression data were normalized against the level of β-actin. Injection of PBS served as control,
396
and fold changes were calculated relative to control group. Data were expressed as mean ± SE,
397
with * indicating p < 0.05.
398 399
Fig. 5. Luciferase assay on the induction of fish IFN promoter activity by On-DDX41. Detection
400
of luciferase activity in EPC cells which were transfected with IFNpro-Luc, 1 pRL-TK together
401
with pcDNA3.1-On-DDX41 or empty pcDNA3.1, with the stimulation of poly(dA:dT) or not.
402
Data were normalized to the Renilla luciferase activity, and fold changes were calculated relative
403
to control group transfected with empty vector pcDNA3.1. Data were expressed as mean ± SE,
404
with * indicating p < 0.05.
405 406 407
1
2 3 4 5
Figure 1
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2 3 4 5 6
Figure 2
7 8
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Figure 3
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12 13
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Figure 4
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Figure 5
22
3
Highlights A DDX41 gene was identified and characterized in tilapia. Structurally, DDX41 is highly conserved from fish to mammals. Tilapia DDX41 was ubiquitously expressed in all tested tissues and up-regulated after poly(I:C) stimulation. Tilapia DDX41 elicits a strong activation of fish IFN promoter with the stimulation of poly(dA:dT).