identification and expression of the laboratory of genetics and ...€¦ · in this study, a...

Journal of Fish Biology (2014)

doi:10.1111/jfb.12541, available online at wileyonlinelibrary.com

Identification and expression of the laboratory of geneticsand physiology 2 gene in common carp Cyprinus carpio

X. L. Cao*†, J. J. Chen†, Y. Cao†, G. X. Nie†, Q. Y. Wan*, L. F. Wang†and J. G. Su*‡

*College of Animal Science and Technology, Northwest A&F University, Yangling 712100,People’s Republic of China and †College of Fisheries, Henan Normal University, Xinxiang

453007, People’s Republic of China

(Received 29 April 2014, Accepted 9 September 2014)

In this study, a laboratory of genetics and physiology 2 gene (lgp2) from common carp Cyprinus car-pio was isolated and characterized. The full-length complementary (c)DNA of lgp2 was 3061 bp andencoded a polypeptide of 680 amino acids, with an estimated molecular mass of 77 341⋅2 Da and apredicted isoelectric point of 6⋅53. The predicted protein included four main overlapping structuraldomains: a conserved restriction domain of bacterial type III restriction enzyme, a DEAD–DEAHbox helicase domain, a helicase super family C-terminal domain and a regulatory domain. Real-timequantitative polymerase chain reaction (PCR) showed widespread expression of lgp2, mitochondrialantiviral signalling protein (mavs) and interferon transcription factor 3 (irf3) in tissues of nine organs.lgp2, mavs and irf3 expression levels were significantly induced in all examined organs by infec-tion with koi herpesvirus (KHV). lgp2, mavs and irf3 messenger (m)RNA levels were significantlyup-regulated in vivo after KHV infection, and lgp2 transcripts were also significantly enhanced in vitroafter stimulation with synthetic, double-stranded RNA polyinosinic polycytidylic [poly(I:C)]. Thesefindings suggest that lgp2 is an inducible protein involved in the innate immune defence against KHVin C. carpio. These results provide the basis for further research into the role and mechanisms of lgp2in fishes.

© 2014 The Fisheries Society of the British Isles

Key words: gene cloning; KHV; lgp2; mRNA expression.

INTRODUCTION

Common carp Cyprinus carpio L. 1758 is one of the most important aquaculture fishspecies worldwide. Viral infections including koi C. carpio herpesvirus (KHV) haverecently become a major problem in the C. carpio aquaculture industry. KHV is knownto cause gill and skin damage in koi and common carp. The disease was first recog-nized in Israel and the U.S.A. (Hedricka et al., 2000), and has since has been reportedin Europe, including Germany (Bretzinger et al., 1999) and the U.K. (Gilad et al.,2003). In Asia, KHV has been reported in Indonesia (Rukyani, 2002), Japan (Sanoet al., 2002) and Taiwan (Tu et al., 2004) and is believed to have been imported intoChina. Rapid spread of the disease is associated with the import and export of ornamen-tal fish. KHV is a linear, double-stranded (ds) DNA virus with a genome of 125–290

‡Author to whom correspondence should be addressed. Tel.: +86 29 87092139; email: [email protected]

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© 2014 The Fisheries Society of the British Isles

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kbp contained within a T = 16 icosahedral capsid and surrounded by a proteinaceousmatrix (the tegument) and a lipid envelope containing membrane-associated proteins(Zhou et al., 2000).

A better understanding of the immune response of C. carpio against this virus isessential to its prevention and the development of a healthy aquaculture industry. Therecognition of viruses by host cells is mediated by pathogen recognition receptors thatsense virus-specific pathogen-associated molecular patterns, typically viral nucleicacids. The nucleic acid sensing transmembrane toll-like receptors (TLR), tlr3, 7, 8,and 9, localize to intracellular compartments and have well-known ligand specificities:tlr3 detects dsRNA, tlr7 and tlr8 detect single-stranded (ss) RNA and tlr9 responds topatterns found in microbial DNA and oligodeoxynucleotides encoding unmethylatedCpG motifs (Takeda & Akira, 2007). In contrast, cytosolic RNA ligands are recog-nized by the retinoic acid-inducible gene-1-like receptor (RLR, rig-I) and melanomadifferentiation-associated gene 5 (mda5) (Yoneyama et al., 2004, 2005). rig-I andmda5 are characterized by a core DExH box domain fused to tandem N-terminalcaspase activation and recruitment domain (CARD) motifs that are essential forpropagating downstream signal transduction. A third RLR-related gene, laboratory ofgenetics and physiology 2 (lgp2), shows high sequence similarity to mda5 and rig-IDExH box domains but lacks N-terminal CARD homology. Expression of lgp2 from aplasmid vector negatively regulates interferon (ifn) production and antiviral signalling(Rothenfusser et al., 2005; Yoneyama et al., 2005; Komuro & Horvath, 2006; Saitoet al., 2007), but analyses using mice deficient in Lgp2 indicated disparate functionsof Lgp2 in response to different viruses (Venkataraman et al., 2007). Recent evidencesuggests that Lgp2 acts as a positive regulator of Ifn responses to diverse viruses andmay act in concert with mda5 and rig-I (Saito et al., 2007). Although the structureand function of lgp2 have been described, its role in the response to DNA viruses iscurrently unknown. Its molecular description and expression profile have not beenreported in C. carpio.

In this study, the C. carpio lgp2 gene was cloned and assayed and its tissue distribu-tion and downstream associated gene mRNA expression profiles (including mavs andirf3 mRNAs, GenBank accession numbers: HQ850440.1 and HQ850443.1) analysedfollowing KHV challenge. The lgp2 mRNA time-dependent expression in epitheliomapapulosum cyprini (EPC) cells after polyinosinic polycytidylic [poly(I:C)] stimulationor KHV infection was also examined. The results of this study will provide the basisfor understanding the function of lgp2 in cellular responses to dsDNA viruses, and willexpand knowledge regarding innate immune mechanisms in teleosts.

MATERIALS AND METHODS

F I S H, V I RU S D E T E C T I O N, I M M U N E C H A L L E N G E A N D S A M P L EC O L L E C T I O N

Nine month-old C. carpio were supplied by the Henan Institute of Aquaculture, Henan, China.They were maintained in indoor tanks equipped with a recirculating water system at a watertemperature of 25∘ C for 7 days prior to experimentation. The fish were handled according tothe guidelines of the China Law for Animal Health Protection and Instructions for GrantingPermits for Animal Experimentation for Scientific Purposes [ethics approval number: SCXK(YU) 2005-0001].

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, doi:10.1111/jfb.12541

L G P2 A N T I V I R A L R E S P O N S E 3

The putative KHV outbreak was found when diseased fish showed clinical signs includinglethargy, swimming at the water surface, dark or reddened skin, sandpaper-like skin lesions anddeep-red gills with pale and white patches. KHV was confirmed by polymerase chain reaction(PCR)-based methods. DNA was extracted from gill, spleen and kidney of infected fish usingDNAzol (Invitrogen; www.lifetechnologies.com), according to the manufacturer’s protocols.The specific primer set, HV-F (forward) and HV-R (reverse), developed by Gray et al. (2002)was used to amplify a 290 bp fragment. DNA amplification was carried out according to theManual of Diagnostic Tests for Aquatic Animals (Anon., 2009). The PCR product was subjectedto electrophoresis, and the results were analysed by gel imaging and analysis.

Cyprinus carpio showing various degrees of gill damage were used for virus isolation. Gill,kidney and spleen of diseased fish were removed aseptically and pooled, the pooled tissue wasground into a smooth paste in a cold sterile mortar, and the resultant homogenate was resus-pended in 9 ml of Hank’s balanced salt solution (HBSS, Gibco; www.lifetechnologies.com)supplemented with 2% foetal bovine serum (FBS). The tissue suspension was centrifuged(1500 g) at 4∘ C for 15 min to pellet tissue debris. The clarified supernatant was diluted1:5 (v/v) with supplemented HBSS and filtered through a 0⋅45 μm membrane filter toremove any bacterial contamination. The filtrate was stored at 4∘ C for KHV challenge.For viral infection, C. carpio weighing 100–150 g were injected intraperitoneally with250–300 μl of KHV at a dose of 104 tissue culture infective dose50 per g body mass. Acontrol group was injected with an equal amount of saline. At 72 h post-infection (hpi),reverse transcription (RT)-PCR and nested PCR were performed to confirm KHV infection.Fish were anaesthetized with 100 mg l−1 of MS-222 and dissected, and tissue samples werecollected at 0, 24, 48 and 72 hpi (three C. carpio for each time interval) for total RNAisolation.

R NA E X T R AC T I O N A N D CD NA S Y N T H E S I S

Total RNA extraction was performed using Triazol reagent, according to the manu-facturer’s instructions (TaKaRa; www.takara-bio.com). The quality and concentration ofthe total RNA were determined by gel electrophoresis and ultraviolet spectrophotometry.Three micrograms of total RNA was incubated with RNase-free DNase I (MBI Fermen-tas; www.thermoscientificbio.com) to remove genomic DNA and then reverse transcribedinto cDNA using Oligo (dT) 20 primer and MMLV reverse transcriptase, according to themanufacturer’s instructions (TaKaRa).

G E N E C L O N I N G A N D S E Q U E N C I N G

The lgp2 cDNA sequence from C. carpio was cloned using degenerate primers designedbased on multiple alignments with DHX58 in zebra fish Danio rerio (Hamilton 1822) (accessionnumber: NM-001257157.1), channel catfish Ictalurus punctatus (Rafinesque 1818) (accessionnumber: JQ008941.1), Japanese flounder Paralichthys olivaceus (Temminck & Schlegel 1846)(accession number: HM070372.1), grass carp Ctenopharyngodon idella (Valenciennes 1844)(accession number: FJ813483.2), Atlantic salmon Salmo salar L. 1758 (accession number:BT045378.1) and goldfish Carassius auratus (L. 1758) (accession number: JF970227.1). PCRwas performed with the degenerate primers LGP2F (forward) and LGP2R (reverse) (Table I),using the cDNA generated from C. carpio spleen. The PCR programme was one cycle at 94∘C for 3 min; 35 cycles at 94∘ C for 30 s, 58∘ C for 30 s and 72∘ C for 1 min and one cycle at72∘ C for 10 min. PCR products were then purified from agarose gels using a DNA Gel Extrac-tion Kit (Axygen Scientific, Inc.; www.axygen.com). The purified DNA fragments were ligatedinto pDM19-Tvectors (TaKaRa), transformed into competent Escherichia coli DH5a cells andincubated on Luria–Bertani (LB) agar plates. Positive colonies were screened by colony PCRand sent to a commercial company (Beijing Genomics Institute; www.economicexpert.com) forsequencing. Full-length cDNA sequences of C. carpio lgp2 were acquired by rapid amplifica-tion of cDNA end (RACE) using the 5′ and 3′ full RACE Kit with TAP (TaKaRa), according tothe manufacturer’s instructions. To obtain the unknown 3′ region, primer pairs LGP2-3-innerand LGP2-3-outer (Table I) were used for primary PCR and nested PCR, respectively. Theamplified PCR product was cloned and sequenced as described above. Similarly, the 5′ end


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Table I. Primer sequences used in this study

Primer name Sequence(5′-3′)

Amplicon length (nt)and primerinformation

lgp2LGP2F (forward) CATHATMTGGCTKCCYAC Gene clone

LGP2R (reverse) AAGGCRTCCABCATDCGSAC 855 bpLGP2-3-inner GGCAATACAACGATGCTCTTCTC 3′ RACELGP2-3-outer ACCACTTGAAGGCAATGATGTT 2133 bpL5-inner CCAGGTGTTTCTTGGTGATGTA 5′ RACEL5-outer GTGACATTCGTCTATTACCA 254 bp3′ RACE outer primer TACCGTCGTTCCACTAGTGATTT3′ RACE inner primer CGCGGATCCTCCACTAGTGATTTCA

CTATAGG5′ RACE outer primer CATGGCTACATGCTGACAGCCTA5′ RACE inner primer CGCGGATCCACAGCCTACTGATGA

TCAGTCGATGCF (forward) GCCTGACGACGTGCTGGAC Confirming sequenceCR (reverse) CTAGATGTGATATACAGACTCAT 2912EF1𝛼-F GTGCCGTGCTGATTGTTGCT ef1𝛼EF1𝛼-R AAAGCCAGGAGGGCGTGTT 91 bpHV-F (forward) GACACCACATCTGCAAGGAG KHVHV-R (reverse) GACACATGTTACAATGGTCGC 292 bpq-PCR𝛽-actin-F GATGATGAAATTGCCGCACTG 𝛽-actin𝛽-actin-R ACCAACCATGACACCCTGATGT 151 bpL-qF CACAGCCAATGCCAAAGTAGLqR GCAGGGTAAAGTCAGTCAGCI-qF CTGTTGGGTTGCCAGTTTTCI-qR CAGCATTGTATGGAGGGTCTIRF-32F CAGGCATACGGAGGACATTIRF-32R TGGCTTCAGGTCTGTTTTTG

Note that H=A/C/T, M=A/C, K=G/T, Y=C/T, R=A/G, B=C/G/T, D=A/G /T, S=C/G. KHV, koiherpesvirus; RACE, rapid amplification of cDNA end; q-PCR, real-time polymerase chain reaction.

of lgp2 was obtained by nested PCR using primer pairs L5-inner and L5-outer (Table I). Thefull-length cDNA sequence was confirmed by sequencing the PCR product amplified by CF(forward) and CR (reverse) primers (Table I) within the predicted 5′ and 3′ untranslated regions(UTR), respectively.

S E Q U E N C E A NA LY S E S

Sequence homology was determined using the basic local alignment search tool (BLAST)programme (www.ncbi.nlm.nih.gov/blast) and matrix global alignment tool (MatGAT)(http://Bitincka.com/ledion/matgat/). Protein structure was predicted using the expert pro-tein analysis system (www.expasy.org) and sequence manipulation suite programmes(www.bioinformatics.org/sms). Protein domain features were predicted using the simplemodular architecture research tool (SMART) (http://smart.embl-heidelberg.de/), Pfam databasesearch (http://pfam.sanger.ac.uk/search/) and putative conserved domain database (Finn et al.,2008). Intra-domain features were predicted by scanning the sequence against the PROSITE



database (http://us.expasy.org/tools/scanprosite/). Multiple alignment of the full-length orpartial protein sequences of known or predicted Rig-I, Mda5 and Lgp2 was generated byCLUSTALW (www.clustal.org) and used for construction of a phylogenetic tree using theneighbour-joining method within the Mega 4.0.2 programme with C. carpio Tlr3 sequence asan outgroup.

R E A L- T I M E Q UA N T I TAT I V E P C R

Real-time quantitative PCR (q-PCR) was conducted using an ABI PRISM 7500 SequenceDetector System (PerkinElmer Applied Biosystems; http://appliedbiosystems.com.cn/) withSYBR Green Master mix (TaKaRa). The housekeeping gene 𝛽-actin served as an internalreference gene, using the gene-specific primers 𝛽-actin-F (forward) and 𝛽-actin-R (reverse)(Table I). Gene-specific primers (Table I) for q-PCR were designed for single, gene-specificamplification of nucleotides. The q-PCR mixture consisted of 2 μl of cDNA sample, 7⋅6 μl ofnuclease-free water, 10 μl of 2× SYBR Green PCR master mix (TaKaRa) and 0⋅2 μl of eachgene-specific primer (10 mM). The PCR cycling conditions were one cycle at 95∘ C for 30 s;40 cycles at 95∘ C for 5 s and 60∘ C for 30 s; one cycle at 95∘ C for 15 s, 60∘ C for 30 s and95∘ C for 15 s; followed by dissociation curve analysis (65–95∘ C at increments of 0⋅5∘ C for5 s) to verify the amplification of a single product. The relative expression ratios of the targetgenes in the treated group v. the control group were calculated by the 2−ΔΔCT method (Livak &Schmittgen, 2001). The expression data obtained from independent biological replicates weresubjected to one-way analysis of variance (ANOVA), followed by unpaired, two-tailed t-tests.Values of P< 0⋅05 were considered statistically significant.

C E L L C U LT U R E A N D T R E AT M E N T

Cyprinus carpio EPC cells were grown in Dulbecco’s modified Eagle medium: nutrient mix-ture F12 (DMEM-F12) medium supplemented with 10% inactivated FBS (Gibco BRL), 100 Uml−1 of penicillin and 100 U ml−1 of streptomycin sulphate. Cells were maintained at 28∘ Cin six-well tissue culture plates. All stimulations and infections were conducted in four paral-lel wells. For synthetic dsRNA stimulation, poly(I:C) (Sigma Aldrich; www.sigmaaldrich.com)dissolved in phosphate-buffered saline (PBS) was heated to 55∘ C for 5 min and allowed tocool to room temperature. The cells (1× 106) were treated with 5 mg ml−1 (terminal concentra-tion) of poly(I:C) or PBS (control). For kinetic studies, cells were harvested at 0, 2, 8 and 24 hpost-stimulation, and RNA was isolated and reverse transcribed. For virus infection, EPC cellswere infected with KHV at a multiplicity of infection of 1. Control cells were treated with PBS.To determine the time-dependent expression profiles, cells were harvested at 0, 2, 8, 16, 24 and48 hpi, and RNA was extracted and reverse transcribed.

RESULTS

C L O N I N G A N D S E Q U E N C E A NA LY S E S

The first fragments of lgp2 were amplified using degenerate primers (Table I).Gene-specific primers (Table I) were designed based on these sequences and usedfor RACE to produce full-length cDNA of the target genes. The full-length cDNAof lgp2 was identified as a 3061 nucleotide cDNA sequence with an open readingframe encoding a putative protein of 680 aa (Fig. 1), a 5′ UTR of 126 nucleotidesand a 3′ UTR of 895 nucleotides, including a poly(A) tail. Analysis of conserveddomains revealed the presence of a DEAD–DEAH box helicase (DExD-H) domain, ahelicase super family C-terminal (HELICc) domain and a regulatory domain (RD) atthe C-terminus.


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1 GCACGGGCCGCGCTGGTCACGCTGCCGGCAAGATTCGCGGGGCACGCGCAGGATGTCGCG

61 ATCCGGGTCGCCGAATTCGCGCCTGACGACGTGCTGGACGAACTGCAGATCGAAGATCCG

121 TTCGAAAATGACCGGACTATACGACGGCATCCCGGTGACCGAGAAATCGGTGGCCGATCA

1 M T G L Y D G I P V T E K S V A D

181 CCCGAAGGGCCGGACATTATCTGGCTGCCCACCGGTGGAGGAAAAACCCGTGCTGCGGT

18 Q P E G P D I I W L P T G G G K T R A A V

241 TTACATCACCAAGAAACACCTGGAGACCACAGCCAATGCCAAAGTAGCAGTGCTCGTCAA

38 Y I T K K H L E T T A N A K V A V L V N K

301 AAGGTGCATCTGGTAGATCAGCACTTTGCAAAAGGATTCAGGCCTTATCTTGGGAGTACAT

58 V H L V D Q H F A K G F R P Y L G S T Y K

361 CAAGATAGCAGCCATCAGTGGGGACAGTAATGAGAAAGACTTGTTTGGGTGTCTAGTCAA

78 I A A I S G D S N E K D L F G C L V K A S

421 GGCCTCAGACCTGGTCATCTGCACAGCTCAGATCTTGGAGAACGCCCTGACCAACATGGA

98 D L V I C T A Q I L E N A L T N M E E E K

481 GGAAGAAAAACACGTGGAGCTGACTGACTTTACCCTGCTGGTAATAGACGAATGTCACCA

118 H V E L T D F T L L V I D E C H H T Q K E

541 TACACAAAAGGAGAGCGTCTATAATAAGATAATGGGCCGCTATGTTGAAAAGAAAGTGAG

138 S V Y N K I M G R Y V E K K V R K E G N L

601 AAAAGAAGGGAATCTTCCTCAGATTTTGGGTCTCACAGCATCGCCTGGTGCAGGAGAAAA

158 P Q I L G L T A S P G A G E N K Q L D K A

661 TAAGCAACTAGATAAAGCTGTTGAACATGTACTGCAGATCTGTGCCAATCTGGATTCAGT

178 V E H V L Q I C A N L D S V I V S T K E

721 AATTGTGTCCACCAAGGAGTTTACTCCAGTGCTGCAGAAAGTCGTTCCCAGACCCAAAAA

198 F T P V L Q K V V P R P K K Q Y D I V E

781 GCAATATGATATTTGACACACCATTTAAACTCAGTCAAAACACTGTCACAGTTACTGTGT

218 R R T L D P F G D H L K A M M L M I H E

841 ATGATGTTGATGATTCATGAGTATATGCCGCCAACGGTGAGTCGCAGCCCGCGAGAGATG

238 Y M P P T V S R S P R E M G T Q E Y E A

901 GGCACCCAGGAGTATGAAGCTGATGTGGTGGAACTAGAAAAAGAAGGTGTAAAGAAAGAG

258 D V V E L E K E G V K K E N R L I A Q C

961 AACAGACTGATTGCTCAGTGTGCTTTGCACCTGCGGCAATACAACGATGCTCTTCTCATT

278 A L H L R Q Y N D A L L I N D A V R M V

1021AATGACGCCGTTCGCATGGTGGACGCCTTCCGGGGTCTAGACGAGTTCTACAATTCAAGG

298 D A F R G L D E F Y N S R K T K L L D G

1081 AAAACCAAATTGCTGGATGGAACAGATATCTTTCTCCAGGGACTTTTTGATGAGAACCGT

318 T D I F L Q G L F D E N R V E L K Q L A

1141 GTGGAGCTTAAACAGCTGGCATCAAATGACCGCTACGAAAACCCCAAACTGGCCCAGTTG

338 S N D R Y E N P K L A Q L Q C T L Q E E

1201CAATGCACATTGCAGGAAGAGTTTAATGATGAAAACTCTCGTGCTATCCTCTTCTCAAAG

358 F N D E N S R A I L F S K T R R G T H C

1261ACCCGCAGGGGCACCCATTGTCTGTTTGACTGGGTGAACTCCAACCCTGAGCTGCAGAGG

378 K T R R G T H C L F D W V N S N P E L Q

1321 GTCAACATCAGAGCTGGCATTCTAACAGGAGCGGGTACAGGTGCAAATCACATGACCCAG

398 R V N I R A G I L T G A G T G A N H M T

Fig. 1. Continued.



1381AATGAACAGAAGGAAACCATAAAACATTTTAGAACTGGAATCCTCAACCTCCTCATCTCC

418 Q N E Q K E T I K H F R T G I L N L L I

1441ACCAGTGTAGCTGAGGAAGGACTTGATATTCCAGAATGCAACTTAGTTGTACGTTATGGG

438 S T S V A E E G L D I P E C N L V V R Y

1501 CTGTTGACCAATGAGATCGCTCAGCAGCAGGCCAGTGGGCGGGCTCGAGCTCTGAACAGC

458 G L L T N E I A Q Q Q A S G R A R A L N

1561 GTCTACTCAGTGGTGGCCGAAGAAGGTGGGCGTGAAATACGCAGGGAACTTACCAATGAG

478 S V Y S V V A E E G G R E I R R E L T N

1621 TATCTAGAAAGTCTGACTGCAAAAGCTATTGAGCAAGTGCAGCAAATGAGTCCAAGAGAG

498 E Y L E S L T A K A I E Q V Q Q M S P R

1681 TTTAGACACAAGATATCCGAGCTCCAGCAAATCGCTGTTCTGATTCGAATCCAGGGCGAG

518 E F R H K I S E L Q Q I A V L I R I Q G

1741 AGGAAAAAGGATGAGAGGAAGCAACGCTATAGTCCTGCTCAGGCTCAGTTTCAGTGCAGA

538 E R K K D E R K Q R Y S P A Q A Q F Q C

1801 GGATGCTTCTTGCCTGTCTGCAGTGGAGAAGATTTAAGGAAAATAGAAAATACACACCAT

558 R G C F L P V C S G E D L R K I E N T H

1861 GTCAACATCAATCCTGAGTTTGTGAGGCATTACAGAACAGGTGGGCAGGTCTTTGTGGGG

578 H V N I N P E F V R H Y R T G G Q V F V

1921 AGGAACTTTGAAGATTGGGAGCCTGGACGGGTTATCAACTGCAACAAATGTGGAAAGGAC

598 G R N F E D W E P G R V I N C N K C G K

1981 TGGGGAATGGAGATTAGATACAGGAATGTGGTAATACTCCCCTGCTTAAAAATAAAAAGC

618 D W G M E I R Y R N V V I L P C L K I K

204 1 TTTTCCTTGAAGACTCCTGAAGGCAAATCCACTCCCAAGCAGTGGAAGGATGTTGAGTTC

638 S F S L K T P E G K S T P K Q W K D V E

2101 CAGGTGAAAGAGTTCGACTTTCTTGAGGACATGAGCAGTCGTTTCCCTGACCTAAACTTA

658 F Q V K E F D F L E D M S S R F P D L N

2161 AACGACTGACACACCATTTAAACTCAGTCAAAACACTGTCACAGTTACTGTGTCAGAATT

678 L N D*

2221 TCTCTGTCTTCAGTGAGTACTTGTGTAACACTGGTTTTCTACTTGTAGGTCATGGAGACT

2281 TTGCAAGTGGCAATTAATTGCTGTTAATTCCAAACTGCTTAGCATCATTTGAATATGACA

2341 TGTTCATTCTAATTATGCCGATTATCCATCGTGGTTGTGATTTGGTCCATTGTGCTTGGT

2401 TGTGAGTGTCAAAAGACATTTAGGAATCTCTGTTCCACATAATATATCTGTACTTGACAT

2461 ATAGTTTTAAATTGTAGTGTTTACTTAGTAGTTTCTGTCATGTGAGAGTTTTGTTTGCTT

2501 TTGTCTGAGAAAGGTGTGTGTGTGTGTGTGTGTGTAAGTGTTGTGATGGTATTTTTATTT

2561 TGAACAGTCTCGTTAATTATTCTGGTGTCCCTCTCTGGGATCAGGTCTGTGCTGCCCAGA

2621 TTATGAGTTATTATAGACATTACTGTGCAATGTGTCTCTAAATGTTTCTGAAGGTCTTAA

2681 TGGCCCTCGTTGAGCTCTAGTCTGCTCAGACTGTCGCTTTCAGATTGCATGAACATCTAC

2741 ATATATTCATTTAACAGATGCTTTTATCCTAAGTGACTTACAAATGAGGAGAAATATGAA

2801 TACGTTTCAAAATCTGTGAGTCTTCATTTAAAGACTGCTTACAGGCTTGTTTATAGCACT

2861 TAAGTATGTGTCTCACCAAACATAAAATTATAATGCGTATAATGCATGCCTTTTTAAAAG

2921 TCTGACCTTTTTACATTTCTGCATTACCACATGAGTCTGTATATCACATCTAGAATATAT

2981 GATTTTTCAAAAATATATAATAAAACAATAATGCACTGTAAAAAAAAAAAAAAAAAAAAA

3061 A

Fig. 1. Nucleotide and deduced amino acid sequences of Cyprinus carpio lgp2. The nucleotide and amino acidsequences are numbered. A termination codon (TAA) in front of the start codon is underlined with a dottedline. The start codon (ATG) is underlined and the stop codon (TGA) is marked (*). Four ATTTA and twoTATAA motifs in the 3′ untranslated region are indicated by boxes. The polyadenylation signals AATAAAare in bold font. In the deduced amino acid sequence, the putative Mg2+-binding sites are in bold (120–124aa). The DExDC domains are boxed (10–221 aa), the Res III domain is underlined with a wavy line (11–176aa), the HELICc domain is underlined in bold (396–473 aa) and the regulatory domain (RD) is indicatedby shading (551–672 aa).


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P H Y L O G E N E T I C A N D H O M O L O G Y A NA LY S I S O F R L R S

To study the molecular evolution and compare the sequence homologues, all theknown and predicted RLR family protein sequences in GenBank were selected to con-struct a phylogenetic tree (Fig. 2) .These results show that the RLR family proteinscould be divided into Rig-I, Mda5 and Lgp2 branches. Each group included severalsub-groups, and the piscine sub-group was separated from other vertebrate membersub-groups in the corresponding branch. Cyprinus carpio Lgp2 had highest amino acididentity to C. idella and C. auratus Lgp2 (77⋅7%), whereas it had lowest identity withthe mouse Mus musculus (46%) (Table II).

T I S S U E D I S T R I B U T I O N O F L G P2 MR NA

lgp2 expression levels in tissues including muscle, spleen, gill, intestine, brain, headkidney, skin, heart and liver were quantified by q-PCR. lgp2 mRNA transcripts weredetected in all examined organs, but expression levels differed among them (Fig. 3).lgp2 mRNA expression was highest in gill, followed by intestine, brain and heart, withthe least expression in skin.

KO I H E R P E S V I RU S D E T E C T I O N

KHV was detected by PCR using specific primers. Infected C. carpio produced asingle band of 290 bp, as expected for KHV DNA. Comparison with the publishedGenBank nucleotide sequence for cyprinid herpesvirus 3 showed 98⋅9% homology.

T I M E- D E P E N D E N T E X P R E S S I O N O F L G P2 , M AV S, A N D I R F3 MR NAF O L L OW I N G K H V I N F E C T I O N

Severe symptoms of KHV infection, including necrotic gill lesions, interstitialnephritis, splenitis and enteritis, were evident at 3 days post-infection. Total RNA wasextracted from muscle, intestine, gill, head kidney, skin, liver, heart, brain and spleenof infected C. carpio. The mRNA expression patterns of lgp2, mavs and irf3 at 24, 48,72 and 0 hpi (control) were examined by q-PCR (Fig. 4).

The expression patterns of lgp2 were similar in all examined organs, with the excep-tion of the intestine [Fig. 4(a)]. lgp2 mRNA expression increased at 24 hpi in muscle,spleen, gill, brain, skin, heart, liver and head kidney, and reached maximum inductionlevels of 4⋅30 fold in muscle, 10⋅00 fold in spleen, 6⋅73 fold in gill, 6⋅70 fold in brain,6⋅84 fold in skin, 5⋅60 fold in heart and 8⋅43 fold in liver at 48 hpi, and 22⋅59 fold inhead kidney at 24 hpi. Levels declined at 72 hpi in all organs except intestine, but did notreturn to the original levels, and remained at an increased level in the intestine at 72 h.

The expression levels of C. carpio mavs were similar in muscle, spleen, gill, intes-tine, brain, skin and head kidney [Fig. 4(b)]. Cyprinus carpio mavs mRNA expressionshowed initial increase at 24 hpi and had returned to the original level at 72 hpi. Cypri-nus carpio mavs mRNA reached maximum induction of 16⋅0 and 2⋅8 fold at 24 hpi inspleen and brain, respectively. Although C. carpio mavs mRNA expression levels wereoriginally highest in liver, these levels reduced following challenge by KHV.

As indicated in Fig. 4(c), constitutive expression of irf3 was ubiquitous in all theexamined tissues. q-PCR showed that irf3 expression levels were similar in all tissues,but higher in the brain. irf3 mRNA was dramatically up-regulated at 24 hpi and reached



Cyprinus carpio Th3

Orcinus orca RIG-1

Lipotes vexillifer RIG-1Balaenoptera acutorostrata scammoni RIG-1Bubalus bubalis RIG-1Sus scrofa RIG-1Pteropus alecto RIG-1Macaca mulatta RIG-1Homo sapiens RIG-1Odobenus rosmarus divergens RIG-1

Leptonychotes weddellii RIG-1Erinaceus europaeus RIG-1Orycteropus afer afer RIG-1Trichechus manatus latirostris RIG-1Sylvilagus bachmani RIG-1Lepus europaeus RIG-1Cricetulus griseus RIG-1Rattus norvegicus RIG-1Mus musculus RIG-1Omithor hynchus anatinus RIG-1Anser anser breed Yangzhou Rig-1Alligator sinensis Rig-1Alligator mississippiensis Rig-1Pelodiscus sinensis Rig-1Chelonis mydas Rig-1Chrysemys picta bellii Rig-1

Xenopus (Silurana) tropicalis Rig-1Callorhinohus milii Rig-1Latimeria chalumnae Rig-1Salmo salar Rig-1Ictalurus punctatus Rig-1Astyanax mexicanus Rig-1Cyprinus carpio Rig-1Cyprinidae sp. EPC Rig-1Ctenopharyngodon idella Rig-1Carassius auratus Rig-1

Salmo salar Lgp2

Ictalurus punctatus Lgp2Danio rerio Lgp2Cyprinus carpio Lgp2Ctenopharyngodon idella Lgp2Carassius auratus Lgp2

Gallus gallus Lgp2Rattus norvegicus LGP2Homo sapiens LGP2Mus musculus LGP2Pteropus alecto LGP2Bos taurus LGP2Sus scrofa LGP2

Oreochromis niloticus Mda5Epinephelus coioides Mda5

Takifugu rubripes Mda5Ictalurus punctatus MDA5Danio rerio Mda5

Ctenopharyngodon idella Mda5Carassius auratus Mda5Chilo scyllium griseum Mda5Chrysemys picta bellii Mda5Chelonia mydas Mda5Alligator mississippiensis Mda5Gallus gallus Mda5

Anser cygnoides Mda5

Mus musculus transcript2 MDA5Mus musculus transcript1 MDA5Rattus norvegicus MDA5

Elephantulus edwardii Mda5Trichechus manatus latirostris Mda5 P

P

P

PP

P

PP

PP

P

P

partialpartial

P

PPP

P

PP

P

PPPP

PPP

Sus scrofa MDA5

Capra hircus MDA5Physetev catodon Mda5Oreochromis niloticus Mda5Pteropus alecto MDA5Sylvilagus bachmani MDA5

Lepus granatensis MDA5Macaca mulatta MDA5Pongo abelii MDA5Homo sapiens MDA569

100

100

100

100

100

100

87

100

100

100

100

100

100

100100

100

100100

100

100

100

10097

4766

50

31

25

64

83

100

84

64

73

84

100

26

98

99

100

95

86

88

2949

100

98

67

8679

99

100

100

75

95

78

100

100100

92

7388

6246

93

77

100

100

100

10022

14

91

91

94

23

34

75

45

Paralichthys olivaceus Mda5Callorhinchus milii Mda5

Paralichthys olivaceus Lgp2Gadus morhua Lgp2 Partial

Oncorhynchus mykiss Lep2 splicing variant1Oncorhynchus mykiss Lep2 splicing variant2

Chrysemys picta bellii Rig-1

FishMda5

Fish Lgp2

Fish Rig-1

Fig. 2. Continued.


10 X . L . C AO E T A L.

a maximum at 48 hpi in spleen, gill and brain, and at 24 hpi in head kidney (27⋅00 fold).Levels of irf3 expression returned to basal levels by 72 hpi in spleen and head kidney,and were decreased to 5⋅50 fold original levels at 72 hpi in intestine.

L G P2 MR NA E X P R E S S I O N P RO F I L E S I N V I T RO A F T E R P O LY( I : C )S T I M U L AT I O N

lgp2 mRNA expression levels were examined in EPC cells at 0, 2, 8 and 24 h afterstimulation with the dsRNA mimic poly(I:C) [Fig. 5(a)]. lgp2 mRNA expression was

Fig. 2. Phylogenetic relationships of retinoic acid-inducible gene-1-like receptor (RLR) family protein sequencesin GenBank, employing common carp Tlr3 sequence as an outgroup. Multiple alignment of the full-lengthor partial protein sequences of known or predicted Rig-I, Mda5 and Lgp2 was generated by CLUSTALWand used for construction of a phylogenetic tree using the neighbour-joining method within the Mega4.0.2 programme. Data were analysed using Poisson correction. The bootstrap values of the branches wereobtained by testing the tree 1000 times. The bar indicated the distance. The ‘P’ after the species nameindicated the sequence was predicted. The ‘partial’ behind the species name meant the protein was a par-tial sequence. The protein IDs were as follows: Homo sapiens Mda5 AF095844.1; Pteropus alecto Mda5JN031515.1; Macaca mulatta Mda5 DQ875603.1; Lepus granatensis Mda5 KF640622.1; Sylvilagus bach-mani Mda5 KF640621.1; Oreochromis niloticus Mda5 XM_003447407.2; Capra hircus XM_005676042.1;Trichechus manatus latirostris Mda5 XM_004375429.1; Elephantulus edwardii XM_006879359.1; Rat-tus norvegicus XM_006234286.1; Mus musculus transcript 1 NM_027835.3; Mus musculus transcript 2NM_001164477.1; Anser cygnoides Mda5 KC282429.1; Gallus gallus MDA5 AB371640.1; Alligator mis-sissippiensis Mda5 XM_006277996.1; Chelonia mydas Mda5 XM_007056641.1; Chrysemys picta belliiMda5 XM_005290355.1; Chiloscyllium griseum Mda5 HG964652.1; Carassius auratus Mda5 JF970226.1;Ctenopharyngodon idella Mda5 FJ542045.2; Danio rerio Mda5 XM_689032.4; Takifugu rubripes Mda5XM_003961960.1; Callorhinchus milii Mda5 XM_007889844.1; Ictalurus punctatus Mda5 JQ008942.1;Oncorhynchus mykiss Mda5 NM_001195179.1; Epinephelus coioides Mda5 HQ880665.1; Paralichthysolivaceus Mda5 HQ401014.1; Oreochromis niloticus XM_003447407.2; Carassius auratus (accessionnumber: JF970227.1); Ctenopharyngodon idella Lgp2 FJ813483.2; Danio rerio Lgp2 NM-001257157.1;Ictalurus punctatus Lgp2 JQ008941.1; Paralichthys olivaceus Lgp2 HM070372.1; Salmo salar Lgp2BT045378.1; Pteropus alecto Lgp2 JN031516.1; Oncorhynchus mykiss Lgp2 partial splicing variant 1FN396358.1; Oncorhynchus mykiss Lgp2 partial splicing variant 2 2FN396359.1; Gallus gallus Lgp2HQ8455773.1; Gadus morhua Lgp2 partial EU371924.1; Mus musculus NM_030150.2; Homo sapiensLgp2 NM-024119.2; Xenopus laevis Lgp2 NP_01085915; Rattus norvegicus Lgp2 XM_006247315.1;Sus scrofa Lgp2 FJ392007.1, Bos taurus Lgp2 BC146128.1; Macaca mulatta Lgp2 XP_001108799;Anas platyrhynchos Lgp2 JQ868804.1; Ctenopharyngodon idella RIG-I ADC81089; Danio rerio Rig-IXP_002666571; Cyprinus carpio Rig-I HQ850439.1; Salmo salar Rig-I NM_001163699.1; Cyprinidae sp.EPC Rig-I FN394062.1; Chiloscyllium griseum Rig-I HG964657.1; Gadus morhua Rig-I HM046436.1;Ictalurus punctatus Rig-I JQ008940.1; Carassius auratus Rig-I JF970225.1; Anas platyrhynchos Rig-IACA61272; Pelodiscus sinensis Rig-I XM_006123091.1; Latimeria chalumnae Rig-1 XM_006009537.1;Callorhinchus milii Rig-I XM_007909503.1; Xenopus (Silurana) tropicalis Rig-1 XM_002935671.2; Chry-semys picta bellii Rig-I XM_005294762.2; Anser anser breed Yangzhou Rig-I HQ829831.1; Latime-ria chalumnae Rig-1 XM_006009537.1; Balaenoptera acutorostrata scammoni Rig-I XM_007183182.1;Bubalus bubalis Rig-I XM_006060270.1; Pteropus alecto Rig-I JN031514.1; Trichechus manatuslatirostris Rig-I XM_004390933.1; Orycteropus afer afer Rig-I XM_007950699.1; Leptonychotes weddel-lii Rig-I XM_006747115.1; Odobenus rosmarus divergens Rig-I XM_004392173.1; Homo sapiens Rig-INP_055129; Macaca mulatta Rig-I NP_001036133; Sus scrofa Rig-I ABV26717; Lipotes vexillifer Rig-IXM_007472300.1; Orcinus orca Rig-I XM_004283615.1 ; Erinaceus europaeus Rig-I XM_007531244.1;Mus musculus Rig-I NP_766277; Rattus norvegicus Rig-I NP_001100115; Lepus europaeus Rig-IKF640636.1; Sylvilagus bachmani Rig-I KF640631.1; Astyanax mexicanus Rig-I XM_007237377.1;Ornithorhynchus anatinus Rig-I XM_007671324.1 and Cyprinus carpio Tlr3 DQ885910.1.



Tab

leII

.H

omol

ogou

sco

mpa

riso

nsof

Lgp

2w

ithot

her

know

npi

scin

e,se

aur

chin

,hum

anan

dm

ouse

Lgp

2pr

otei

nse

quen

ces

(%)

Sequ

ence

sC

.C

.C

.D

.I.

S.P

.O

.H

.S

RM

.ca

rpio

idel

laau

ratu

sre

rio

punc

tatu

ssa

lar

oliv

aceu

sm

ykis

ssa

pien

ssc

rofa

norv

egic

usm

uscu

lus

Cyp

rinu

sca

rpio

ID77

⋅777

⋅775

⋅565

⋅163

⋅860

⋅360

⋅047

⋅647

⋅547

⋅246

⋅0C

teno

phar

yngo

don

idel

laID

99⋅7

76⋅8

66⋅0

66⋅1

62⋅4

61⋅8

48⋅7

49⋅9

49⋅6

49⋅0

Car

assi

usau

ratu

sID

76⋅6

65⋅9

65⋅9

62⋅0

61⋅6

48⋅7

49⋅7

50⋅0

49⋅0

Dan

iore

rio

ID65

⋅464

⋅962

⋅261

⋅548

⋅448

⋅347

⋅546

⋅4Ic

talu

rus

punc

tatu

sID

64⋅6

59⋅4

61⋅7

49⋅0

48⋅3

48⋅3

47⋅ 5

Salm

osa

lar

ID68

⋅086

⋅947

⋅847

⋅376

⋅246

⋅5Pa

rali

chth

ysol

ivac

eus

ID63

⋅347

⋅146

⋅345

⋅945

⋅3O

ncor

hync

hus

myk

iss

ID45

⋅044

⋅644

⋅044

⋅1H

omo

sapi

ens

ID82

⋅279

⋅276

⋅8Su

ssc

rofa

ID76

⋅275

⋅3R

attu

sno

rveg

icus

ID91

⋅6M

usm

uscu

lus

ID

ID,i

dent

ity.


12 X . L . C AO E T A L.

Muscle0

0·5

1

1·5

2

Fold

cha

nge

of lg

p2-b

-act

in m

RN

Aex

pans

ion

tissu

e

2·5

3

3·5

4

4·5

5

Spleen Gill Intestines Brain Head kidneyTissue

Skin Heart Liver

Fig. 3. Tissue distribution of lgp2 mRNA in healthy Cyprinus carpio. lgp2 mRNA levels were measured byquantitative real-time polymerase chain reaction (q-PCR) and normalized against the housekeeping gene𝛽-actin. Values are mean± s.d. (n= 5).

rapidly and significantly elevated at 2 h (13⋅63-fold, P< 0⋅05), and then graduallydecreased. The transcripts returned to control levels at 24 h post-stimulation (P< 0⋅05).

T E M P O R A RY E X P R E S S I O N PAT T E R N O F L G P2 I N V I T RO A F T E RK H V I N F E C T I O N

Plaques were observed at 8 hpi with KHV, and then spread gradually. lgp2 expressionin EPC cells was significantly enhanced at 8 h (1⋅77 fold, P< 0⋅05), reached a peak at24 h (2⋅16 fold, P< 0⋅05) and returned to control levels at 48 h (P> 0⋅05) [Fig. 5(b)].There were no significant differences in lgp2 mRNA expression levels among the dif-ferent time intervals in corresponding control samples (P> 0⋅05).

DISCUSSION

lgp2 is known to play an essential role in antiviral responses (Honda & Taniguchi,2006). Cyprinus carpio lgp2 has been cloned from C. auratus, D. rerio (Sun et al.,2011), pufferfish Takifugu rubripes (Temminck & Schlegel 1850) (Zou et al., 2009)and C. idella (Huang et al., 2010). Gene cloning in this study allowed full-length lgp2cDNA sequences to be obtained, which were predicted to encode a 680 aa protein (the-oretical pI/MW: 6⋅53/77 kDa). Six AU-rich instability motifs that might be involvedin RNA instability and translational control were found in the 3′ UTR of lgp2. Stud-ies have shown that these motifs played a role in the 3′ end processing of pre-mRNAand mRNA deadenylation and decay (Beyer et al., 1997; Key & Pagano, 1997; Xuet al., 1997). In addition, four alternative splicing variants of lgp2 were obtained bygene cloning in a recent study (unpubl. data), which might explain why these motifsconfer lgp2 mRNA instability, although their involvement in the antiviral response inC. carpio remains to be determined. The predicted Lgp2 protein sequence includedthe conserved DExDc, ResIII, HELICc and RD domains, implying similar fundamen-tal functions. The Asp-Glu-X-Asp–His (DExD-H) motif in Lgp2 is highly conserved



00

10

20

30

40

24

a a a a a

b

aa

e e

d

d ec d

c d

f

c

d

c c

c

c

cc

d d

b b b b

a

d d

d

d

e

d

d ee

e

d

e

a

b c cb

b b

b

e ee

a

e

g

c

aa

d

b b

c c

b

a

cc

b

e ff

f

f

b

48

Muscle Spleen Gill Intestine Brain Head kidneyTime (h) post KHV injection in examined tissues

Skin Heart Liver72 0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72

0

10

20

Fold

cha

nge

of in

f3 b

-act

in m

RN

A e

xpre

ssio

n in

tiss

ues

30

40

0

10

20

30

40

(a)

(b)

(c)

d

ccccccc d

d

ba

cb

ba

de

c

a

c

d

ba

bb

aa

b

c c c

aaaa

f

Fig. 4. Expression of (a) lgp2, (b) mavs and (c) irf3 mRNA in muscle, intestine, gill, head kidney, skin, liver, heart,brain and spleen of koi herpesvirus (KHV)-infected Cyprinus carpio at 0, 24, 48 and 72 h post-infection.Expression levels of lgp2, mavs and irf3 mRNA were measured in tissues from KHV-infected fish by quan-titative real-time polymerase chain reaction (q-PCR) at 0, 24, 48 and 72 hpi, and were normalized against thehousekeeping gene 𝛽-actin. Values are mean± s.d. (n= 5). Different lower-case letters indicate significantdifferences, P< 0⋅05.

in fishes and mammals, with the consensus sequence Asp-Glu-Cys-His (DECH). Thelgp2 gene showed high homology with lgp2 in C. idella and C. auratus (77⋅7%). Thephylogenetic tree also showed that C. carpio, D. rerio, C. idella and C. auratus belongto the same cluster, which is most closely related to the cluster including I. punctatus,rainbow trout Oncorhynchus mykiss (Walbaum 1792) and S. salar, and thus formed aseparate cluster of fish Lgp2 differing from other vertebrate Lgp2s.

Rig-I, Mda5 and Lgp2 have recently been identified as essential for the recog-nition of and response to viral pathogens, establishing their importance for innateantiviral immunity (Yoneyama et al., 2005; Kato et al., 2006; Takahasi et al., 2008).The significant role of these proteins in antiviral signalling makes them primetargets for virus-encoded host invasion and antagonism. The role of Lgp2, which


14 X . L . C AO E T A L.

00

5

10

20

Fold

cha

nge

of lg

p2–e

f1a

mR

NA

expr

essi

on in

infe

cted

cel

ls

15

2 8

*

**

*

*

16Time (h) post KHV infected

24 48

00

5

10

20

Fold

cha

nge

of lg

p2–

ef1a

mR

NA

expr

essi

on in

stim

ulat

ed c

ells

15

2 8

Time (h) post poly(I:C) stimulation

(b)

(a)

24

Fig. 5. (a) lgp2 expression in epithelioma papulosum cyprini (EPC) cells stimulated by poly(I:C). lgp2 expressionwas measured at 0, 2, 8 and 24 h post-stimulation and the mRNA levels were normalized to elongation factor1𝛼 (ef1𝛼) expression. The poly(I:C) concentration was 5 mg ml−1. (b) lgp2 expression in Cyprinus carpioEPC cells infected with koi herpesvirus (KHV). lgp2 mRNA levels were measured at 0, 2, 8, 16, 24 and48 h post-stimulation and normalized to ef1a expression. *P< 0⋅05 between experimental group and controlgroup. Values are mean± s.d.

shows high sequence similarity to Rig-I and Mda5 and selectively binds dsRNAbut lacks caspase activation and recruitment domain (CARD) effector domains formavs signalling, has been under intense investigation. When LGP2 was discoveredin mammals, in vitro studies suggested that it functioned as a negative regulator(Rothenfusser et al., 2005; Yoneyama et al., 2005; Saito et al., 2007) in Rig-I sig-nalling. In addition, LGP2−/− M. musculus displayed enhanced resistance to intranasalinfection with a lethal inoculum of vesicular stomatitis virus, known to be sensed byRig-I. In contrast to the above mechanism, Lgp2 deficiency resulted in a suppressedIfn response against encephalomyocarditis virus, which triggered Mda5-mediatedantiviral signalling (Gitlin et al., 2006; Venkataraman et al., 2007). These resultssuggest that Lgp2 has both negative and positive regulatory effects on Rig-I andMda5 signalling. The generation of LGP2−/− M. musculus and M. musculus witha point mutation, D30A, that disrupts the ATPase activity of Lgp2, revealed thatLgp2 positively regulates the production of type I Ifn in response to RNA virusesrecognized by both Rig-I and Mda5 (Satoh et al., 2010). Nevertheless, Lgp2 is dis-pensable for type I Ifn production following transfection by synthetic RNAs. Theseresults suggest that Lgp2 may modify viral RNA by removing proteins from viralribonucleoprotein complexes or unwinding complex RNA structures to facilitateMda5-mediated and Rig-I-mediated recognition of dsRNA (Takeuchi & Akira, 2010).Pollpeter et al. (2011) surmised that Lgp2 may function upstream of Rig-I and Mda5



and its adaptor, Mavs, as a positive regulator in viral DNA-mediated responses.In bony fishes such as P. olivaceus, C. idella and O. mykiss, expression levels of thelgp2 gene and type I ifn gene were dramatically induced by poly(I:C) stimulation andviral infection (Ohtani et al., 2010; Huang et al., 2010; Chang et al., 2011; Hikimaet al., 2012). There is conflicting evidence, however, for example, in C. auratus thatlgp2 inhibits poly(I:C)-induced activation of D. rerio type I ifn production (Sun et al.,2011). These data suggest that lgp2 antiviral function may vary among species ofteleosts. Further studies are needed to clarify the role of lgp2; however, the presentdata expand the role of lgp2 to DNA-pathogen detection within cells, and furthercorroborate the idea of a positive regulatory role for lgp2 in vivo.

KHV is a dsDNA herpesvirus-like pathogen. Risk of the disease is high in popula-tions of common carp and koi. The virus may enter the body through the gills, replicatethere, and induce mucosal sloughing and necrosis. To test the involvement of lgp2 incellular innate immune responses to pathogens with DNA genomes, lgp2, mavs and irf3mRNA levels were measured after KHV challenge. The temporary expression patternof lgp2 in vitro after infection was observed. lgp2 was constitutively expressed in var-ious organs, as expected given its crucial role in antiviral immunity. Although spleen,liver and head kidney are important organs in terms of innate immunity, the lgp2 genewas not highly expressed in these tissues. Transient expression of lgp2 was observedafter intraperitoneal injection of KHV, infection of EPC with KHV and poly(I:C) stim-ulation. lgp2 transcripts were significantly elevated at 24 h (P< 0⋅05), and continued tobe elevated at 48 h (P< 0⋅05), in all tissues after KHV injection, while lgp2 transcriptsin EPC cells were rapidly up-regulated following poly(I:C) stimulation, subsequentlyrecovering to control levels. lgp2 mRNA expression in EPC cells was significantlyup-regulated at 8 hpi (P< 0⋅05) and returned to control levels at 48 hpi (P> 0⋅05).Accordingly, the dramatic up-regulation of lgp2 transcription could play a key positiverole in the antiviral response. Consistent with cytoplasmic viral sensor lgp2 expression,the expression profiles of the adaptor molecules mavs [also known as IFN-𝛽 promoterstimulator 1 (ips-1), virus-induced signalling adaptor (visa) and CARD adaptor induc-ing interferon-𝛽 (cardifs)] and irf3 were similar in some examined tissues with respectto time-dependent induction by KHV. mavs is a CARD family protein that mediatesCARD-dependent interactions with rig-I and mda5. These interactions stimulate mavssignalling of downstream irf3 and nuclear factor-𝜅B transcription factors (nf-𝜅b) thatinduce IFN-a/𝛽 production and IFN-stimulated genes that suppress virus infection.lgp2, however, lacks the two CARDs present at the N-termini of rig-I and mda5 whichmediate homotypic interactions involved in the assembly of a complex with the essen-tial RLR adapter Mavs and other downstream molecules. Given that mavs and irf3expression levels were dramatically induced, it was surmised that RLR participates insensing and signalling in response to viral DNA. Based on overall results, it was spec-ulated that lgp2 might contribute to the positive regulation of dsDNA-virus-mediatedcellular innate immune responses. There are two possible mechanisms whereby lgp2up-regulation could be involved in the antiviral innate immune response: lgp2 couldwork upstream of rig-I and mda5 to potentiate viral DNA-induced signalling, or lgp2may be a node or branch point for co-ordinating crosstalk among seemingly diverseinnate responses that are activated in infected cells.

In summary, this study identified lgp2 as a component of innate cellular responsesmediated by DNA pathogens, and demonstrated, both in vitro and in vivo, that lgp2plays an unanticipated role in the positive regulation of downstream responses. Further


16 X . L . C AO E T A L.

investigation is needed to explore the antiviral mechanisms and regulatory pathway oflgp2 in C. carpio. Understanding these regulatory pathways might help in the devel-opment of new methods for preventing infections in C. carpio culture.

These studies were supported by the National Natural Science Foundation of China(NSFC-Henan Joint Training Fund for Fostering Talents) (number U1204329).

References

Anon (2009). Manual of Diagnostic Tests for Aquatic Animals. Paris: Office International DesEpizooties.

Beyer, K., Dandekar, T. & Keller, W. (1997). RNA ligands selected by cleavage stimulationfactor contain distinct sequence motifs that function as downstream elements in 3 ́ -endprocessing of pre-mRNA. Journal of Biological Chemistry 272, 26769–26779.

Bretzinger, A., Fischer-Scherl, T., Oumouna, M., Hoffmann, R. & Truyen, U. (1999). Massmortalities in koi, Cyprinus carpio, associated with gill and skin disease. Bulletin of theEuropean Association of Fish Pathologists 19, 182–185.

Chang, M., Collet, B., Nie, P., Lester, K., Campbell, S. & Secombes, C. J. (2011). Expressionand functional characterization of the RIG-I-like receptors MDA5 and LGP2 in rainbowtrout Oncorhynchus mykiss. Journal of Virology 85, 8403–8412.

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