- Email: [email protected]
A 15 nucleotide deletion mutation in coding region of the RIG-I lowers grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus
Fish & Shellfish Immunology 33 (2012) 442e447
Contents lists available at SciVerse ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Short communication
A 15 nucleotide deletion mutation in coding region of the RIG-I lowers grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus Quanyuan Wan, Jianguo Su*, Lan Wang, Lijun Chen, Xiaohui Chen College of Animal Science and Technology, Northwest A&F University, Shaanxi Key Laboratory of Molecular Biology for Agriculture, No. 22 Xinong Rd, Yangling 712100, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 January 2012 Received in revised form 19 April 2012 Accepted 7 May 2012 Available online 21 May 2012
RIG-I (Retinoic acid-inducible gene I) is a pivotal receptor that detects numerous RNA and DNA viruses and plays crucial roles in the induction of type I interferons. In the present study, a deletion mutation in CiRIG-I (Ctenopharyngodon idella RIG-I) coding region was detected, its association with resistance/ susceptibility to grass carp reovirus (GCRV) was examined, and possible mechanism was analyzed. A 15bp deletion mutation was found, and the mutation results in a deletion of five amino acids. To investigate the genotypes and alleles, the relevant PCR products were electrophoresed on 2.5% agarose gel. Three genotypes and two alleles were discovered. The general allele was named as A and the deletion mutation allele was named as B. The deletion mutation cancels a predicted phosphorylation site and changes the secondary structure and the probability of peroxisomal targeting signal 1 in CiRIG-I. To explore the correlation between these genotypes and the resistance of grass carp to GCRV, a challenge experiment was carried out. The cumulative mortality in genotype AA (40.70%) and AB (52.73%) was significantly lower than that in genotype BB (71.43%) (P ¼ 0.032). The result demonstrated that genotype AA and AB were resistant to GCRV, while genotype BB was susceptible. The 15-bp deletion mutation lowers the resistance of grass carp to GCRV. This result might provide a potential genetic marker for further investigation of selective breeding of resistant grass carp to GCRV. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Grass carp (Ctenopharyngodon idella) RIG-I Deletion mutation Grass carp reovirus
1. Introduction Grass carp (Ctenopharyngodon idella) is a momentous economic aquaculture species in China, but the fractional yield is impeded by grass carp reovirus (GCRV), a double strand RNA (dsRNA) virus [1]. Since GCRV was characterized in 1970s [2], researches have been going on continuously. Some researchers focused on the genomic sequence of GCRV [3,4], and others were more concerned with the innate immunity in host cells after GCRV infection. In innate immunity, pattern recognition receptors (PRRs) can recognize the specific conserved structures among pathogens that called pathogen associated molecular patterns (PAMPs) [5]. PRRs mainly include Toll-like receptors (TLRs), RIG-I (Retinoic acid-inducible gene I) -like receptors (RLRs), Nucleotide-oligomerization domain (NOD)-like receptors (NLRs), etc. Some members of the three PRRs had been reported to play roles in response to GCRV, such as TLR3 [6], NOD1 and NOD2 [7] and RIG-I [8].
* Corresponding author. Tel.: þ86 29 87092139; fax: þ86 29 87092164. E-mail address: [email protected] (J. Su). 1050-4648/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2012.05.010
RIG-I (also called DDX58), MDA5 (Melanoma differentiation associated gene 5, also known as IFIH1 or Helicard) and LGP2 (Laboratory of genetics and physiology 2, also referred to as DHX58) constitute RLRs. RIG-I belongs to the DExD/H box RNA helicase family and consists of two N-terminal caspase recruitment domains (CARDs), a signature of DExD/H box RNA helicase domain with the ATP-hydrolyzing function and the capacity to change the conformation of substrate dsRNA, and a C-terminal repression domain (RD) that can control RIG-I multimerization [9e11]. When viral RNA in the cytoplasm are detected by RIG-I, the adapter protein named IPS1 (interferon-b promoter stimulator 1, also known as MAVS, VISA or CARDIF) is activated, via downstream transcription factor to boost type 1 interferon (IFN) production and antiviral gene expression to control virus infection [9,12]. As a PRR in antiviral immunity, RIG-I has received wide attention [13,14]. And also, RIG-I was identified in some fishes, such as Salmo salar (accession NO., CAX48607) [15], Carassius auratus (accession NO., JF970225) [16], Cyprinus carpio (accession NO., HQ850439) [17] and C. idella (accession NO., GQ478334) [8]. The expression of CiRIG-I gene (C. idella RIG-I) can be significantly up-regulated by poly(I:C) and GCRV [8]. As more and more immune-related genes are characterized, the relevance of polymorphism and spontaneous deletion
Q. Wan et al. / Fish & Shellfish Immunology 33 (2012) 442e447
mutation to certain diseases becomes a growing focus point. The polymorphism research in Japanese flounder MHC gene reveals that nine alleles are related to the resistance to Vibrio anguillarum [18]. In common carp, three single nucleotide polymorphisms (SNPs) in IL-10a gene are associated with resistance/susceptibility to cyprinid herpesvirus-3 infection [19]. Some polymorphisms of TLR3 [20], Mx2 [21] and TLR22 [22] in grass carp have significant association with resistance/susceptibility to GCRV. Deletion mutations in the 50 part of the pol gene of Moloney murine leukemia virus result in variation of its virulence [23]. In mice, a 17-base deletion in exon 4 of SLC12A6 is associated with neuromuscular disease [24]. Some insertion/deletion mutations are also related to disease in human. For instance, a 9 bp insertion/deletion polymorphism in the 30 untranslated region of b-transducin repeat-containing protein (bTrCP) is associated with susceptibility for hepatocellular carcinoma in Chinese people [25]. One deletion polymorphism (rs885945) neighboring the gene encoding MHC IF is significantly associated with the risk of Avellino corneal dystrophy [26]. In a Pakistani family with autosomal dominant hypercholesterolemia, a recurrent insertion mutation in the low density lipoprotein receptor (LDLR) gene is identified [27]. In the present study, CiRIG-I was employed to detect delete mutations in coding region and to analyze the association with resistance/susceptibility to GCRV. 2. Materials and methods 2.1. Sample preparation and deletion mutation discovery 40 grass carp were collected from the fish farm in Rougu (Shaanxi province, China), and approximate 1 g muscle of each fish was cut and kept at 80 C until DNA isolation. About 200 mg sample was homogenized, and DNA was extracted with traditional phenol-chloroform method and was stored at 20 C. The incipient primers used to explore CiRIG-I genomic structure were designed from the CiRIG-I cDNA sequence (GenBank accession NO., GQ478334), using Primer Premier 5.0 software. The reaction volume of PCR was 25 mL, containing 100 ng genomic DNA, 10 pM of each primer, Buffer (including MgCl2), 200 mM dNTPs and 2 units of Taq DNA polymerase (MBI). The PCR protocol was 3 min at 95 C, 35 cycles of 94 C for 30 s, n oC (according to the annealing temperatures of the different primers), annealing for 30 s, 72 C for 2 min, with a final extension at 72 C for 5 min. The products were purified by using the DNA Fragment Purification Kit (TIANGEN Corp., Beijing, China) and then ligated with pMD18-T vector (Takara). Three positive clones of each fragment were sequenced (Nanjing GeneScript Corp., China). Sequencing results were aligned by Vector NTI Suite 11.0 (Invitrogen). After obtaining the full length genomic sequence of CiRIG-I gene by PCR amplification with sequencespecific primers and genomic walking, DNA pool sequencing of 20 grass carp was carried out to detect polymorphisms or deletion/ insertion mutations. Very excited to find a 15 nucleotide deletion mutation in the coding region of CiRIG-I gene. In order to confirm the deletion mutation, another pair of primers was designed, forward primer RF186rc (50 -AAGGGCTGCGGATCGGAT-30 ) and reverse primer RR505 (50 -CCTGCCTGACAGACATTTATTGC-30 ). The size of expected PCR product was 745 bp. PCR was performed using genomic DNA of the 40 grass carp as template. The PCR products were purified and sequenced directly, meanwhile, the corresponding PCR products were electrophoresed on 2.5% agarose gels.
443
using the PTS1 predictor (http://mendel.imp.ac.at/mendeljsp/sat/ pts1/PTS1predictor.jsp). The subsequent forecast of possible phosphorylation sites was implemented using the tool (http://www.cbs. dtu.dk/services/NetPhos/). 2.3. Challenge experiment Average 10 cm in body length of 300 grass carp were collected from three fish farms (Shaanxi province, China), where hemorrhagic disease of grass carp was not found in recent years. The juvenile fish of the three fish farms were bought from the same fish hatchery, and the mating of parent fishes in this fish hatchery is randomly. Then, the 300 grass carp were fostered in aerated freshwater at 28 C for one week before processing. For the viral infection experiment, grass carp were divided into five groups (60 animals in each group). The conditions were the same among tanks and the fish were randomly distributed into different tanks. Four groups were cultured in four aquariums and intraperitoneally injected with 100 mL of GCRV (097 strain, 3.63 107 TCID50/ml), suspended in PBS, per gram body weight. The control group was reared in another aquarium and injected with PBS. All the fish were observed every 6 h to survey the mortality and collect samples until the termination of the experiment at 168 h after challenge. Grass carp died in the first 72 h post challenge were classified as susceptible individuals for their high sensitivity to GCRV and obvious symptoms of hemorrhage disease of grass carp, while the animals that survived over 168 h post challenge were considered as resistant group, and the rest were discarded for their ambiguity to GCRV. Approximate 1 g muscle was cut and kept at 80 C until DNA isolation. 2.4. Association analysis between deletion mutation and susceptibility/resistance to GCRV The genomic DNA of susceptible individuals and resistant individuals were used as templates in PCR. The PCR products were electrophoresed, and the agarose electrophoresis results were photographed by quantity one system (Bio-Rad) as preparation for statistical analysis. Allele and genotype frequencies and their associations with susceptibility/resistance to GCRV were analysed with SHEsis software (http://analysis.bio-x.cn/SHEsisMain.htm). The P value less than 0.05 was considered statistically significant. 3. Results 3.1. Deletion mutation in CiRIG-I A 15 nucleotide deletion mutation was found in the coding region and was described as g. 665-679 del AAA CCA CGA CGA CAG (Fig. 1). Certainly, there are two alleles: the general allele named as A and the deletion mutation allele named as B. And the deletion mutation in CiRIG-I coincidently caused a deletion of five amino acids (Fig. 2). After agarose electrophoresis, the result had three genotypes in CiRIG-I: homozygous genotypes AA and BB, heterozygous genotype AB (Fig. 3). The predicted results showed the deletion mutation may change the secondary structure of CiRIG-I protein (Fig. 4) and may delete a phosphorylation site which was called T215 (Fig. 5), also may change the probability of peroxisomal targeting signal 1 (PTS1) (Table 1) [28].
2.2. Deduced protein analysis 3.2. Analysis of challenge experiment Secondary structure of the deduced proteins was predicted in the website (http://bioinf.cs.ucl.ac.uk/psipred/submit). Prediction of putative peroxisomal targeting signals (PTS) was done
In the challenge experiment, the first dead grass carp was observed at 12 h post GCRV injection. Then the mortality gradually increased
444
Q. Wan et al. / Fish & Shellfish Immunology 33 (2012) 442e447
Fig. 1. The sequencing maps and comparison of 15-bp deletion mutation site in CiRIG-I. The general allele named A, and the deletion mutation allele named B. (AA) The sequencing result of CiRIG-I-AA genotype. (AB) The sequencing result of heterozygote CiRIG-I-AB genotype. (BB) The sequencing result of CiRIG-I-BB genotype. The deletion mutation region was showed by underline with phrase. The deletion start site was indicated by triangle and phrase.
with the highest level at 72 h. About 65% of fish after GCRV injection died at termination of the experiment at 168 h after challenge. 79 grass carp that died in the first 72 h were considered as the susceptible individuals, and the 83 survivors were regarded as the resistant individuals. No dead fish was found in the control group.
Fig. 2. Comparison of corresponding cDNA and amino acid sequences of allele A and B in CiRIG-I. Allele A was showed in the top row, the sequence of allele B was in the middle, and the bottom row was the corresponding amino acid sequence. The 15-bp nucleotide absent in allele B was marked with gray background in allele A and corresponding amino acids was under wavy line.
Fig. 3. The 2.5% agarose electrophoresis profile of PCR products containing the mutant in CiRIG-I gene. M: marker D2000. AA indicated the genotype AA, and the fragment length was 745 bp; AB marked the heterozygous genotype AB, and the fragment lengths were 745 bp and 730 bp, respectively; BB demonstrated the genotype BB, and the fragment was 730 bp.
3.3. Association between the 15 nucleotide deletion and susceptibility/resistance to GCRV 162 PCR products were electrophoresed and the photographs were stored for analyzing the association between the nucleotide deletion and susceptibility/resistance to GCRV. Statistic analysis showed that the frequencies of genotype AA/AB/BB in susceptible group were 0.4430, 0.3671, 0.1899 and 0.6145, 0.3132, 0.0723 in resistant group, respectively (Table 2). The chi-square test indicated the cumulative mortality in AA (40.70%) and AB (52.73%) genotypes
Fig. 4. The predicted secondary structures of two types of CiRIG-I. (A): AA. (B): BB. ‘Conf’ indicated the possibility of the homologous configuration, the higher of the pillar the more possibility; ‘Pred’ showed the predicted type of secondary structure (C standed for coil; E indicated strand which was showed with yellow arrowhead; pink cylinder represented H, helix). The red frame was the deletion mutation region and corresponding secondary structure. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Q. Wan et al. / Fish & Shellfish Immunology 33 (2012) 442e447
445
Fig. 5. The predicted result of phosphorylation sites. The deletion amino acids were in the red frames. The red arrows pointed the possible phosphorylation site in the deletion. (A) All predicted phosphorylation sites in normal grass carp RIG-I. (B) The partial Threonine phosphorylation sites in normal grass carp RIG-I. Asterisks marked the quite possible phosphorylation sites, and the score of T215 was 0.660. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 1 Predicted scores of PTS1 in CiRIG-I-AA genotype and CiRIG-I-BB genotype.
were significantly lower than that in BB (71.43%) genotype (P < 0.05) (Fig. 6).
Genotype
Profile score
Sppt (Non-accessibility)
Sppt (Accessibility)
Total score
False positive
4. Discussion
AA BB
0.610 39.994
1.944 9.045
0.086 0.212
1.420 49.251
0.84% 73.06%
RIG-I was first found as an all-trans retinoic acid-inducible gene in an acute promyelocytic leukemia cell line [29]. In 2004, RIG-I was identified as a key receptor in detecting the replication of dsRNA virus genomes, and it is also an upstream signal of transcription factor nuclear factor-kB (NF-kB) and interferon regulatory factor-3 (IRF-3) in human [9]. Subsequently, RIG-I was also confirmed to recognize uncapped 50 -Triphosphate RNA [30]. Some scientists
Note: Profile score is a composite profile term that evaluates concordance with amino acid type preferences in the heterogeneous learning set. Sppt is a sum of independently parametrised terms. Any sequence yielding total score below 10 will be classified as ‘not targeted’, total score between 10 and 0 will be termed ‘twilight zone’ PTS1 signal-containing protein, and total score above 0 will be considered ‘targeted’.
446
Q. Wan et al. / Fish & Shellfish Immunology 33 (2012) 442e447
Table 2 Distribution of 15-bp deletion mutation of CiRIG-I in susceptible and resistant groups. Genotype
Susceptible N (%)
Resistant N (%)
AA AB BB
35 (44.30) 29 (36.71) 15 (18.99)
51 (61.45) 26 (31.32) 6 (7.23)
c2 6.903
P
Allele
Susceptible N (%)
Resistant N (%)
c2
P
0.032*
A B
99 (62.66) 59 (37.34)
128 (77.11) 38 (22.89)
8.059
0.005*
*The deletion mutation distribution between susceptible and resistant groups was significantly different (P < 0.05).
focused on the expression and antiviral function of RIG-I in some species, like human [30], duck [31], mouse [32] and so on. As to fish, RIG-I was mentioned to be important to antiviral immunity in Atlantic salmon and fathead minnow [15], grass carp [8], crucian carp [16] and common carp [17]. A frameshift mutation (P229fs) and a mutation (S183I) in human RIG-I gene drastically inhibit antiviral signaling and can be potential genetic determinants of viral susceptibility [33]. Some RIG-I polymorphisms in Caucasians have association with variations in both measles-specific IFN-g and IL-2 secretion [34]. In the present study, a 15-bp deletion was identified in coding region of CiRIG-I. Interestedly, the base before the 15-bp deletion is guanine, same as the last one in the deletion, which results in a transformation from a seeming frameshift mutation to a deletion mutation in CiRIG-I (Fig. 2). The deletion mutation does not locate in the major functional domains, it sites at the link between the second CARD and helicase domain. Around this region, a missense mutation in the first CARD of human RIG-I modifies the innate immune response to viral infection [35], and a missense mutation in the second CARD of human RIG-I results in the second CARD suppression and a dominant inhibitory function [36]. 55 amino acid linker between helicase domain and RD of RIG-I in human is responsible for holding RIG-I in suppression state in uninfected cells [37]. Thus, the influence of the five amino acids deletion mutation may not be ignored. In general, sequence is the foundation of the structure and function of one protein. To deduce the influences of the deletion mutation, the predicted structure, protein modification and signal transduction of the general and the mutant CiRIG-I were compared. 1) The deletion mutation changes the helix in E203 and A204 into coil, and changes the coil in M208 into strand (Fig. 4). The alteration may change the function(s) of CiRIG-I. 2) The deletion mutation cancels a possible threonine phosphorylation site (Fig. 5). Phosphorylation is an ordinary way in protein modification and has great influence in function of proteins. The test of phosphorylation and dephosphorylation in RIG-I illustrates phosphorylation can inhibit antiviral response [38]. 3) The normal CiRIG-I is ‘twilight zone’ PTS1 signal-containing protein while the mutant is ‘not targeted’ according to the total scores in Table 1. IPS-1 as an adapter
protein of RIG-I is located on peroxisome and mitochondria [39], and PTS1 is a guidance that leads proteins to import into the peroxisomal matrix smoothly [40,41]. This may demonstrate that the mutational CiRIG-I is less capable of recognising potential PTS1s [28], in another words, the mutational CiRIG-I may be less efficient to access peroxisome without PTS1, which may result in the less contact of RIG-I with peroxisomal IPS-1. As peroxisomal IPS-1 can trigger IFN-independent signaling pathway [39], the deletion mutation may affect immune response. These predicted results and inferences may hint that the deletion mutation is able to affect the resistance of grass carp to GCRV. On the other hand, the statistic analysis of the challenge test reveals that the deletion mutation significantly lowers the resistance of grass carp to GCRV (P ¼ 0.032). The AA genotype rate in resistant group (0.614) is notable higher than that in susceptible group (0.443), the BB genotype rate in resistant group (0.072) is about triplicate as low as that in susceptible group (0.190), and the AB genotype rates are no difference between resistant group (0.313) and susceptible group (0.367) (Table 2). In short, the AA, AB genotypes and A allele in CiRIG-I are more helpful for grass carp resistance to GCRV, and B allele can lower it. Thereupon, the above deductive effects of the deletion mutation were partially verified by the challenge experiment. In summary, a 15 nucleotide deletion mutation in coding region of the CiRIG-I was found and confirmed to be associated with the resistance of grass carp to GCRV. The specific mechanism of the association was deduced. The present study may provide a potential genetic marker for the selective breeding of resistant grass carp to GCRV. Acknowledgements The authors would like to thank Miss Limin Peng, Miss Qingmei Li and Miss Yixuan Zhang for technical advice and assistance in challenge experiment. The study was supported by Program for New Century Excellent Talents in University (NCET-08-0466), National Natural Science Foundation of China (30871917) and Chinese Universities Scientific Fund (QN2009022). References
Fig. 6. Cumulative mortality of grass carp in genotype AA, AB, BB after GCRV injection. 240 animals were injected with GCRV. The number of dead/survival individuals was 35/51, 29/26 and 15/6 for each genotype, and the numbers inside columns were the rates of cumulative mortality. The asterisk indicates statistically significant difference of cumulative mortality among the three genotypes (P < 0.05).
[1] Rangel AAC, Rockemann DD, Hetrick FM, Samal SK. Identification of grass carp haemorrhage virus as a new genogroup of aquareovirus. J Gen Virol 1999;80: 2399e402. [2] Chen Y, Jiang Y. Morphological and physic-chemical characterization of the hemorrhagic virus of grass carp. KeXue TongBao 1984;29:832e5. [3] Fang Q, Attoui H, Cantaloube JF, Biagini P, Zhu Z, de Micco P, et al. Sequence of genome segments 1, 2, and 3 of the grass carp reovirus (Genus Aquareovirus, family Reoviridae). Biochem Biophys Res Commun 2000;274:762e6. [4] Ye X, Tian YY, Deng GC, Chi YY, Jiang XY. Complete genomic sequence of a reovirus isolated from grass carp in China. Virus Res 2012;163:275e83. [5] Dostert C, Meylan E, Tschopp J. Intracellular pattern-recognition receptors. Adv Drug Deliver Rev 2008;60:830e40. [6] Su J, Jang S, Yang C, Wang Y, Zhu Z. Genomic organization and expression analysis of Toll-like receptor 3 in grass carp (Ctenopharyngodon idella). Fish Shellfish Immunol 2009;27:433e9. [7] Chen W, Xu Q, Chang M, Nie P, Peng K. Molecular characterization and expression analysis of nuclear oligomerization domain proteins NOD1 and NOD2 in grass carp Ctenopharyngodon idella. Fish Shellfish Immunol 2010;28: 18e29.
Q. Wan et al. / Fish & Shellfish Immunology 33 (2012) 442e447 [8] Yang C, Su J, Huang T, Zhang R, Peng L. Identification of a retinoic acid-inducible gene I from grass carp (Ctenopharyngodon idella) and expression analysis in vivo and in vitro. Fish Shellfish Immunol 2011;30:936e43. [9] Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, et al. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol 2004;5:730e7. [10] Saito T, Hirai R, Loo YM, Owen D, Johnson CL, Sinha SC, et al. Regulation of innate antiviral defenses through a shared repressor domain in RIG-I and LGP2. Proc Natl Acad Sci USA 2007;104:582e7. [11] Yoneyama M, Fujita T. Structural mechanism of RNA recognition by the RIG-Ilike receptors. Immunity 2008;29:178e81. [12] Loo YM, Gale Jr M. Immune signaling by RIG-I-like receptors. Immunity 2011; 34:680e92. [13] Zou J, Chang M, Nie P, Secombes CJ. Origin and evolution of the RIG-I like RNA helicase gene family. BMC Evol Biol 2009;9:85. [14] Sarkar D, DeSalle R, Fisher P. Evolution of MDA-5/RIG-I-dependent innate immunity: independent evolution by domain grafting. Proc Natl Acad Sci USA 2008;105:17040e5. [15] Biacchesi S, LeBerre M, Lamoureux A, Louise Y, Lauret E, Boudinot P, et al. Mitochondrial antiviral signaling protein plays a major role in induction of the fish innate immune response against RNA and DNA viruses. J Virol 2009;83: 7815e27. [16] Sun F, Zhang YB, Liu TK, Shi J, Wang B, Gui JF. Fish MITA serves as a mediator for distinct fish IFN gene activation dependent on IRF3 or IRF7. J Immunol 2011;187:2531e9. [17] Feng H, Liu H, Kong R, Wang L, Wang Y, Hu W, et al. Expression profiles of carp IRF-3/-7 correlate with the up-regulation of RIG-I/MAVS/TRAF3/TBK1, four pivotal molecules in RIG-I signaling pathway. Fish Shellfish Immunol 2011; 30:1159e69. [18] Xu T, Chen S, Ji X, Tian Y. MHC polymorphism and disease resistance to Vibrio anguillarum in 12 selective Japanese flounder (Paralichthys olivaceus) families. Fish Shellfish Immunol 2008;25:213e21. [19] Kongchum P, Sandel E, Lutzky S, Hallerman EM, Hulata G, David L, et al. Association between IL-10a single nucleotide polymorphisms and resistance to Cyprinid herpesvirus-3 infection in common carp (Cyprinus carpio). Aquaculture 2011;315:417e21. [20] Heng J, Su J, Huang T, Dong J, Chen L. The polymorphism and haplotype of TLR3 gene in grass carp (Ctenopharyngodon idella) and their associations with susceptibility/resistance to grass carp reovirus. Fish Shellfish Immunol 2011; 30:45e50. [21] Wang L, Su J, Peng L, Heng J, Chen L. Genomic structure of grass carp Mx2 and the association of its polymorphisms with susceptibility/resistance to grass carp reovirus. Mol Immunol 2011;49:359e66. [22] Su J, Heng J, Huang T, Peng L, Yang C, Li Q. Identification, mRNA expression and genomic structure of TLR22 and its association with GCRV susceptibility/ resistance in grass carp (Ctenopharyngodon idella). Dev Comp Immunol 2012; 36:450e62. [23] Crawford S, Goff SP. A deletion mutation in the 5’ part of the pol gene of moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins. J Virol 1985;53:899e907. [24] Jiao Y, Jin X, Yan J, Zhang C, Jiao F, Li X, et al. A deletion mutation in SLC12A6 is associated with neuromuscular disease in gaxp mice. Genomics 2008;91: 407e14.
447
[25] Chen S, He Y, Ding J, Jiang Y, Jia S, Xia W, et al. An insertion/deletion polymorphism in the 3’ untranslated region of b-transducin repeat-containing protein (bTrCP) is associated with susceptibility for hepatocellular carcinoma in Chinese. Biochem Biophys Res Commun 2010;391:552e6. [26] Park M, Kim DJ, Kim KJ, Hong CB, Kim YJ, Cheong HS, et al. Genetic associations of common deletion polymorphisms in families with Avellino corneal dystrophy. Biochem Biophys Res Commun 2009;387:688e93. [27] Ajmal M, Ahmed W, Sadeque A, Ali SHB, Bokhari SH, Ahmed N, et al. Identification of a recurrent insertion mutation in the LDLR gene in a Pakistani family with autosomal dominant hypercholesterolemia. Mol Biol Rep 2010; 37:3869e75. [28] Neuberger G, Maurer-Stroh S, Eisenhaber B, Hartig A, Eisenhaber F. Prediction of peroxisomal targeting signal 1 containing proteins from amino acid sequence. J Mol Biol 2003;328:581e92. [29] Hornung V, Ellegast J, Kim S, Brzózka K, Jung A, Kato H, et al. 5’-Triphosphate RNA is the ligand for RIG-I. Science 2006;314:994e7. [30] Barber MR, Aldridge Jr JR, Webster RG, Magor KE. Association of RIG-I with innate immunity of ducks to influenza. Proc Natl Acad Sci USA 2010;107: 5913e8. [31] Sun Y. RIG-I, a human homolog gene of RNA helicase, is induced by retinoic acid during the differentiation of acute promyelocytic leukemia cell. Thesis (1997) Shanghai Institute of Haematology, Rui-Jin Hospital, Shanghai Second Medical University. [32] Nazmi A, Dutta K, Basu A. RIG-I mediates innate immune response in mouse neurons following Japanese encephalitis virus infection. PLoS One 2011;6: e21761. [33] Pothlichet J, Burtey A, Kubarenko AV, Caignard G, Solhonne B, Tangy F, et al. Study of human RIG-I polymorphisms identifies two variants with an opposite impact on the antiviral immune response. PLos One 2009;4:e7582. [34] Haralambieva IH, Ovsyannikova IG, Umlauf BJ, Vierkant RA, Shane Pankratz V, Jacobson RM, et al. Genetic polymorphisms in host antiviral genes: associations with humoral and cellular immunity to measles vaccine. Vaccine 2011; 29:8988e97. [35] Hu J, Nistal-Villán E, Voho A, Ganee A, Kumar M, Ding Y, et al. A common polymorphism in the caspase recruitment domain of RIG-I modifies the innate immune response of human dendritic cells. J Immunol 2010;185:424e32. [36] Shigemoto T, Kageyama M, Hirai R, Zheng JP, Yoneyama M, Fujita T. Identification of loss of function mutations in human genes encoding RIG-I and MDA5: implications for resistance to type I diabetes. J Biol Chem 2009;284: 13348e54. [37] Kageyama M, Takahasi K, Narita R, Hirai R, Yoneyama M, Kato H, et al. 55 Amino acid linker between helicase and carboxyl terminal domains of RIG-I functions as a critical repression domain and determines inter-domain conformation. Biochem Biophys Res Commun 2011;415:78e81. [38] Sun Z, Ren H, Liu Y, Teeling JL, Gu J. Phosphorylation of RIG-I by casein kinase II inhibits its antiviral response. J Virol 2011;85:1036e47. [39] Dixit E, Boulant S, Zhang Y, Lee AS, Odendall C, Shum B, et al. Peroxisomes are signaling platforms for antiviral innate immunity. Cell 2010;141:668e81. [40] Gould SJ, Keller GA, Subramani S. Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase. J Cell Biol 1987;105: 2923e31. [41] Subramani S. Targeting of proteins into the peroxisomal matrix. J Membr Biol 1992;125:99e106.
Contents lists available at SciVerse ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Short communication
A 15 nucleotide deletion mutation in coding region of the RIG-I lowers grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus Quanyuan Wan, Jianguo Su*, Lan Wang, Lijun Chen, Xiaohui Chen College of Animal Science and Technology, Northwest A&F University, Shaanxi Key Laboratory of Molecular Biology for Agriculture, No. 22 Xinong Rd, Yangling 712100, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 January 2012 Received in revised form 19 April 2012 Accepted 7 May 2012 Available online 21 May 2012
RIG-I (Retinoic acid-inducible gene I) is a pivotal receptor that detects numerous RNA and DNA viruses and plays crucial roles in the induction of type I interferons. In the present study, a deletion mutation in CiRIG-I (Ctenopharyngodon idella RIG-I) coding region was detected, its association with resistance/ susceptibility to grass carp reovirus (GCRV) was examined, and possible mechanism was analyzed. A 15bp deletion mutation was found, and the mutation results in a deletion of five amino acids. To investigate the genotypes and alleles, the relevant PCR products were electrophoresed on 2.5% agarose gel. Three genotypes and two alleles were discovered. The general allele was named as A and the deletion mutation allele was named as B. The deletion mutation cancels a predicted phosphorylation site and changes the secondary structure and the probability of peroxisomal targeting signal 1 in CiRIG-I. To explore the correlation between these genotypes and the resistance of grass carp to GCRV, a challenge experiment was carried out. The cumulative mortality in genotype AA (40.70%) and AB (52.73%) was significantly lower than that in genotype BB (71.43%) (P ¼ 0.032). The result demonstrated that genotype AA and AB were resistant to GCRV, while genotype BB was susceptible. The 15-bp deletion mutation lowers the resistance of grass carp to GCRV. This result might provide a potential genetic marker for further investigation of selective breeding of resistant grass carp to GCRV. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Grass carp (Ctenopharyngodon idella) RIG-I Deletion mutation Grass carp reovirus
1. Introduction Grass carp (Ctenopharyngodon idella) is a momentous economic aquaculture species in China, but the fractional yield is impeded by grass carp reovirus (GCRV), a double strand RNA (dsRNA) virus [1]. Since GCRV was characterized in 1970s [2], researches have been going on continuously. Some researchers focused on the genomic sequence of GCRV [3,4], and others were more concerned with the innate immunity in host cells after GCRV infection. In innate immunity, pattern recognition receptors (PRRs) can recognize the specific conserved structures among pathogens that called pathogen associated molecular patterns (PAMPs) [5]. PRRs mainly include Toll-like receptors (TLRs), RIG-I (Retinoic acid-inducible gene I) -like receptors (RLRs), Nucleotide-oligomerization domain (NOD)-like receptors (NLRs), etc. Some members of the three PRRs had been reported to play roles in response to GCRV, such as TLR3 [6], NOD1 and NOD2 [7] and RIG-I [8].
* Corresponding author. Tel.: þ86 29 87092139; fax: þ86 29 87092164. E-mail address: [email protected] (J. Su). 1050-4648/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2012.05.010
RIG-I (also called DDX58), MDA5 (Melanoma differentiation associated gene 5, also known as IFIH1 or Helicard) and LGP2 (Laboratory of genetics and physiology 2, also referred to as DHX58) constitute RLRs. RIG-I belongs to the DExD/H box RNA helicase family and consists of two N-terminal caspase recruitment domains (CARDs), a signature of DExD/H box RNA helicase domain with the ATP-hydrolyzing function and the capacity to change the conformation of substrate dsRNA, and a C-terminal repression domain (RD) that can control RIG-I multimerization [9e11]. When viral RNA in the cytoplasm are detected by RIG-I, the adapter protein named IPS1 (interferon-b promoter stimulator 1, also known as MAVS, VISA or CARDIF) is activated, via downstream transcription factor to boost type 1 interferon (IFN) production and antiviral gene expression to control virus infection [9,12]. As a PRR in antiviral immunity, RIG-I has received wide attention [13,14]. And also, RIG-I was identified in some fishes, such as Salmo salar (accession NO., CAX48607) [15], Carassius auratus (accession NO., JF970225) [16], Cyprinus carpio (accession NO., HQ850439) [17] and C. idella (accession NO., GQ478334) [8]. The expression of CiRIG-I gene (C. idella RIG-I) can be significantly up-regulated by poly(I:C) and GCRV [8]. As more and more immune-related genes are characterized, the relevance of polymorphism and spontaneous deletion
Q. Wan et al. / Fish & Shellfish Immunology 33 (2012) 442e447
mutation to certain diseases becomes a growing focus point. The polymorphism research in Japanese flounder MHC gene reveals that nine alleles are related to the resistance to Vibrio anguillarum [18]. In common carp, three single nucleotide polymorphisms (SNPs) in IL-10a gene are associated with resistance/susceptibility to cyprinid herpesvirus-3 infection [19]. Some polymorphisms of TLR3 [20], Mx2 [21] and TLR22 [22] in grass carp have significant association with resistance/susceptibility to GCRV. Deletion mutations in the 50 part of the pol gene of Moloney murine leukemia virus result in variation of its virulence [23]. In mice, a 17-base deletion in exon 4 of SLC12A6 is associated with neuromuscular disease [24]. Some insertion/deletion mutations are also related to disease in human. For instance, a 9 bp insertion/deletion polymorphism in the 30 untranslated region of b-transducin repeat-containing protein (bTrCP) is associated with susceptibility for hepatocellular carcinoma in Chinese people [25]. One deletion polymorphism (rs885945) neighboring the gene encoding MHC IF is significantly associated with the risk of Avellino corneal dystrophy [26]. In a Pakistani family with autosomal dominant hypercholesterolemia, a recurrent insertion mutation in the low density lipoprotein receptor (LDLR) gene is identified [27]. In the present study, CiRIG-I was employed to detect delete mutations in coding region and to analyze the association with resistance/susceptibility to GCRV. 2. Materials and methods 2.1. Sample preparation and deletion mutation discovery 40 grass carp were collected from the fish farm in Rougu (Shaanxi province, China), and approximate 1 g muscle of each fish was cut and kept at 80 C until DNA isolation. About 200 mg sample was homogenized, and DNA was extracted with traditional phenol-chloroform method and was stored at 20 C. The incipient primers used to explore CiRIG-I genomic structure were designed from the CiRIG-I cDNA sequence (GenBank accession NO., GQ478334), using Primer Premier 5.0 software. The reaction volume of PCR was 25 mL, containing 100 ng genomic DNA, 10 pM of each primer, Buffer (including MgCl2), 200 mM dNTPs and 2 units of Taq DNA polymerase (MBI). The PCR protocol was 3 min at 95 C, 35 cycles of 94 C for 30 s, n oC (according to the annealing temperatures of the different primers), annealing for 30 s, 72 C for 2 min, with a final extension at 72 C for 5 min. The products were purified by using the DNA Fragment Purification Kit (TIANGEN Corp., Beijing, China) and then ligated with pMD18-T vector (Takara). Three positive clones of each fragment were sequenced (Nanjing GeneScript Corp., China). Sequencing results were aligned by Vector NTI Suite 11.0 (Invitrogen). After obtaining the full length genomic sequence of CiRIG-I gene by PCR amplification with sequencespecific primers and genomic walking, DNA pool sequencing of 20 grass carp was carried out to detect polymorphisms or deletion/ insertion mutations. Very excited to find a 15 nucleotide deletion mutation in the coding region of CiRIG-I gene. In order to confirm the deletion mutation, another pair of primers was designed, forward primer RF186rc (50 -AAGGGCTGCGGATCGGAT-30 ) and reverse primer RR505 (50 -CCTGCCTGACAGACATTTATTGC-30 ). The size of expected PCR product was 745 bp. PCR was performed using genomic DNA of the 40 grass carp as template. The PCR products were purified and sequenced directly, meanwhile, the corresponding PCR products were electrophoresed on 2.5% agarose gels.
443
using the PTS1 predictor (http://mendel.imp.ac.at/mendeljsp/sat/ pts1/PTS1predictor.jsp). The subsequent forecast of possible phosphorylation sites was implemented using the tool (http://www.cbs. dtu.dk/services/NetPhos/). 2.3. Challenge experiment Average 10 cm in body length of 300 grass carp were collected from three fish farms (Shaanxi province, China), where hemorrhagic disease of grass carp was not found in recent years. The juvenile fish of the three fish farms were bought from the same fish hatchery, and the mating of parent fishes in this fish hatchery is randomly. Then, the 300 grass carp were fostered in aerated freshwater at 28 C for one week before processing. For the viral infection experiment, grass carp were divided into five groups (60 animals in each group). The conditions were the same among tanks and the fish were randomly distributed into different tanks. Four groups were cultured in four aquariums and intraperitoneally injected with 100 mL of GCRV (097 strain, 3.63 107 TCID50/ml), suspended in PBS, per gram body weight. The control group was reared in another aquarium and injected with PBS. All the fish were observed every 6 h to survey the mortality and collect samples until the termination of the experiment at 168 h after challenge. Grass carp died in the first 72 h post challenge were classified as susceptible individuals for their high sensitivity to GCRV and obvious symptoms of hemorrhage disease of grass carp, while the animals that survived over 168 h post challenge were considered as resistant group, and the rest were discarded for their ambiguity to GCRV. Approximate 1 g muscle was cut and kept at 80 C until DNA isolation. 2.4. Association analysis between deletion mutation and susceptibility/resistance to GCRV The genomic DNA of susceptible individuals and resistant individuals were used as templates in PCR. The PCR products were electrophoresed, and the agarose electrophoresis results were photographed by quantity one system (Bio-Rad) as preparation for statistical analysis. Allele and genotype frequencies and their associations with susceptibility/resistance to GCRV were analysed with SHEsis software (http://analysis.bio-x.cn/SHEsisMain.htm). The P value less than 0.05 was considered statistically significant. 3. Results 3.1. Deletion mutation in CiRIG-I A 15 nucleotide deletion mutation was found in the coding region and was described as g. 665-679 del AAA CCA CGA CGA CAG (Fig. 1). Certainly, there are two alleles: the general allele named as A and the deletion mutation allele named as B. And the deletion mutation in CiRIG-I coincidently caused a deletion of five amino acids (Fig. 2). After agarose electrophoresis, the result had three genotypes in CiRIG-I: homozygous genotypes AA and BB, heterozygous genotype AB (Fig. 3). The predicted results showed the deletion mutation may change the secondary structure of CiRIG-I protein (Fig. 4) and may delete a phosphorylation site which was called T215 (Fig. 5), also may change the probability of peroxisomal targeting signal 1 (PTS1) (Table 1) [28].
2.2. Deduced protein analysis 3.2. Analysis of challenge experiment Secondary structure of the deduced proteins was predicted in the website (http://bioinf.cs.ucl.ac.uk/psipred/submit). Prediction of putative peroxisomal targeting signals (PTS) was done
In the challenge experiment, the first dead grass carp was observed at 12 h post GCRV injection. Then the mortality gradually increased
444
Q. Wan et al. / Fish & Shellfish Immunology 33 (2012) 442e447
Fig. 1. The sequencing maps and comparison of 15-bp deletion mutation site in CiRIG-I. The general allele named A, and the deletion mutation allele named B. (AA) The sequencing result of CiRIG-I-AA genotype. (AB) The sequencing result of heterozygote CiRIG-I-AB genotype. (BB) The sequencing result of CiRIG-I-BB genotype. The deletion mutation region was showed by underline with phrase. The deletion start site was indicated by triangle and phrase.
with the highest level at 72 h. About 65% of fish after GCRV injection died at termination of the experiment at 168 h after challenge. 79 grass carp that died in the first 72 h were considered as the susceptible individuals, and the 83 survivors were regarded as the resistant individuals. No dead fish was found in the control group.
Fig. 2. Comparison of corresponding cDNA and amino acid sequences of allele A and B in CiRIG-I. Allele A was showed in the top row, the sequence of allele B was in the middle, and the bottom row was the corresponding amino acid sequence. The 15-bp nucleotide absent in allele B was marked with gray background in allele A and corresponding amino acids was under wavy line.
Fig. 3. The 2.5% agarose electrophoresis profile of PCR products containing the mutant in CiRIG-I gene. M: marker D2000. AA indicated the genotype AA, and the fragment length was 745 bp; AB marked the heterozygous genotype AB, and the fragment lengths were 745 bp and 730 bp, respectively; BB demonstrated the genotype BB, and the fragment was 730 bp.
3.3. Association between the 15 nucleotide deletion and susceptibility/resistance to GCRV 162 PCR products were electrophoresed and the photographs were stored for analyzing the association between the nucleotide deletion and susceptibility/resistance to GCRV. Statistic analysis showed that the frequencies of genotype AA/AB/BB in susceptible group were 0.4430, 0.3671, 0.1899 and 0.6145, 0.3132, 0.0723 in resistant group, respectively (Table 2). The chi-square test indicated the cumulative mortality in AA (40.70%) and AB (52.73%) genotypes
Fig. 4. The predicted secondary structures of two types of CiRIG-I. (A): AA. (B): BB. ‘Conf’ indicated the possibility of the homologous configuration, the higher of the pillar the more possibility; ‘Pred’ showed the predicted type of secondary structure (C standed for coil; E indicated strand which was showed with yellow arrowhead; pink cylinder represented H, helix). The red frame was the deletion mutation region and corresponding secondary structure. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Q. Wan et al. / Fish & Shellfish Immunology 33 (2012) 442e447
445
Fig. 5. The predicted result of phosphorylation sites. The deletion amino acids were in the red frames. The red arrows pointed the possible phosphorylation site in the deletion. (A) All predicted phosphorylation sites in normal grass carp RIG-I. (B) The partial Threonine phosphorylation sites in normal grass carp RIG-I. Asterisks marked the quite possible phosphorylation sites, and the score of T215 was 0.660. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 1 Predicted scores of PTS1 in CiRIG-I-AA genotype and CiRIG-I-BB genotype.
were significantly lower than that in BB (71.43%) genotype (P < 0.05) (Fig. 6).
Genotype
Profile score
Sppt (Non-accessibility)
Sppt (Accessibility)
Total score
False positive
4. Discussion
AA BB
0.610 39.994
1.944 9.045
0.086 0.212
1.420 49.251
0.84% 73.06%
RIG-I was first found as an all-trans retinoic acid-inducible gene in an acute promyelocytic leukemia cell line [29]. In 2004, RIG-I was identified as a key receptor in detecting the replication of dsRNA virus genomes, and it is also an upstream signal of transcription factor nuclear factor-kB (NF-kB) and interferon regulatory factor-3 (IRF-3) in human [9]. Subsequently, RIG-I was also confirmed to recognize uncapped 50 -Triphosphate RNA [30]. Some scientists
Note: Profile score is a composite profile term that evaluates concordance with amino acid type preferences in the heterogeneous learning set. Sppt is a sum of independently parametrised terms. Any sequence yielding total score below 10 will be classified as ‘not targeted’, total score between 10 and 0 will be termed ‘twilight zone’ PTS1 signal-containing protein, and total score above 0 will be considered ‘targeted’.
446
Q. Wan et al. / Fish & Shellfish Immunology 33 (2012) 442e447
Table 2 Distribution of 15-bp deletion mutation of CiRIG-I in susceptible and resistant groups. Genotype
Susceptible N (%)
Resistant N (%)
AA AB BB
35 (44.30) 29 (36.71) 15 (18.99)
51 (61.45) 26 (31.32) 6 (7.23)
c2 6.903
P
Allele
Susceptible N (%)
Resistant N (%)
c2
P
0.032*
A B
99 (62.66) 59 (37.34)
128 (77.11) 38 (22.89)
8.059
0.005*
*The deletion mutation distribution between susceptible and resistant groups was significantly different (P < 0.05).
focused on the expression and antiviral function of RIG-I in some species, like human [30], duck [31], mouse [32] and so on. As to fish, RIG-I was mentioned to be important to antiviral immunity in Atlantic salmon and fathead minnow [15], grass carp [8], crucian carp [16] and common carp [17]. A frameshift mutation (P229fs) and a mutation (S183I) in human RIG-I gene drastically inhibit antiviral signaling and can be potential genetic determinants of viral susceptibility [33]. Some RIG-I polymorphisms in Caucasians have association with variations in both measles-specific IFN-g and IL-2 secretion [34]. In the present study, a 15-bp deletion was identified in coding region of CiRIG-I. Interestedly, the base before the 15-bp deletion is guanine, same as the last one in the deletion, which results in a transformation from a seeming frameshift mutation to a deletion mutation in CiRIG-I (Fig. 2). The deletion mutation does not locate in the major functional domains, it sites at the link between the second CARD and helicase domain. Around this region, a missense mutation in the first CARD of human RIG-I modifies the innate immune response to viral infection [35], and a missense mutation in the second CARD of human RIG-I results in the second CARD suppression and a dominant inhibitory function [36]. 55 amino acid linker between helicase domain and RD of RIG-I in human is responsible for holding RIG-I in suppression state in uninfected cells [37]. Thus, the influence of the five amino acids deletion mutation may not be ignored. In general, sequence is the foundation of the structure and function of one protein. To deduce the influences of the deletion mutation, the predicted structure, protein modification and signal transduction of the general and the mutant CiRIG-I were compared. 1) The deletion mutation changes the helix in E203 and A204 into coil, and changes the coil in M208 into strand (Fig. 4). The alteration may change the function(s) of CiRIG-I. 2) The deletion mutation cancels a possible threonine phosphorylation site (Fig. 5). Phosphorylation is an ordinary way in protein modification and has great influence in function of proteins. The test of phosphorylation and dephosphorylation in RIG-I illustrates phosphorylation can inhibit antiviral response [38]. 3) The normal CiRIG-I is ‘twilight zone’ PTS1 signal-containing protein while the mutant is ‘not targeted’ according to the total scores in Table 1. IPS-1 as an adapter
protein of RIG-I is located on peroxisome and mitochondria [39], and PTS1 is a guidance that leads proteins to import into the peroxisomal matrix smoothly [40,41]. This may demonstrate that the mutational CiRIG-I is less capable of recognising potential PTS1s [28], in another words, the mutational CiRIG-I may be less efficient to access peroxisome without PTS1, which may result in the less contact of RIG-I with peroxisomal IPS-1. As peroxisomal IPS-1 can trigger IFN-independent signaling pathway [39], the deletion mutation may affect immune response. These predicted results and inferences may hint that the deletion mutation is able to affect the resistance of grass carp to GCRV. On the other hand, the statistic analysis of the challenge test reveals that the deletion mutation significantly lowers the resistance of grass carp to GCRV (P ¼ 0.032). The AA genotype rate in resistant group (0.614) is notable higher than that in susceptible group (0.443), the BB genotype rate in resistant group (0.072) is about triplicate as low as that in susceptible group (0.190), and the AB genotype rates are no difference between resistant group (0.313) and susceptible group (0.367) (Table 2). In short, the AA, AB genotypes and A allele in CiRIG-I are more helpful for grass carp resistance to GCRV, and B allele can lower it. Thereupon, the above deductive effects of the deletion mutation were partially verified by the challenge experiment. In summary, a 15 nucleotide deletion mutation in coding region of the CiRIG-I was found and confirmed to be associated with the resistance of grass carp to GCRV. The specific mechanism of the association was deduced. The present study may provide a potential genetic marker for the selective breeding of resistant grass carp to GCRV. Acknowledgements The authors would like to thank Miss Limin Peng, Miss Qingmei Li and Miss Yixuan Zhang for technical advice and assistance in challenge experiment. The study was supported by Program for New Century Excellent Talents in University (NCET-08-0466), National Natural Science Foundation of China (30871917) and Chinese Universities Scientific Fund (QN2009022). References
Fig. 6. Cumulative mortality of grass carp in genotype AA, AB, BB after GCRV injection. 240 animals were injected with GCRV. The number of dead/survival individuals was 35/51, 29/26 and 15/6 for each genotype, and the numbers inside columns were the rates of cumulative mortality. The asterisk indicates statistically significant difference of cumulative mortality among the three genotypes (P < 0.05).
[1] Rangel AAC, Rockemann DD, Hetrick FM, Samal SK. Identification of grass carp haemorrhage virus as a new genogroup of aquareovirus. J Gen Virol 1999;80: 2399e402. [2] Chen Y, Jiang Y. Morphological and physic-chemical characterization of the hemorrhagic virus of grass carp. KeXue TongBao 1984;29:832e5. [3] Fang Q, Attoui H, Cantaloube JF, Biagini P, Zhu Z, de Micco P, et al. Sequence of genome segments 1, 2, and 3 of the grass carp reovirus (Genus Aquareovirus, family Reoviridae). Biochem Biophys Res Commun 2000;274:762e6. [4] Ye X, Tian YY, Deng GC, Chi YY, Jiang XY. Complete genomic sequence of a reovirus isolated from grass carp in China. Virus Res 2012;163:275e83. [5] Dostert C, Meylan E, Tschopp J. Intracellular pattern-recognition receptors. Adv Drug Deliver Rev 2008;60:830e40. [6] Su J, Jang S, Yang C, Wang Y, Zhu Z. Genomic organization and expression analysis of Toll-like receptor 3 in grass carp (Ctenopharyngodon idella). Fish Shellfish Immunol 2009;27:433e9. [7] Chen W, Xu Q, Chang M, Nie P, Peng K. Molecular characterization and expression analysis of nuclear oligomerization domain proteins NOD1 and NOD2 in grass carp Ctenopharyngodon idella. Fish Shellfish Immunol 2010;28: 18e29.
Q. Wan et al. / Fish & Shellfish Immunology 33 (2012) 442e447 [8] Yang C, Su J, Huang T, Zhang R, Peng L. Identification of a retinoic acid-inducible gene I from grass carp (Ctenopharyngodon idella) and expression analysis in vivo and in vitro. Fish Shellfish Immunol 2011;30:936e43. [9] Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, et al. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol 2004;5:730e7. [10] Saito T, Hirai R, Loo YM, Owen D, Johnson CL, Sinha SC, et al. Regulation of innate antiviral defenses through a shared repressor domain in RIG-I and LGP2. Proc Natl Acad Sci USA 2007;104:582e7. [11] Yoneyama M, Fujita T. Structural mechanism of RNA recognition by the RIG-Ilike receptors. Immunity 2008;29:178e81. [12] Loo YM, Gale Jr M. Immune signaling by RIG-I-like receptors. Immunity 2011; 34:680e92. [13] Zou J, Chang M, Nie P, Secombes CJ. Origin and evolution of the RIG-I like RNA helicase gene family. BMC Evol Biol 2009;9:85. [14] Sarkar D, DeSalle R, Fisher P. Evolution of MDA-5/RIG-I-dependent innate immunity: independent evolution by domain grafting. Proc Natl Acad Sci USA 2008;105:17040e5. [15] Biacchesi S, LeBerre M, Lamoureux A, Louise Y, Lauret E, Boudinot P, et al. Mitochondrial antiviral signaling protein plays a major role in induction of the fish innate immune response against RNA and DNA viruses. J Virol 2009;83: 7815e27. [16] Sun F, Zhang YB, Liu TK, Shi J, Wang B, Gui JF. Fish MITA serves as a mediator for distinct fish IFN gene activation dependent on IRF3 or IRF7. J Immunol 2011;187:2531e9. [17] Feng H, Liu H, Kong R, Wang L, Wang Y, Hu W, et al. Expression profiles of carp IRF-3/-7 correlate with the up-regulation of RIG-I/MAVS/TRAF3/TBK1, four pivotal molecules in RIG-I signaling pathway. Fish Shellfish Immunol 2011; 30:1159e69. [18] Xu T, Chen S, Ji X, Tian Y. MHC polymorphism and disease resistance to Vibrio anguillarum in 12 selective Japanese flounder (Paralichthys olivaceus) families. Fish Shellfish Immunol 2008;25:213e21. [19] Kongchum P, Sandel E, Lutzky S, Hallerman EM, Hulata G, David L, et al. Association between IL-10a single nucleotide polymorphisms and resistance to Cyprinid herpesvirus-3 infection in common carp (Cyprinus carpio). Aquaculture 2011;315:417e21. [20] Heng J, Su J, Huang T, Dong J, Chen L. The polymorphism and haplotype of TLR3 gene in grass carp (Ctenopharyngodon idella) and their associations with susceptibility/resistance to grass carp reovirus. Fish Shellfish Immunol 2011; 30:45e50. [21] Wang L, Su J, Peng L, Heng J, Chen L. Genomic structure of grass carp Mx2 and the association of its polymorphisms with susceptibility/resistance to grass carp reovirus. Mol Immunol 2011;49:359e66. [22] Su J, Heng J, Huang T, Peng L, Yang C, Li Q. Identification, mRNA expression and genomic structure of TLR22 and its association with GCRV susceptibility/ resistance in grass carp (Ctenopharyngodon idella). Dev Comp Immunol 2012; 36:450e62. [23] Crawford S, Goff SP. A deletion mutation in the 5’ part of the pol gene of moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins. J Virol 1985;53:899e907. [24] Jiao Y, Jin X, Yan J, Zhang C, Jiao F, Li X, et al. A deletion mutation in SLC12A6 is associated with neuromuscular disease in gaxp mice. Genomics 2008;91: 407e14.
447
[25] Chen S, He Y, Ding J, Jiang Y, Jia S, Xia W, et al. An insertion/deletion polymorphism in the 3’ untranslated region of b-transducin repeat-containing protein (bTrCP) is associated with susceptibility for hepatocellular carcinoma in Chinese. Biochem Biophys Res Commun 2010;391:552e6. [26] Park M, Kim DJ, Kim KJ, Hong CB, Kim YJ, Cheong HS, et al. Genetic associations of common deletion polymorphisms in families with Avellino corneal dystrophy. Biochem Biophys Res Commun 2009;387:688e93. [27] Ajmal M, Ahmed W, Sadeque A, Ali SHB, Bokhari SH, Ahmed N, et al. Identification of a recurrent insertion mutation in the LDLR gene in a Pakistani family with autosomal dominant hypercholesterolemia. Mol Biol Rep 2010; 37:3869e75. [28] Neuberger G, Maurer-Stroh S, Eisenhaber B, Hartig A, Eisenhaber F. Prediction of peroxisomal targeting signal 1 containing proteins from amino acid sequence. J Mol Biol 2003;328:581e92. [29] Hornung V, Ellegast J, Kim S, Brzózka K, Jung A, Kato H, et al. 5’-Triphosphate RNA is the ligand for RIG-I. Science 2006;314:994e7. [30] Barber MR, Aldridge Jr JR, Webster RG, Magor KE. Association of RIG-I with innate immunity of ducks to influenza. Proc Natl Acad Sci USA 2010;107: 5913e8. [31] Sun Y. RIG-I, a human homolog gene of RNA helicase, is induced by retinoic acid during the differentiation of acute promyelocytic leukemia cell. Thesis (1997) Shanghai Institute of Haematology, Rui-Jin Hospital, Shanghai Second Medical University. [32] Nazmi A, Dutta K, Basu A. RIG-I mediates innate immune response in mouse neurons following Japanese encephalitis virus infection. PLoS One 2011;6: e21761. [33] Pothlichet J, Burtey A, Kubarenko AV, Caignard G, Solhonne B, Tangy F, et al. Study of human RIG-I polymorphisms identifies two variants with an opposite impact on the antiviral immune response. PLos One 2009;4:e7582. [34] Haralambieva IH, Ovsyannikova IG, Umlauf BJ, Vierkant RA, Shane Pankratz V, Jacobson RM, et al. Genetic polymorphisms in host antiviral genes: associations with humoral and cellular immunity to measles vaccine. Vaccine 2011; 29:8988e97. [35] Hu J, Nistal-Villán E, Voho A, Ganee A, Kumar M, Ding Y, et al. A common polymorphism in the caspase recruitment domain of RIG-I modifies the innate immune response of human dendritic cells. J Immunol 2010;185:424e32. [36] Shigemoto T, Kageyama M, Hirai R, Zheng JP, Yoneyama M, Fujita T. Identification of loss of function mutations in human genes encoding RIG-I and MDA5: implications for resistance to type I diabetes. J Biol Chem 2009;284: 13348e54. [37] Kageyama M, Takahasi K, Narita R, Hirai R, Yoneyama M, Kato H, et al. 55 Amino acid linker between helicase and carboxyl terminal domains of RIG-I functions as a critical repression domain and determines inter-domain conformation. Biochem Biophys Res Commun 2011;415:78e81. [38] Sun Z, Ren H, Liu Y, Teeling JL, Gu J. Phosphorylation of RIG-I by casein kinase II inhibits its antiviral response. J Virol 2011;85:1036e47. [39] Dixit E, Boulant S, Zhang Y, Lee AS, Odendall C, Shum B, et al. Peroxisomes are signaling platforms for antiviral innate immunity. Cell 2010;141:668e81. [40] Gould SJ, Keller GA, Subramani S. Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase. J Cell Biol 1987;105: 2923e31. [41] Subramani S. Targeting of proteins into the peroxisomal matrix. J Membr Biol 1992;125:99e106.