Molecular Characterization and Expression Analysis of ftr01, ftr42, and ftr58 in Zebrafish (Danio rerio)

  • Wanmeng Liu
  • Ming Kuang
  • Ze Zhang
  • Yuanan Lu
  • Xueqin LiuEmail author
Research Article


Tripartite motif (TRIM) proteins were shown to play an important role in innate antiviral immunity. FinTRIM (ftr) is a new subset of TRIM genes that do not possess obvious orthologs in higher vertebrates. However, little is known about its function. In this study, we used bioinformatic analysis to examine the phylogenetic relationships and conserved domains of zebrafish (Danio rerio) ftr01, ftr42, and ftr58, as well as qualitative real-time PCR to examine their expression patterns in zebrafish embryonic fibroblast (ZF4) cells and zebrafish tissues. Sequence analysis showed that the three finTRIMs are highly conserved, and all contain a RING domain, B-box domain, and SPRY-PRY domain. In addition, ftr42 and ftr58 had one coiled-coil domain (CCD), whereas ftr01 had two CCDs. Tissue expression analysis revealed that the mRNA level of ftr01 was the highest in the liver, whereas those of ftr42 and ftr58 were the highest in the gill; the expression of these finTRIMs was clearly upregulated not in the eyes, but in the liver, spleen, kidney, gill, and brain of zebrafish following spring viremia of carp virus (SVCV) infection. Similarly, the expression of these three finTRIM genes also increased in ZF4 cells after SVCV infection. Our study revealed that ftr01, ftr42, and ftr58 may play an important role in antiviral immune responses, and these findings validate the need for more in-depth research on the finTRIM family in the future.


FinTRIM Tissue distribution Expression patterns Bioinformatics analysis Spring viremia of carp virus (SVCV) Zebrafish 



This work was supported by the National Training Program of Innovation and Entrepreneurship for Undergraduates of Huazhong Agricultural University (2015308200403), the National Key Research and Development Program of China (2018YFD0900505), the Natural Science Foundation of China (31172433), and the Fundamental Research Funds for the Central Universities (2662018YJ022).

Author Contributions

LX, LW and KM conceived the project, wrote and revised the manuscript. KM performed the experiments. LW, KM and ZZ did data analysis. LY, LX checked and finalized the manuscript. All authors read and approved the final manuscript.

Compliance with Ethics Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Animal and Human Rights Statement

All animal procedures were conducted strictly in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All the animals used for viral infection were not endangered or protected species. Zebrafish were employed in in vivo experimental tests under the approval of the Animal Ethics Committee of Huazhong Agricultural University (HZAU). The infection and dissection experiments were performed under anesthesia with 3-aminobenzoic acid ethyl ester methane sulfonate (MS-222) (Sigma, USA) to minimize suffering in the fishes.

Supplementary material

12250_2019_112_MOESM1_ESM.pdf (329 kb)
Supplementary material 1 (PDF 329 kb)


  1. Ahne W, Bjorklund HV, Essbauer S, Fijan N, Kurath G, Winton JR (2002) Spring viremia of carp (svc). Dis Aquat Organ 52:261–272CrossRefGoogle Scholar
  2. Alvarez-Pellitero P (2008) Fish immunity and parasite infections: from innate immunity to immunoprophylactic prospects. Vet Immunol Immunopathol 126:171–198CrossRefGoogle Scholar
  3. Barr SD, Smiley JR, Bushman FD (2008) The interferon response inhibits hiv particle production by induction of trim22. PLoS Pathog 4:e1000007CrossRefGoogle Scholar
  4. Bell JL, Malyukova A, Holien JK, Koach J, Parker MW, Kavallaris M, Marshall GM, Cheung BB (2012) Trim16 acts as an e3 ubiquitin ligase and can heterodimerize with other trim family members. PLoS ONE 7:e37470CrossRefGoogle Scholar
  5. Beutler B (2004) Innate immunity: an overview. Mol Immunol 40:845–859CrossRefGoogle Scholar
  6. Borden KL (1998) Ring fingers and b-boxes: zinc-binding protein-protein interaction domains. Cell Biol 76:351–358Google Scholar
  7. Campbell EM, Weingart J, Sette P, Opp S, Sastri J, O’Connor SK, Talley S, Diazgriffero F, Hirsch V, Bouamr F (2015) Trim5α-mediated ubiquitin chain conjugation is required for inhibition of hiv-1 reverse transcription and capsid destabilization. J Virol 90:1849–1857CrossRefGoogle Scholar
  8. Chang TH (2008) Trim family proteins and their emerging roles in innate immunity. Nat Rev Immunol 8:849–860CrossRefGoogle Scholar
  9. Chang HW, Wang WD, Chiu CC, Chen CH, Wang YS, Chen ZY, Liu W, Tai MH, Wen ZH, Wu CY (2017) Ftr82 is critical for vascular patterning during zebrafish development. J Mol Sci 18:156CrossRefGoogle Scholar
  10. Chen B, Huo S, Liu W, Wang F, Lu Y, Xu Z, Liu X (2019) Fish-specific finTRIM FTR36 triggers IFN pathway and mediates inhibition of viral replication. Fish Shellfish Immunol 84:876–884CrossRefGoogle Scholar
  11. Cui Z, Liu Y, Luan W, Li Q, Wu D, Wang S (2010) Molecular cloning and characterization of a heat shock protein 70 gene in swimming crab (portunus trituberculatus). Fish Shellfish Immunol 28:56–64CrossRefGoogle Scholar
  12. Dawidziak DM, Sanchez JG, Wagner JM, Ganser-Pornillos BK, Pornillos O (2017) Structure and catalytic activation of the trim23 ring e3 ubiquitin ligase. Proteins 85:1957–1961CrossRefGoogle Scholar
  13. Ebner P, Versteeg GA, Ikeda F (2017) Ubiquitin enzymes in the regulation of immune responses. Crit Rev Biochem Mol Biol 52:425–460CrossRefGoogle Scholar
  14. Eldin P, Papon L, Oteiza A, Brocchi E, Lawson TG, Mechti N (2009) Trim22 e3 ubiquitin ligase activity is required to mediate antiviral activity against encephalomyocarditis virus. J Gen Virol 90:536–545CrossRefGoogle Scholar
  15. Esposito D, Koliopoulos MG, Rittinger K (2017) Structural determinants of trim protein function. Biochem Soc Trans 45:183–191CrossRefGoogle Scholar
  16. Fan W, Wu M, Qian S, Zhou Y, Chen H, Li X, Qian P (2016) Trim52 inhibits japanese encephalitis virus replication by degrading the viral ns2a. Sci Rep 6:33698CrossRefGoogle Scholar
  17. Fearon DT, Locksley RM (1996) The instructive role of innate immunity in the acquired immune response. Science 272:50–53CrossRefGoogle Scholar
  18. Germain RN (2004) An innately interesting decade of research in immunology. Nat Med 10:1307–1320CrossRefGoogle Scholar
  19. Gong XY, Zhang QM, Gui JF, Zhang YB (2018) SVCV infection triggers fish IFN response through RLR signaling pathway. Fish Shellfish Immunol 86:1058–1063CrossRefGoogle Scholar
  20. Huang Y, Yu Y, Yang Y, Yang M, Zhou L, Huang X, Qin Q (2016a) Fish trim8 exerts antiviral roles through regulation of the proinflammatory factors and interferon signaling. Fish Shellfish Immunol 54:435–444CrossRefGoogle Scholar
  21. Huang Y, Yang M, Yu Y, Yang Y, Zhou L, Huang X, Qin Q (2016b) Grouper trim13 exerts negative regulation of antiviral immune response against nodavirus. Fish Shellfish Immunol 55:106–115CrossRefGoogle Scholar
  22. Kumar S, Stecher G, Tamura K (2016) Mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874CrossRefGoogle Scholar
  23. Langevin C, Aleksejeva E, Houel A, Briolat V, Torhy C, Lunazzi A, Levraud JP, Boudinot P (2017) Ftr83, a member of the large fish-specific fintrim family, triggers ifn pathway and counters viral infection. Front Immunol 8:617CrossRefGoogle Scholar
  24. Letunic I, Bork P (2017) 20 years of the smart protein domain annotation resource. Nucleic Acids Res 46:D493–D496CrossRefGoogle Scholar
  25. Li Y, Wu H, Wu W, Zhuo W, Liu W, Zhang Y, Cheng M, Chen YG, Gao N, Yu H (2014) Structural insights into the trim family of ubiquitin e3 ligases. Cell Res 24:762–765CrossRefGoogle Scholar
  26. Liu B, Li NL, Wang J, Shi PY, Wang T, Miller MA, Li K (2014) Overlapping and distinct molecular determinants dictating the antiviral activities of trim56 against flaviviruses and coronavirus. J Virol 88:13821–13835CrossRefGoogle Scholar
  27. Liu L, Zhu B, Wu S, Lin L, Liu G, Zhou Y, Wang W, Asim M, Yuan J, Li L (2015) Spring viraemia of carp virus induces autophagy for necessary viral replication. Cell Microbiol 17:595–605CrossRefGoogle Scholar
  28. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method. Methods 25:402–408CrossRefGoogle Scholar
  29. Luo K, Li Y, Xia L, Hu W, Gao W, Guo L, Tian G, Qi Z, Yuan H, Qiaoqing X (2017) Analysis of the expression patterns of the novel large multigene trim gene family (fintrim) in zebrafish. Fish Shellfish Immunol 66:224–230CrossRefGoogle Scholar
  30. Matthew S, Owens CM, Perron MJ, Michael K, Patrick A, Joseph S (2004) The cytoplasmic body component trim5alpha restricts hiv-1 infection in old world monkeys. Nature 427:848–853CrossRefGoogle Scholar
  31. Meroni G, Diez-Roux G (2005) Trim/rbcc, a novel class of ‘single protein ring finger’ e3 ubiquitin ligases. BioEssays 27:1147–1157CrossRefGoogle Scholar
  32. Novoa B, Romero A, Mulero V, Rodríguez I, Fernández I, Figueras A (2006) Zebrafish (danio rerio) as a model for the study of vaccination against viral haemorrhagic septicemia virus (vhsv). Vaccine 24:5806–5816CrossRefGoogle Scholar
  33. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) Ucsf chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612CrossRefGoogle Scholar
  34. Pietro AD, Kajasterudnitski A, Oteiza A, Nicora L, Towers GJ, Mechti N, Vicenzi E (2013) Trim22 inhibits influenza a virus infection by targeting the viral nucleoprotein for degradation. J Virol 87:4523–4533CrossRefGoogle Scholar
  35. Pizzi M (1950) Sampling variation of the fifty per cent end-point, determined by the reedmuench (behrens) method. Hum Biol 22:151–190Google Scholar
  36. Rajsbaum R, García-Sastre A, Versteeg GA (2014) Trimmunity: the roles of the trim e3-ubiquitin ligase family in innate antiviral immunity. J Mol Biol 426:1265–1284CrossRefGoogle Scholar
  37. Reddy BA, Etkin LD (1991) A unique bipartite cysteine-histidine motif defines a subfamily of potential zinc-finger proteins. Nucleic Acids Res 19:6330CrossRefGoogle Scholar
  38. Reddy BA, Etkin LD, Freemont PS (1992) A novel zinc finger coiled-coil domain in a family of nuclear proteins. Trends Biochem Sci 17:344–345CrossRefGoogle Scholar
  39. Reymond A, Meroni G, Fantozzi A, Merla G, Cairo S, Luzi L, Riganelli D, Zanaria E, Messali S, Cainarca S (2001) The tripartite motif family identifies cell compartments. EMBO J 20:2140–2151CrossRefGoogle Scholar
  40. Sanchez JG, Okreglicka K, Chandrasekaran V, Welker JM, Sundquist WI, Pornillos O (2014) The tripartite motif coiled-coil is an elongated antiparallel hairpin dimer. Proc Natl Acad Sci U S A 111:2494–2499CrossRefGoogle Scholar
  41. Sanders GE, Batts WN, Winton JR (2003) Susceptibility of zebrafish (danio rerio) to a model pathogen, spring viremia of carp virus. Comp Med 53:514–521Google Scholar
  42. Smyth MJ, Godfrey DI, Trapani JA (2001) A fresh look at tumor immunosurveillance and immunotherapy. Nat Immunol 2:293–299CrossRefGoogle Scholar
  43. Thompson JD, Higgins DG, Gibson TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefGoogle Scholar
  44. Uchil PD, Quinlan BD, Chan WT, Luna JM, Mothes W (2008) Trim e3 ligases interfere with early and late stages of the retroviral life cycle. PLoS Pathog 4:e16CrossRefGoogle Scholar
  45. Uchil PD, Hinz A, Siegel S, Coenen-Stass A, Pertel T, Luban J, Mothes W (2013) TRIM protein-mediated regulation of inflammatory and innate immune signaling and its association with antiretroviral activity. J Virol 87:257–272CrossRefGoogle Scholar
  46. Van der Aa LM, Levraud JP, Yahmi M, Lauret E, Briolat V, Herbomel P, Benmansour A, Boudinot P (2009) A large new subset of TRIM genes highly diversified by duplication and positive selection in teleost fish. BMC Biol 7:7CrossRefGoogle Scholar
  47. Van der Aa LM, Jouneau L, Laplantine E, Bouchez O, Van KL, Boudinot P (2012) FinTRIMs, fish virus-inducible proteins with E3 ubiquitin ligase activity. Dev Comp Immunol 36:433–441CrossRefGoogle Scholar
  48. Versteeg GA, Benke S, Garcíasastre A, Rajsbaum R (2014) Intrimsic immunity: positive and negative regulation of immune signaling by tripartite motif proteins. Cytokine Growth Factor Rev 25:563–576CrossRefGoogle Scholar
  49. Wang W, Huang Y, Yu Y, Yang Y, Xu M, Chen X, Ni S, Qin Q, Huang X (2016a) Fish trim39 regulates cell cycle progression and exerts its antiviral function against iridovirus and nodavirus. Fish Shellfish Immunol 50:1–10CrossRefGoogle Scholar
  50. Wang Y, Li Z, Lu Y, Hu G, Li L, Zeng L, Yong Z, Liu X (2016b) Molecular characterization, tissue distribution and expression, and potential antiviral effects of trim32 in the common carp (cyprinus carpio). Int J Mol Sci 17:E1693CrossRefGoogle Scholar
  51. Wang Y, Zhang H, Lu Y, Wang F, Liu L, Liu J, Liu X (2017) Comparative transcriptome analysis of zebrafish (Danio rerio) brain and spleen infected with spring viremia of carp virus (SVCV). Fish Shellfish Immunol 69:35–45CrossRefGoogle Scholar
  52. Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L (2018) Swiss-model: homology modelling of protein structures and complexes. Nucleic Acids Res 46:W296–W303CrossRefGoogle Scholar
  53. Weinert C, Morger D, Djekic A, Grütter MG, Mittl PRE (2015) Crystal structure of trim20 c-terminal coiled-coil/b30.2 fragment: implications for the recognition of higher order oligomers. Sci Rep 5:10819CrossRefGoogle Scholar
  54. Yang Y, Huang Y, Yu Y, Yang M, Zhou S, Qin Q, Huang X (2016a) Ring domain is essential for the antiviral activity of trim25 from orange spotted grouper. Fish Shellfish Immunol 55:304–314CrossRefGoogle Scholar
  55. Yang Y, Huang Y, Yu Y, Zhou S, Wang S, Yang M, Qin Q, Huang X (2016b) Negative regulation of the innate antiviral immune response by trim62 from orange spotted grouper. Fish Shellfish Immunol 57:68–78CrossRefGoogle Scholar
  56. Ye Y, Rape M (2009) Building ubiquitin chains: E2 enzymes at work. Nat Rev Mol Cell Biol 10:755–764CrossRefGoogle Scholar
  57. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinform 13:134CrossRefGoogle Scholar
  58. Yu Y, Huang X, Zhang J, Liu J, Yin H, Ying Y, Jia C, Huang Y, Qin Q (2016) Fish trim16l exerts negative regulation on antiviral immune response against grouper iridoviruses. Fish Shellfish Immunol 59:256–267CrossRefGoogle Scholar
  59. Yu Y, Huang X, Liu J, Zhang J, Yin H, Ying Y, Huang Y, Qin Q (2017) Fish trim32 functions as a critical antiviral molecule against iridovirus and nodavirus. Fish Shellfish Immunol 60:33–43CrossRefGoogle Scholar
  60. Zhang S, Guo JT, Wu JZ, Yang G (2013) Identification and characterization of multiple trim proteins that inhibit hepatitis b virus transcription. PLoS ONE 8:e70001CrossRefGoogle Scholar
  61. Zhou Z, Jia X, Xue Q, Dou Z, Ma Y, Zhao Z, Jiang Z, He B, Jin Q, Wang J (2014) Trim14 is a mitochondrial adaptor that facilitates retinoic acid-inducible gene-i-like receptor-mediated innate immune response. Proc Natl Acad Sci U S A 111:E245–E254CrossRefGoogle Scholar

Copyright information

© Wuhan Institute of Virology, CAS 2019

Authors and Affiliations

  1. 1.College of FisheriesHuazhong Agricultural UniversityWuhanChina
  2. 2.Freshwater Aquaculture Collaborative Innovation Center of Hubei ProvinceWuhanChina
  3. 3.Key Lab of Freshwater Animal BreedingMinistry of AgricultureWuhanChina
  4. 4.School of Life SciencesBeijing Normal UniversityBeijingChina
  5. 5.National Institute of Biological SciencesZhongguancun Life Science ParkBeijingChina
  6. 6.Department of Public Health SciencesUniversity of Hawaii at ManoaHonoluluUSA

Personalised recommendations