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Molecular cloning, characterization and expression analysis of woodchuck retinoic acid-inducible gene I

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Summary

Cytosolic retinoic acid-inducible gene I (RIG-I) is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-β (IFN-β). Innate immune response to hepatitis B virus (HBV) plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I (wRIG-I), we analyzed the complete coding sequences (CDSs) of wRIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced wRIG-I protein was 106.847 kD with a theoretical isoelectric point (pI) of 6.07, and contained three important functional structures [caspase activation and recruitment domains (CARDs), DExD/H-box helicases, and a repressor domain (RD)]. In woodchuck fibroblastoma cell line (WH12/6), wRIG-I-targeted small interfering RNA (siRNA) down-regulated RIG-I and its downstrean effector–IFN-β transcripts under RIG-I’ ligand, 5’-ppp double stranded RNA (dsRNA) stimulation. We also measured mRNA levels of wRIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus (WHV)-infected woodchucks. The basal expression levels of wRIG-I were abundant in the kidney and liver. Importantly, wRIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection.

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References

  1. Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol, 1995, 13: 29–60

    Article  CAS  PubMed  Google Scholar 

  2. Ganem D, Prince AM. Hepatitis B virus infection—natural history and clinical consequences. N Engl J Med, 2004, 350(11): 1118–1129

    Article  CAS  PubMed  Google Scholar 

  3. Law YF. Prevention and surveillance of hepatitis B virus-related hepatocellular carcinoma. Semin Liver Dis, 2005, 25(Suppl 1): 40–47

    Article  Google Scholar 

  4. Xu CL, Hao YH, Lu YP, et al. Upregulation of toll-like receptor 4 on T cells in PBMCs is associated with disease aggravation of HBV-related acute-on-chronic liver failure. J Huazhong Univ Sci Technolog Med Sci, 2015, 35(6): 910–915

    Article  CAS  PubMed  Google Scholar 

  5. Thimme R, Wieland S, Steiger C, et al. CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection. J Virol, 2003, 77(1): 68–76

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Bertoletti A, Maini MK, Ferrari C. The host-pathogen interaction during HBV infection: immunological controversies. Antiviral Ther, 2010, 15(Suppl 3): 15–24

    Article  CAS  Google Scholar 

  7. Guo H, Jiang D, Ma D, et al. Activation of pattern recognition receptor-mediated innate immunity inhibits the replication of hepatitis B virus in human hepatocyte-derived cells. J Virol, 2009, 83(2): 847–858

    Article  CAS  PubMed  Google Scholar 

  8. Luangsay S, Ait-Goughoulte M, Michelet M, et al. Expression and functionality of Toll-and RIG-like receptors in HepaRG cells. J Hepatol, 2015, 63(5): 1077–1085

    Article  CAS  PubMed  Google Scholar 

  9. Sharma S, Fitzgerald KA. Innate immune sensing of DNA. PLoS Pathog, 2011, 7: e1001310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Yoneyama M, Kikuchi M, Natsukawa T, et al. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol, 2004, 5(7): 730–737

    Article  CAS  PubMed  Google Scholar 

  11. Yoneyama M, Kikuchi M, Matsumoto K, et al. Shared and unique functions of the DExD/H box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity. J Immunol, 2005, 175(5): 2851–2858

    Article  CAS  PubMed  Google Scholar 

  12. Yoneyama M, Fujita T. Function of RIG-I-like receptors in antiviral innate immunity. J Biol Chem, 2007, 282(21): 15315–15318

    Article  CAS  PubMed  Google Scholar 

  13. Hornung V, Ellegast J, Kim S, et al. 5'-Triphosphate RNA is the ligand for RIG-I. Science, 2006, 314(5801): 994–997

    Article  PubMed  Google Scholar 

  14. Murali A, Li X, Ranjith-Kumar CT, et al. Structure and function of LGP2, a DEX(D/H) helicase that regulates the innate immunity response. J Biol Chem, 2008, 283(23): 825–15 833

    Article  Google Scholar 

  15. Sarkar D, Desalle R, Fisher PB. Evolution of MDA-5/RIG-I-dependent innate immunity: independent evolution by domain grafting. Proc Natl Acad Sci USA, 2008, 105(44): 17040–17045

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Schmidt A, Schwerd T, Hamm W, et al. 5'-triphosphate RNA requires base-paired structures to activate antiviral signaling via RIG-I. Proc Natl Acad Sci USA, 2009, 106(29): 12067–12072

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zou J, Chang M, Nie P, et al. Origin and evolution of the RIG-I like RNA helicase gene family. BMC Evol Biol, 2009, 9: 85

    Article  PubMed  PubMed Central  Google Scholar 

  18. Takahasi K, Yoneyama M, Nishihori T, et al. Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses. Mol Cell, 2008, 29(4): 428–440

    Article  CAS  PubMed  Google Scholar 

  19. Saito T, Hirai R, Loo YM, et al. Regulation of innate antiviral defenses through a shared repressor domain in RIG-I and LGP2. Proc Natl Acad Sci USA, 2007, 104(2): 582–587

    Article  CAS  PubMed  Google Scholar 

  20. Jiang F, Ramanathan A, Miller MT, et al. Structural basis of RNA recognition and activation by innate immune receptor RIG-I. Nature, 2011, 479(7373): 423–427

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wu B, Peisley A, Richards C, et al. Structural basis for dsRNA recognition, filament formation, and antiviral signal activation by MDA5. Cell, 2013, 152(1-2): 276–289

    Article  CAS  PubMed  Google Scholar 

  22. Loo YM, Fornek J, Crochet N, et al. Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity. J Virol, 2008, 82(1): 335–345

    Article  CAS  PubMed  Google Scholar 

  23. Saito T, Owen DM, Jiang F, et al. Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA. Nature, 2008, 54(7203): 523–527

    Article  Google Scholar 

  24. Arnaud N, Dabo S, Akazawa D, et al. Hepatitis C virus reveals a novel early control in acute immune response. PLoS Pathog, 2011, 7(10): e1002289

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Chen X, Qian Y, Yan F, et al. 5'-triphosphate-siRNA activates RIG-I-dependent type I interferon production and enhances inhibition of hepatitis B virus replication in HepG2.2.15 cells. Eur J Pharmacol, 2013, 721(1-3): 86–95

    Article  CAS  PubMed  Google Scholar 

  26. Sato S, Li K, Kameyama T, et al. The RNA sensor RIG-I dually functions as an innate sensor and direct antiviral factor for hepatitis B virus. Immunity, 2015, 42(1): 123–132

    Article  CAS  PubMed  Google Scholar 

  27. Chen MF, Lin Y, Xia YC, et al. Establishment and application of hepatitis B virus persistent replication model in IFNAR(-/-) mouse. J Huazhong Univ Sci Technolog Med Sci, 2013, 33(3): 392–327

    Article  CAS  PubMed  Google Scholar 

  28. Hao Y, Yang D. Cloning, eukaryotic expression of human ISG20 and preliminary study on the effect of its anti-HBV. J Huazhong Univ Sci Technolog Med Sci, 2008, 28(1): 11–13

    Article  PubMed  Google Scholar 

  29. Chayama K, Hayes CN, Hiraga N, et al. Animal model for study of human hepatitis viruses. J Gastroenterol Hepatol, 2011, 26(1): 13–18

    Article  PubMed  Google Scholar 

  30. Roggendorf M, Tolle TK. The woodchuck: an animal model for hepatitis B virus infection in man. Intervirology, 1995, 38(1-2): 100–112

    CAS  PubMed  Google Scholar 

  31. Tennant BC, Gerin JL. The woodchuck model of hepatitis B virus infection. ILAR J, 2001, 42(2): 89–102

    Article  CAS  PubMed  Google Scholar 

  32. Wang Y, Menne S, Jacob JR, et al. Role of type 1 versus type 2 immune responses in liver during the onset of chronic woodchuck hepatitis virus infection. Hepatology, 2003, 37(4): 771–780

    Article  CAS  PubMed  Google Scholar 

  33. Wang BJ, Tian YJ, Meng ZJ, et al. Establishing a new animal model for hepadnaviral infection: susceptibility of Chinese Marmota-species to woodchuck hepatitis virus infection. J Gen Virol, 2011, 92(Pt 2): 681–691

    Article  CAS  PubMed  Google Scholar 

  34. Yang X, Hao Y, Liu Z, et al. Frequencies and characterization of HBV-specific cytotoxic Tlymphocytes in self-limited and chronic hepatitis B viral infection in China. J Huazhong Univ Sci Technolog Med Sci, 2009, 29(5): 567–574

    Article  PubMed  Google Scholar 

  35. Wang J, Michalak TI. Inhibition by woodchuck hepatitis virus of class I major histocompatibility complex presentation on hepatocytes is mediated by virus envelope pre-S2 protein and can be reversed by treatment with gamma interferon. J Virol, 2006, 80(17): 8541–8553

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lu M, Menne S, Yang D, et al. Immunomodulation as an option for the treatment of chronic hepatitis B virus infection: preclinical studies in the woodchuck model. Expert Opin Investig Drugs, 2007, 16(6): 787–801

    Article  CAS  PubMed  Google Scholar 

  37. Kosinska AD, Zhang E, Johrden L, et al. Combination of DNA prime—adenovirus boost immunization with entecavir elicits sustained control of chronic hepatitis B in the woodchuck model. PLoS Pathog, 2013, 9(6): e1003391

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Tamura K, Dudley J, Nei M, et al. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol, 2007, 24(8): 1596–1599

    Article  CAS  PubMed  Google Scholar 

  39. Lu M, Lohrengel B, Hilken G, et al. Woodchuck gamma interferon upregulates major histocompatibility complex class I transcription but is unable to deplete woodchuck hepatitis virus replication intermediates and RNAs in persistently infected woodchuck primary hepatocytes. J Virol, 2002, 76(1): 58–67

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Michalak TI, Hodgson PD, Churchill ND. Post transcriptional inhibition of class I major histocompatibility complex presentation on hepatocytes and lymphoid cells in chronic woodchuck hepatitis virus infection. J Virol, 2000, 74(10): 4483–4494

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Bamming D, Horvath CM. Regulation of signal transduction by enzymatically inactive antiviral RNA helicase proteins MDA5, RIG-I, and LGP2. J Biol Chem, 2009, 284(15): 9700–9712

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lech M, Avila-Ferrufino A, Skuginna V, et al. Quantitative expression of RIG-like helicase, NOD-like receptor and inflammasome-related mRNAs in humans and mice. Int Immunol, 2010, 22(9): 717–728

    Article  CAS  PubMed  Google Scholar 

  43. Kubota K, Sakaki H, Imaizumi T, et al. Retinoic acid-inducible gene-I is induced in gingival fibroblasts by lipopolysaccharide or poly IC: possible roles in interleukin-1 beta, -6 and -8 expression. Oral Microbiol Immunol, 2006, 21(6): 399–406

    Article  CAS  PubMed  Google Scholar 

  44. Kuniyoshi K, Takeuchi O, Pandey S, et al. Pivotal role of RNA-binding E3 ubiquitin ligase MEX3C in RIG-I-mediated antiviral innate immunity. Proc Natl Acad Sci USA, 2014, 111(15): 5646–5651

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Riedl T, Egly JM. Phosphorylation in transcription: the CTD and more. Gene Expr, 2000, 9(1-2): 3–13

    CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Infectious Diseases, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China

    Qi Yan  (严 琦), Qin Liu  (刘 钦), Meng-meng Li  (李蒙蒙), Fang-hui Li  (李芳慧), Bin Zhu  (朱 彬), Jun-zhong Wang  (王俊忠), Yin-ping Lu  (卢银平), Jia Liu  (刘 嘉), Jun Wu  (吴 珺), Xin Zheng  (郑 昕), Bao-ju Wang  (王宝菊) & Dong-liang Yang  (杨东亮)

  2. Institute of Virology, University of Duisburg-Essen, Essen, 45149, Germany

    Meng-ji Lu  (陆蒙吉)

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  1. Qi Yan  (严 琦)
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  8. Jia Liu  (刘 嘉)
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  11. Meng-ji Lu  (陆蒙吉)
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Corresponding authors

Correspondence to Bao-ju Wang  (王宝菊) or Dong-liang Yang  (杨东亮).

Additional information

This project was supported by grants from the National Science and Technology Major Project for Infectious Diseases of China (No. 2012ZX10004503), the National Natural Science Foundation of China (No. 81101248, No. 81261120397 and No. 81371828), and the Deutsche Forschungs-gemeinschaft (Transregio TRR60).

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Yan, Q., Liu, Q., Li, Mm. et al. Molecular cloning, characterization and expression analysis of woodchuck retinoic acid-inducible gene I. J. Huazhong Univ. Sci. Technol. [Med. Sci.] 36, 335–343 (2016). https://doi.org/10.1007/s11596-016-1588-5

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  • DOI: https://doi.org/10.1007/s11596-016-1588-5

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