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Characterization and functional assay of apsup (Lyxy105) from Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV)

Abstract

The baculoviral anti-apoptotic genes, p35 and iap (inhibitor of apoptosis), play important roles in the initiation of viral infection. Recently, a new anti-apoptotic gene (apoptosis suppressor, apsup) was identified in Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). An apsup homolog gene, Lyxy105 (ly-apsup), was also predicted in the Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) genome. In this study, we attempt to perform a gene expression analysis and a functional assay of ly-apsup to demonstrate its anti-apoptotic activity and identify the functional domain of this protein. The transcription of the ly-apsup gene region was detected from 12 h post-infection (hpi) and increased significantly after 24–72 hpi. Comparison of the putative amino acid sequences to those of 18 baculoviral homolog proteins showed high amino acid identity to the LdMNPV sequences. Moreover, five conserved protein domains (named as domains I–V) were found. Therefore, protein functional assays were conducted on full-length proteins and different truncation clones. The overexpression of each clone was confirmed by western blot analysis, and the data revealed that a cleavage of ~ 5 kDa at the N-terminal region of the full-length, domains I–IV (1–241) and I–III (1–178), proteins occurred. The results of the functional analysis showed that full-length Ly-apsup and Ly-apsup with domain I (1–70) could inhibit Drosophila-RPR protein (D-RPR)-induced and actinomycin D (ActD)-induced apoptoses. In addition, the domains I and I–II (1–126) regions showed higher anti-apoptotic activity than the other domains in both D-RPR-induced and ActD-induced cell apoptoses. In conclusion, domain I of Ly-apsup may play an important role in the anti-apoptotic activity of this protein; cleavage of the Ly-apsup N-terminus may lead to decreased anti-apoptotic activity.

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References

  1. T.C. Shen, C.M. Tseng, L.C. Guan, S.Y. Hwang, J. Econ. Entomol. 99, 714–721 (2006)

    Article  PubMed  CAS  Google Scholar 

  2. C.C. Yu, H.H. Kao, J.T. Chao, S.S. Lu, C.H. Wang, Chin. J. Entomol. 17, 23–32 (1997)

    CAS  Google Scholar 

  3. Y.S. Nai, C.Y. Wu, T.C. Wang, Y.R. Chen, W.H. Lau, C.F. Lo, M.F. Tsai, C.H. Wang, BMC Genom. 11, 116 (2010)

    Article  CAS  Google Scholar 

  4. C.Y. Wu, C.H. Wang, J. Invertebr. Pathol. 88, 238–246 (2005)

    Article  PubMed  CAS  Google Scholar 

  5. Y.S. Nai, T.C. Wang, Y.R. Chen, C.F. Lo, C.H. Wang, J. Invertebr. Pathol. 102, 110–119 (2009)

    Article  PubMed  CAS  Google Scholar 

  6. T.D. Morris, L.K. Miller, J. Virol. 66, 7397–7405 (1992)

    PubMed  PubMed Central  CAS  Google Scholar 

  7. C. Wu, Z. Deng, Z. Long, Y. Cai, Z. Ying, H. Yin, M. Yuan, R.J. Clem, K. Yang, Y. Pang, Sci. Rep. 6, 28072 (2016)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. H. Everett, G. McFadden, Virology 288, 1–7 (2001)

    Article  PubMed  CAS  Google Scholar 

  9. S.M. Thiem, X.W. Cheng, Virologica Sinica 24, 436–457 (2009)

    Article  Google Scholar 

  10. R.J. Clem, The Baculoviruses The Viruses (Springer, Berlin, 1997)

    Google Scholar 

  11. R.J. Clem, M. Fechheimer, L.K. Miller, Science 254, 1388–1390 (1991)

    Article  PubMed  CAS  Google Scholar 

  12. R.J. Clem, Curr Drug Targets 8, 1069–1074 (2007)

    Article  PubMed  CAS  Google Scholar 

  13. N.E. Crook, R.J. Clem, L.K. Miller, J Virol 67, 2168–2174 (1993)

    PubMed  PubMed Central  CAS  Google Scholar 

  14. N.J. Bump, M. Hackett, M. Hugunin, S. Seshagiri, K. Brady, P. Chen, C. Ferenz, S. Franklin, T. Ghayur, P. Li et al., Science 269, 1885–1888 (1995)

    Article  PubMed  CAS  Google Scholar 

  15. Q. Du, D. Lehavi, O. Faktor, Y. Qi, N. Chejanovsky, J. Virol. 73, 1278–1285 (1999)

    PubMed  PubMed Central  CAS  Google Scholar 

  16. M.J. Eddins, D. Lemongello, P.D. Friesen, A.J. Fisher, Acta Crystallogr. Sect. D 58, 299–302 (2002)

    Article  CAS  Google Scholar 

  17. Y.R. Chen, C.Y. Wu, S.T. Lee, Y.J. Wu, C.F. Lo, M.F. Tsai, C.H. Wang, J. Gen. Virol. 89, 2315–2330 (2008)

    Article  PubMed  CAS  Google Scholar 

  18. M.D. Ayres, S.C. Howard, J. Kuzio, M. Lopez-Ferber, R.D. Possee, Virology 202, 586–605 (1994)

    Article  PubMed  CAS  Google Scholar 

  19. R.L. Harrison, B.C. Bonning, J. Gen. Virol. 84, 1827–1842 (2003)

    Article  PubMed  CAS  Google Scholar 

  20. S. Gomi, K. Majima, S. Maeda, J. Gen. Virol. 80(Pt 5), 1323–1337 (1999)

    Article  PubMed  CAS  Google Scholar 

  21. Y. Pang, J. Yu, L. Wang, X. Hu, W. Bao, G. Li, C. Chen, H. Han, S. Hu, H. Yang, Virology 287, 391–404 (2001)

    Article  PubMed  CAS  Google Scholar 

  22. T. Luque, R. Finch, N. Crook, D.R. O’Reilly, D. Winstanley, J. Gen. Virol. 82, 2531–2547 (2001)

    Article  PubMed  CAS  Google Scholar 

  23. T. Maguire, P. Harrison, O. Hyink, J. Kalmakoff, V.K. Ward, J. Gen. Virol. 81, 2803–2811 (2000)

    Article  PubMed  CAS  Google Scholar 

  24. M.J. Birnbaum, R.J. Clem, L.K. Miller, J. Virol. 68, 2521–2528 (1994)

    PubMed  PubMed Central  CAS  Google Scholar 

  25. M. Ikeda, K. Yanagimoto, M. Kobayashi, Virology 321, 359–371 (2004)

    Article  PubMed  CAS  Google Scholar 

  26. M.P. Carpes, M.E. de Castro, E.F. Soares, A.G. Villela, F.J. Pinedo, B.M. Ribeiro, Arch Virol 150, 1549–1562 (2005)

    Article  PubMed  CAS  Google Scholar 

  27. Y.S. Kim, H.Z. Xiao, E.Q. Du, G.S. Cai, S.Y. Lu, Y.P. Qi, J. Biochem. Mol. Biol. 40, 571–576 (2007)

    PubMed  CAS  Google Scholar 

  28. C. Liang, J. de Lange, X. Chen, M.M. van Oers, J.M. Vlak, M. Westenberg, Virus Res. 165, 107–111 (2012)

    Article  PubMed  CAS  Google Scholar 

  29. L. Vilaplana, D.R. O’Reilly, Virus Res. 92, 107–111 (2003)

    Article  PubMed  CAS  Google Scholar 

  30. J.C. Means, I. Muro, R.J. Clem, J. Virol. 77, 4481–4488 (2003)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. F. Yan, X. Deng, J. Yan, J. Wang, L. Yao, S. Lv, Y. Qi, H. Xu, J Microbiol 48, 199–205 (2010)

    Article  PubMed  CAS  Google Scholar 

  32. Y.L. Wu, C.P. Wu, C.Y. Liu, S.T. Lee, H.P. Lee, Y.C. Chao, J Virol 85, 6856–6866 (2011)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. X. Zeng, F. Nan, C. Liang, J. Song, Q. Wang, J.M. Vlak, X. Chen, Sci China C 52, 761–770 (2009)

    Article  Google Scholar 

  34. H. Yamada, M. Shibuya, M. Kobayashi, M. Ikeda, J. Virol. 85, 5237–5242 (2011)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. H. Yamada, K. Kitaguchi, R. Hamajima, M. Kobayashi, M. Ikeda, J Virol 87, 12925–12934 (2013)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. C.Y. Wu, C.H. Wang, J. Invertebr. Pathol. 93, 186–191 (2006)

    Article  PubMed  Google Scholar 

  37. Y.S. Nai, Y.T. Yang, J.S. Kim, C.Y. Wu, Y.W. Chen, C.H. Wang, Appl. Entomol. Zool. 51, 305–316 (2016)

    Article  CAS  Google Scholar 

  38. J.C. Chang, S.J. Lee, J.S. Kim, C.H. Wang, Y.S. Nai, J Vis Exp (JoVE) (2018). https://doi.org/10.3791/56693

    Article  Google Scholar 

  39. J.L. Vaughn, R.H. Goodwin, G.J. Tompkins, P. McCawley, In Vitro 13, 213–217 (1977)

    Article  PubMed  CAS  Google Scholar 

  40. W.F. Hink, E. Strauss, Growth of the Trichoplusia ni (TN-368) cell line in suspension culture (Academic Press, New York, 1976)

    Book  Google Scholar 

  41. S.C. Yeh, S.T. Lee, C.Y. Wu, C.H. Wang, J. Invertebr. Pathol. 96, 138–146 (2007)

    Article  PubMed  CAS  Google Scholar 

  42. J. Bikandi, R. San Millan, A. Rementeria, J. Garaizar, Bioinformatics 20, 798–799 (2004)

    Article  PubMed  CAS  Google Scholar 

  43. M. Kallberg, H. Wang, S. Wang, J. Peng, Z. Wang, H. Lu, J. Xu, Nat Protoc 7, 1511–1522 (2012)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. B.G. Pierce, K. Wiehe, H. Hwang, B.H. Kim, T. Vreven, Z. Weng, Bioinformatics 30, 1771–1773 (2014)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. M.D. Summers, G.E. Smith, Texas Agric Exp Station Bull 1555, 1–57 (1987)

    Google Scholar 

  46. A. Untergasser, I. Cutcutache, T. Koressaar, J. Ye, B.C. Faircloth, M. Remm, S.G. Rozen, Nucleic Acids Res 40, e115 (2012)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. J.H. Leu, Y.C. Kuo, G.H. Kou, C.F. Lo, Dev. Comp. Immunol. 32, 121–133 (2008)

    Article  PubMed  CAS  Google Scholar 

  48. R.J. Clem, L.K. Miller, Mol. Cell. Biol 14, 5212–5222 (1994)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. E.N. Shiozaki, Y. Shi, Trends Biochem. Sci. 29, 486–494 (2004)

    Article  PubMed  CAS  Google Scholar 

  50. T. Tenev, A. Zachariou, R. Wilson, M. Ditzel, P. Meier, Nat. Cell Biol. 7, 70–77 (2005)

    Article  PubMed  CAS  Google Scholar 

  51. G.F. Rohrmann, Baculovirus Molecular Biology (National Center for Biotechnology Information (US), Bethesda, 2013)

    Google Scholar 

  52. K. Xing, R. Deng, J. Wang, J. Feng, M. Huang, X. Wang, Virus Res. 113, 64–71 (2005)

    Article  PubMed  CAS  Google Scholar 

  53. M. Shokhen, T. Traube, S. Vijayakumar, M. Hirsch, N. Uritsky, A. Albeck, Chembiochem 12(7), 1023–1026 (2011)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. J.C. Reed, K.S. Doctor, A. Godzik, Sci. STKE 2004, re9 (2004)

    PubMed  Google Scholar 

  55. D. Vucic, W.J. Kaiser, A.J. Harvey, L.K. Miller, Proc. Natl. Acad. Sci. USA 94, 10183–10188 (1997)

    Article  PubMed  CAS  Google Scholar 

  56. A.J. Harvey, H. Soliman, W.J. Kaiser, L.K. Miller, Cell Death Differ. 4, 733–744 (1997)

    Article  PubMed  CAS  Google Scholar 

  57. S. Seshagiri, D. Vucic, J. Lee, V.M. Dixit, J. Biol. Chem. 274, 36769–36773 (1999)

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

This research was supported by Grant No. 106-2311-B-197-001- from the Ministry of Science and Technology (MOST). The authors thank Professor Wang Chung-Hsiung, Entomology Department of National Taiwan University, who gifted LY cell line and LyxyMNPV.

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Authors and Affiliations

Authors

Contributions

JCC, ZTC, SJL, JSK and YSN designed and performed the experiments; JCC, YFH and YSN performed the protein structure predictions; JCC, JSK and YSN wrote and edited the manuscript.

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Correspondence to Yu-Shin Nai.

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The authors declare there is no conflict of interest involved in this work.

Research involving human and/or animals participants

There is no research involving human participants and/or animals in this study.

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Edited by A. Lorena Passarelli.

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11262_2018_1580_MOESM1_ESM.tiff

Supplementary material 1 Prediction of Ly-apsup docking to (A) Ld-Dronc and (B) D-RPR revealed a reduction in the flexibility of the docked protein complex. The prediction of the Ly-apsup protein structure revealed two major domains (domains A and B). Domain A ranged from amino acids 1 to 220 at the N-terminus, and domain B (boxed) ranged from amino acids 221 to 333 at the C-terminus. A longer coil structure between domain A and domain B was predicted and supports the higher flexibility of the Ly-apsup protein structure, and the docked protein complex showed that this structure might influence the flexibility (TIFF 8054 KB)

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Chang, JC., Chang, ZT., Huang, YF. et al. Characterization and functional assay of apsup (Lyxy105) from Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV). Virus Genes 54, 578–586 (2018). https://doi.org/10.1007/s11262-018-1580-1

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  • DOI: https://doi.org/10.1007/s11262-018-1580-1

Keywords

  • LyxyMNPV
  • Apoptosis
  • Apsup
  • Drosophila rpr
  • Functional assay