Skip to main content
SpringerLink
Log in

Role of the DxxDxD motif in the assembly and stability of betanodavirus particles

  • Original Article
  • Published:
Archives of Virology Aims and scope Submit manuscript

Abstract

Piscine betanodavirus possesses a bipartite genome of single-stranded (+)RNAs. RNA2 cDNA of dragon grouper nervous necrosis virus (DGNNV) has been expressed previously to form virus-like particles (VLPs), which are highly similar to the native virion. Experiments with calcium-chelating or reducing/oxidizing reagents showed that the DGNNV VLPs required only calcium for particle assembly. With the recombinant VLPs, site-directed mutagenesis can be employed to investigate the roles of calcium-binding ligands in particle formation. The results of mutational analysis of DxxDxD, which is putatively involved in the coordination of calcium ions, showed that the D133N mutation significantly disrupted the assembly of VLPs, while D130N and D135N mutants produced heterogeneous VLPs with broken shapes. The thermal stability of the VLP-forming fractions demonstrated that VLPs of the D135N mutant were stable at a temperature of 85°C, which is slightly higher than that for wild-type, whereas VLPs of the D130N mutant could not tolerate the thermal effects at a temperature higher than 60°C. It was deduced that the three aspartate residues of the motif DxxDxD are all important for the efficient formation of DGNNV VLPs and that, among them, the DxxD provides a more stable coordinate of calcium ligand than DxD.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Abell BA, Brown DT (1993) Sindbis virus membrane fusion is mediated by reduction of glycoprotein disulfide bridges at the cell surface. J Virol 67:5496–5501

    PubMed  CAS  Google Scholar 

  2. Alder AJ, Greenfield NJ, Fasman GD (1973) Circular dichroism and optical rotatory dispersion of proteins and polypeptides. Methods Enzymol 27:675–735

    Article  Google Scholar 

  3. Braman J, Papworth C, Greener A (1996) Site-directed mutagenesis using double-stranded plasmid DNA templates. Methods Mol Biol 57:31–44

    PubMed  CAS  Google Scholar 

  4. Campbell JW, Clifton IJ, Greenhough TJ, Hajdu J, Harrison SC, Liddington RC, Shrive AK (1990) Calcium binding sites in Tomato bushy stunt virus visualized by Laue crystallography. J Mol Biol 214:627–632

    Article  PubMed  CAS  Google Scholar 

  5. Chemello ME, Aristimuno OC, Michelangeli F, Ruiz MC (2002) Requirement for vacuolar H+-ATPase activity and Ca2+ gradient during entry of rotavirus into MA104 cells. J Virol 76:13083–13087

    Article  PubMed  CAS  Google Scholar 

  6. Chen PL, Wang M, Ou WC, Lii CK, Chen LS, Chang D (2001) Disulfide bonds stabilize JC virus capsid-like structure by protecting calcium ions from chelation. FEBS Lett 500:109–113

    Article  PubMed  CAS  Google Scholar 

  7. Ishizu KI, Watanabe H, Han SI, Kanesashi SN, Hoque M, Yajima H, Kataoka K, Handa H (2001) Roles of disulfide linkage and calcium ion-mediated interactions in assembly and disassembly of virus-like particles composed of simian virus 40 VP1 capsid protein. J Virol 75:61–72

    Article  PubMed  CAS  Google Scholar 

  8. Li PP, Naknanishi A, Tran MA, Ishizu K, Kawano M, Phillips M, Handa H, Liddington RC, Kasamatsu H (2003) Importance of Vp1 calcium-binding residues in assembly, cell entry, and nuclear entry of simian virus 40. J Virol 77:7527–7538

    Article  PubMed  CAS  Google Scholar 

  9. Liddington RC, Yan Y, Moulai J, Sahli R, Benjamin TL, Harrison SC (1991) Structure of simian virus 40 at 3.8-Å resolution. Nature 354:278–284

    Article  PubMed  CAS  Google Scholar 

  10. Lin CS, Lu MW, Tang L, Liu W, Chao CB, Lin CJ, Krishna NK, Johnson JE, Schneemann A (2001) Characterization of virus-like particles assembled in a recombinant baculovirus system expressing the capsid protein of a fish nodavirus. Virology 290:50–58

    Article  PubMed  CAS  Google Scholar 

  11. Lu MW, Liu W, Lin CS (2003) Infection competition against grouper nervous necrosis virus by virus-like particles produced in Escherichia coli. J Gen Virol 84:1577–1582

    Article  PubMed  CAS  Google Scholar 

  12. Mézeth KB, Nylund S, Henriksen H, Patel S, Nerland AH, Szilvay AM (2007) RNA-dependent RNA polymerase from Atlantic halibut nodavirus contains two signals for localization to the mitochondria. Virus Res 130:43–52

    Article  PubMed  Google Scholar 

  13. Michelangeli F, Liprandi F, Chemello ME, Ciarlet M, Ruiz MC (1995) Selective depletion of stored calcium by thapsigargin blocks rotavirus maturation but not the cytopathic effect. J Virol 69:3838–3847

    PubMed  CAS  Google Scholar 

  14. Munday BL, Kwang J, Moody N (2002) Betanodavirus infections of teleost fish: a review. J Fish Dis 25:127–142

    Article  Google Scholar 

  15. Paintsil J, Muller M, Picken M, Gissmann L, Zhou J (1998) Calcium is required in reassembly of bovine papillomavirus in vitro. J Gen Virol 79:1133–1141

    PubMed  CAS  Google Scholar 

  16. Ruiz MC, Cohen J, Michelangeli F (2000) Role of Ca2+ in the replication and pathogenesis of rotavirus and other viral infections. Cell Calcium 28:137–149

    Article  PubMed  CAS  Google Scholar 

  17. Sangita V, Lokesh GL, Satheshkumar PS, Vijay CS, Saravanan V, Savithri HS, Murthy MRN (2004) T = 1 Capsid structures of Sesbania mosaic virus coat protein mutants: determinants of T = 3 and T = 1 capsid assembly. J Mol Biol 342:987–999

    Article  PubMed  CAS  Google Scholar 

  18. Satheshkumar PS, Lokesh GL, Sangita V, Saravanan V, Vijay CS, Murthy MRN, Savithri HS (2004) Role of metal ion-mediated interactions in the assembly and stability of Sesbania mosaic virus T = 3 and T = 1 capsids. J Mol Biol 342:1001–1014

    Article  PubMed  CAS  Google Scholar 

  19. Sherman MB, Guenther RH, Tama F, Sit TL, Brooks CL, Mikhailov AM, Orlova EV, Baker TS, Lommel SA (2006) Removal of divalent cations induces structural transitions in Red clover necrotic mosaic virus, revealing a potential mechanism for RNA release. J Virol 80:10395–10406

    Article  PubMed  CAS  Google Scholar 

  20. Tang L, Johnson KN, Ball LA, Lin T, Yeager M, Johnson JE (2001) The structure of Pariacoto virus reveals a dodecahedral cage of duplex RNA. Nat Struct Biol 8:77–83

    Article  PubMed  CAS  Google Scholar 

  21. Tang L, Lin CS, Neel KK, Yeager M, Schneemann A, Johnson JE (2002) Virus-like particles of a fish nodavirus display a capsid subunit domain organization different from that of insect nodaviruses. J Virol 76:6370–6375

    Article  PubMed  CAS  Google Scholar 

  22. Tars K, Zeltins A, Liljas L (2003) The three-dimensional structure of Cocksfoot mottle virus at 2.7 Å resolution. Virology 310:287–297

    PubMed  CAS  Google Scholar 

  23. Victoria P, Pavel I, Carlos FA, Susana L (2002) Influence of calcium on the early steps of rotavirus infection. Virology 295:190–200

    Article  Google Scholar 

  24. Wery JP, Reddy VS, Hosur MV, Johnson JE (1994) The refined three-dimensional structure of an insect virus at 2.8 Å resolution. J Mol Biol 235:565–586

    Article  PubMed  CAS  Google Scholar 

  25. Zhao R, Hadfield AT, Kremer MJ, Rossmann MG (1997) Cations in human rhinoviruses. Virology 227:13–23

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was partially supported by grants from the National Science Council, Taiwan (NSC95-2313-B-110-002 and NSC96-2313-B-110-002-MY3), the Ministry of Education (96C031701, 95C030312 and 94C100303), and Academia Sinica (AS95lC1 to WHC). We also thank the reviewers and Mr. Simon White for editing the manuscript.

Author information

Authors and Affiliations

  1. Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, 80424, Taiwan, ROC

    Yi-Min Wu, Chi-Hsin Hsu, Chun-Hsiung Wang, Wangta Liu & Chan-Shing Lin

  2. Marine Biotechnology Group, Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung, Taiwan, ROC

    Chan-Shing Lin

  3. Institute of Chemistry, Academia Sinica, Taipei, 11529, Taiwan, ROC

    Yi-Min Wu & Wei-hau Chang

Authors
  1. Yi-Min Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chi-Hsin Hsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chun-Hsiung Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wangta Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wei-hau Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chan-Shing Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Wei-hau Chang or Chan-Shing Lin.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wu, YM., Hsu, CH., Wang, CH. et al. Role of the DxxDxD motif in the assembly and stability of betanodavirus particles. Arch Virol 153, 1633–1642 (2008). https://doi.org/10.1007/s00705-008-0150-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00705-008-0150-6

Keywords

Search

Navigation