Structural studies of orbivirus particles

  • D. I. Stuart
  • P. Gouet
  • J. Grimes
  • R. Malby
  • J. Diprose
  • S. Zientara
  • J. N. Burroughs
  • P. P. C. Mertens
Conference paper


We are using crystallographic methods to investigate the structure of AHSV and BTV. Our initial approach was to investigate the structure of the major protein component of the viral core, VP7(T13). This trimeric protein has been studied in several crystal forms from both orbiviruses and reveals a structure made up of conserved domains, capable of conformational changes and possessing a cleavage site. Further crystallographic analyses of native particles have provided a picture of the VP7(T13) and VP3(T2) layers of the BTV core. The VP7(T13) layer consists of 260 trimers arranged rather symmetrically and possessing very similar structures, thereby following the rules of quasi equivalence. The VP3(T2) layer is thin and contains 120 copies of 100 kDa protein arranged as 60 approximate dimers. This type of icosahedral construction has not been observed before and appears to contain a genome which is highly ordered. We anticipate that all of these features will be common to AHSV.


Core Particle Apical Domain Cryoelectron Microscopy European Synchrotron Radiation Facility Bluetongue Virus 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Bacon D, Anderson WF (1988) A fast algorithm for rendering space-filling molecule pictures. J Mol Graphics 6: 219–220CrossRefGoogle Scholar
  2. 2.
    Bartlett NM, Gillies SC, Bullivant S, Bellamy AR (1974) Electron-microscopy of reovirus reaction cores. J Virol 14: 315–326PubMedGoogle Scholar
  3. 3.
    Basak AK, Grimes JM, Gouet P, Roy P, Stuart DI (1997) Structures of orbivirus VP7: implications for the role of this protein in the viral life cycle. Structure 5: 871–883PubMedCrossRefGoogle Scholar
  4. 4.
    Basak AK, Gouet P, Grimes J, Roy P, Stuart D (1996) Crystal-structure of the top domain of African horse sickness virus VP7 — comparisons with bluetongue virus VP7. J Virol 70: 3797–3806PubMedGoogle Scholar
  5. 5.
    Bourgeois D, Nurizzo D. Kahn R, Cambillau C (1998) An integration routine based on profile fitting with optimized fitting area for the evaluation of weak and/or overlapped two-dimensional Laue or monochromatic patterns. J Appl Cryst 31 (in press)Google Scholar
  6. 6.
    Brookes SM, Hyatt AD, Eaton BT (1993) Characterization of virus inclusion bodies in bluetongue virus-infected cells. J Gen Virol 74: 525–530PubMedCrossRefGoogle Scholar
  7. 7.
    Brunger AT (1992) X-PLOR Version 3.1 (Yale University Press)Google Scholar
  8. 8.
    Burroughs JN, Grimes JM, Mertens PPC, Stuart DI (1995) Crystallization and preliminary X-ray analysis of the core particle of bluetongue virus. Virology 210: 217–220PubMedCrossRefGoogle Scholar
  9. 9.
    Burroughs JN, O’Hara RS, Smale CJ, Hamblin C, Walton A, Armstrong R, Mertens PPC (1994) Purification and properties of virus particles, infectious sub viral particles, cores and VP7 crystals of African horse sickness virus serotype 9. J Gen Virol 75: 1849–1857PubMedCrossRefGoogle Scholar
  10. 10.
    Butcher SJ, Dokland T, Ojala PM, Bamford DH, Fuller SD (1997) Intermediates in the assembly pathway of the double-stranded RNA virus phi 6. EMBO J 16: 4477–4487PubMedCrossRefGoogle Scholar
  11. 11.
    Calisher CH, Mertens PPC (1998) Taxonomy of African horse sickness virus. In: Mellor PS, Baylis M, Hamblin C, Calisher CH, Mertens PPC (eds) African horse sickness. Springer, Wien New York, pp 3–11 (Arch Virol [Suppl] 14 )Google Scholar
  12. 12.
    Caspar DLD, Klug A (1962) Physical principles in the construction of regular viruses. Cold Spring Harbor Sym Quant Biol 27: 1–24Google Scholar
  13. 13.
    Chiu W, Burnett RM, Garcea RL (eds) (1997) Structural biology of viruses. Oxford University Press, New YorkGoogle Scholar
  14. 14.
    Dai R, Wu A, Shen X, Qian L, Sun Y (1982) Isolation of the genome-enzyme complex from cytoplasmic polyhedrosis virus of silkworm Bombyx mori. Sci Sin B 25: 29–35PubMedGoogle Scholar
  15. 15.
    Delano WL, Brunger AT (1995) the direct rotation function — rotational Patterson correlationb search applied to molecular replacement. Acta Crystallogr D 51: 740–748Google Scholar
  16. 16.
    Devi R, Burroughs JN, Mertens PPC, Roy P (1998) Transmethylase 1 and transmethylase 2 activities associated with the Cap protein (VP4) of BTV, in native cores, core like particles and as a purified, baculovirus expressed protein. Proc Natl Acad Sci USA (in press)Google Scholar
  17. 17.
    Eaton BT, Hyatt AD, Brookes SM (1990) The replication of bluetongue virus. Curr Top Microbiol Immunol 162: 89–118PubMedGoogle Scholar
  18. 18.
    Esnouf RM (1997) An extensively modified version of MolScript that includes greatly enhanced colouring capabilities. J Mol Graphics 15: 133–138Google Scholar
  19. 19.
    Fu H, Bland AP, Wade-Evans A M, Mertens PPC, Mellor PS (1998) Ultra structural comparison of bluetongue virus replication in mammalian and Culicoides cells. (Manuscript submitted)Google Scholar
  20. 20.
    Fukusho A, Yu Y, Yamaguchi S, Roy P (1989) Completion of the sequence of blue-tongue virus serotype 10 by the characterization of the structural protein, VP6, and a non-structural protein, NS2. J Gen Virol 70: 1677–1689PubMedCrossRefGoogle Scholar
  21. 21.
    Foulser D (1995) IRIS EXPLORER; A framework for investigation. Comp Graphics 29: 13–16CrossRefGoogle Scholar
  22. 22.
    Gillies S, Bullivant S, Bellamy AR (1971) Viral RNA polymerases: electron microscopy of reovirus reaction cores. Science 174: 694–696PubMedCrossRefGoogle Scholar
  23. 23.
    Gouet P, Grimes JM, Diprose JM, Malby R, Burroughs JN, Zeintara S, Stuart, DI, Mertens PPC (1998) Crystallographic studies show ordered double-stranded RNA in bluetongue virus. (submitted)Google Scholar
  24. 24.
    Grimes JM, Basak AK, Roy P, Stuart DI (1995) The crystal structure of bluetongue virus VP7. Nature 373: 167–170PubMedCrossRefGoogle Scholar
  25. 25.
    Grimes JM, Jakana J, Ghosh M, Basak AK, Roy P, Chiu W, Stuart DI, Prasad BVV (1997) An atomic model of the outer layer of the bluetongue virus core derived from X-ray crystallography and electron cryomicroscopy. Structure 5: 885–893PubMedCrossRefGoogle Scholar
  26. 26.
    Grimes JM, Burroughs JN, Gouet P, Diprose JM, Malby R, Zeintara S, Mertens PPC, Stuart DI (1998) The atomic structure of the bluetongue virus core. Nature (in press)Google Scholar
  27. 27.
    Hadfield AT, Lee WM, Zhao R, Oliveira MA, Minor I, Rueckert RR, Rossmann MG (1997) The refined structure of human rhinovirus 16 at 2.15 A resolution: Implications for the viral life cycle. Structure 5: 427–441PubMedCrossRefGoogle Scholar
  28. 28.
    Harlos K (1992) Micro-bridges for sitting-drop crystallizations J Appl Crystallogr 25: 536–538Google Scholar
  29. 29.
    Hewat EA, Booth TF, Loudon PT, Roy P (1992) Three-dimensional reconstruction of baculovirus expressed bluetongue virus core-like particles by cryo-electron microscopy. Virology 189: 10–20PubMedCrossRefGoogle Scholar
  30. 30.
    Hewat EA, Booth TF, Roy P (1992) Structure of bluetongue virus particles by cryoelectron microscopy. J Struct Biol 109: 61–69PubMedCrossRefGoogle Scholar
  31. 31.
    Hill, CL (1997) Analysis of the structure and function of a cytoplasmic polyhedrosis virus. Ph.D. Thesis, Oxford UniversityGoogle Scholar
  32. 32.
    Holmes IH, Boccardo G, Estes MK, Furuichi MK, Hoshino Y, Joklik WK, McCrae M, Mertens PPC, Milne RG, Samal KSK, Shiata E, Winton JR, Uyeda I (1995) Family Reoviridae. In: Murphy FA, Fauquet CM, Bishop DHL, Ghabrial SA, Jarvis AW, Martelli GP, Mayo MA, Summers MD (eds) Virus Taxonomy. Classification and Nomenclature of Viruses. Sixth Report of the International Committee on Taxonomy of Viruses. Springer, Wien New York, pp 208–237 (Arch Virol [Suppl] 10 )Google Scholar
  33. 33.
    Huismans H, Van Dijk AA, Els HJ (1987) Uncoating of parental bluetongue virus to core and subcore particles in infected L cells. Virology 157: 180–188PubMedCrossRefGoogle Scholar
  34. 34.
    Johnson JE, Speir JA (1997) Quasi-equivalent viruses: a paradigm for protein assemblies. J Mol Biol 269: 665–675PubMedCrossRefGoogle Scholar
  35. 35.
    Joklik WK (1983) In: Fraenkel-Conrat H, Wagner RR (eds) The Reoviridae. Plenum Press, New YorkGoogle Scholar
  36. 36.
    Juuti JT, Ravantti JJ, Bamford DH (1997) The common and uncommon among dsRNA viruses. In: Arias CF, Lopez S (eds) 6th International Symposium on dsRNA Viruses. Cocoyoc, Mexico, pp Al-Al0Google Scholar
  37. 37.
    Lawton JA, Estes MK, Prasad BV (1997) Three-dimensional visualisation of mRNA release from actively transcribing rotavirus particles. Nature Struct Biol 4: 118–121PubMedCrossRefGoogle Scholar
  38. 38.
    Lawton JA, Zeng CQY, Mukherjee SK, Cohen J, Estes MK, Prasad BVV (1997) Three-dimensional structural analysis of recombinant rotavirus-like particles with intact and amino-terminal deleted VP2: implications for the architecture of the VP2 capsid layer. J Virol 71: 7363–7360Google Scholar
  39. 39.
    Liddington RC, Yan Y, Moulai J, Sahli R, Benjamin TL, Harrison SC (1991) Structure of simian virus 40 at 3.8-A resolution. Nature 354: 278–284PubMedCrossRefGoogle Scholar
  40. 40.
    Loudon PT, Roy P (1991) Assembly of five bluetongue virus proteins expressed by recombinant baculoviruses: inclusion of the largest protein VP1 in the core and virus-like particles. Virology 180: 798–802PubMedCrossRefGoogle Scholar
  41. 41.
    Loudon PT, Roy P (1992) Interaction of nucleic acids with core-like and subcore-like particles of bluetongue virus. Virology 191: 231–236PubMedCrossRefGoogle Scholar
  42. 42.
    Merritt EA, Murphy MEP (1994) Raster 3D version 2.0. A program for photorealistic molecular graphics. Acta Crystallog D 50: 869–873Google Scholar
  43. 43.
    Mertens PPC, Burroughs JN, Anderson J (1987) Purification and properties of virus particles, infectious subviral particles, and cores of bluetongue virus serotypes 1 and 4. Virology 157: 375–386PubMedCrossRefGoogle Scholar
  44. 44.
    Mertens PPC, Burroughs JN, Wade-Evans AM, Le Blois H, Oldfield S, Basak A, Loudon P, Roy P (1992) Analysis of guanylyltransferase and transmethylase activities associated with bluetongue virus cores and recombinant baculovirus-expressed core-like particles. Bluetongue, African horse sickness and related orbiviruses. In: Walton TE, Osburn BI (eds) Second International Symposium on bluetongue, African horse sickness and related orbiviruse. CRC Press, Boca Raton, pp 404–415Google Scholar
  45. 45.
    Mertens PPC, Burroughs JN, Walton A, Wellby MP, Fu H, O’Hara RS, Brookes SM, Mellor PS (1996) Enhanced infectivity of modified bluetongue virus particles for two insect cell lines and for two Culicoides vector species. Virology 217: 582–593PubMedCrossRefGoogle Scholar
  46. 46.
    Mertens PPC et al (in preparation) Family Reoviridae. In: Seventh Report of the International Committee on Taxonomy of VirusesGoogle Scholar
  47. 47.
    Moss SR, Nuttall PA (1994) Subcore-and core-like particles of Broadhaven virus (BRDV), a tick-bourne orbivirus, synthesized from baculovirus expressed VP2 and VP7, the major core proteins of BRDV. Virus Res 32: 401–407PubMedCrossRefGoogle Scholar
  48. 48.
    Oliveira MA, Zhao R, Lee WM, Kremer MJ, Minor I, Rueckert RR, Diana GD, Pevear DC, Dutko FJ, Mckinlay MA, Rossmann MG (1993) The structure of human rhinovirus 16. Structure 1: 51–68PubMedCrossRefGoogle Scholar
  49. 49.
    Otwinoski ZO (1993) DENZO. In: Saywer L, Isaacs N, Bailey S (eds) Proceedings of CCP4 study weekend: Data collection and reductionGoogle Scholar
  50. 50.
    Prasad BVV, Yamaguchi S, Roy P (1992) Three-dimensional structure of single-shelled bluetongue virus. J Virol 66: 2135–2142PubMedGoogle Scholar
  51. 51.
    Prasad BVV, Rothnagel R, Zeng CQY, Jakana J, Lawton JA, Chiu W, Estes MK (1996) Visualisation of ordered genomic RNA and localisation of transcriptional complexes in rotavirus. Nature 382: 471–473PubMedCrossRefGoogle Scholar
  52. 52.
    Roussel A, Inisan AG, Cambillau C (1991) AFMB and biographies. Marseille, FranceGoogle Scholar
  53. 53.
    Roy P (1989) Bluetongue virus genetics and genome structure. Virus Res 13: 179–206PubMedCrossRefGoogle Scholar
  54. 54.
    Roy P (1989) Bluetongue virus proteins. J Gen Virol 73: 3051–3064CrossRefGoogle Scholar
  55. 55.
    Roy P, Fukusho A, Ritter D G, Lyons D (1988) Evidence for genetic relationship between RNA and DNA viruses from the sequence homology of a putative polymerase gene of bluetongue virus with that of vaccinia virus: conservation of RNA polymerase genes from diverse species. Nucleic Acids Res 16: 11759–11767PubMedCrossRefGoogle Scholar
  56. 56.
    Schoehn G, Moss SR, Nuttall PA, Hewat EA (1997) Structure of Broadhaven virus by cryoelectron microscopy: correlation of structural and antigenic properties of Broad-haven virus and bluetongue virus outer capsid proteins. Virology 235: 191–200PubMedCrossRefGoogle Scholar
  57. 57.
    Stuart D (1993) Viruses. Curr Opin Struct Biol 3: 167–174CrossRefGoogle Scholar
  58. 58.
    Stauber N, Martinez-Costas J, Sutton G, Monastyrskaya K, Roy P (1997) Bluetongue virus VP6 protein binds ATP and exhibits an RNA-dependent ATPase function and a helicase activity that catalyze the unwinding of double-stranded RNA substrates. J Virol 71: 7220–7226PubMedGoogle Scholar
  59. 59.
    Urakawa T, Ritter DG, Roy P (1989) Expression of largest RNA segment and synthesis of VP1 protein of bluetongue virus in insect cells by recombinant baculovirus; association of VP1 protein with RNA polymerase activity. Nucleic Acids Res 17: 7395–7401PubMedCrossRefGoogle Scholar
  60. 60.
    Van Dijk AA, Huismans H (1980) The in vitro activation and further characterization of the bluetongue virus-associated transcriptase. Virology 104: 347–356PubMedCrossRefGoogle Scholar
  61. 61.
    Yazaki K, Miura K (1980) Relation of the structure of cytoplasmic polyhedrosis virus and the synthesis of its messenger RNA. Virology 105: 467–479PubMedCrossRefGoogle Scholar
  62. 62.
    Zhu Y, Hemmings AM, Iwasaki K, Fujiyoshi Y, Zhong B, Yan J, Isogai M, Omura T (1997) Details of the arrangement of the outer capsid of rice dwarf phytoreovirus, as visualized by two-dimensional crystallography. J Virol 71: 8899–8901PubMedGoogle Scholar

Copyright information

© Springer-Verlag/Wien 1998

Authors and Affiliations

  • D. I. Stuart
    • 1
    • 2
  • P. Gouet
    • 1
  • J. Grimes
    • 1
  • R. Malby
    • 1
  • J. Diprose
    • 1
  • S. Zientara
    • 3
  • J. N. Burroughs
    • 4
  • P. P. C. Mertens
    • 5
  1. 1.Laboratory of Molecular BiophysicsOxfordUK
  2. 2.New Chemistry LaboratoryThe Oxford Centre for Molecular SciencesOxfordUK
  3. 3.Laboratoire Central de Recherches VétérinairesCentre National d’Études Vétérinaires et AlimentairesMaisons-AlfortFrance
  4. 4.Pirbright LaboratoryInstitute for Animal HealthPirbright, SurreyUK
  5. 5.Pirbright LaboratoryInstitute for Animal HealthPirbright, Woking SurreyUK

Personalised recommendations