1887

Abstract

A DNA clone of RNA segment 8 (S8) of bluetongue virus type 10 (BTV-10), an orbivirus member of the family has been expressed to high levels (20 mg/1 × l0 cells) using an nuclear polyhedrosis virus expression vector (pA- cYMl). The expressed protein is similar to the authentic BTV phosphoprotein NS2, in its size, antigenicity, and also the manner of phosphorylation (e.g. same peptides and residues). Both mammalian and insect cell-derived NS2 proteins are phosphory- lated at serine residues only. Using affinity column chromatography and a gel retardation assay, the expressed protein has been shown to possess ssRNA- binding ability, a property which is shown to be independent of the phosphorylation state of the protein. In immunoelectron micrographic studies, gold-labelled anti-expressed NS2 antibodies have been used to localize the NS2 protein within the viral inclusion bodies (VIBs) in BTV-infected mammalian cells. Large inclusion bodies, morphologically similar to VIBs, have been identified in the recombinant virus- infected cells. These structures have been shown to react with gold-labelled anti-BTV- 10 antisera, demonstrating the first direct evidence of the origin of inclusion bodies in orbivirus infection.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-71-9-2073
1990-09-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/71/9/JV0710092073.html?itemId=/content/journal/jgv/10.1099/0022-1317-71-9-2073&mimeType=html&fmt=ahah

References

  1. Bowne J. G., Jochim M. M. 1967; Cytopathologic changes and development of inclusion bodies in cultured cells infected with bluetongue virus. American Journal of Veterinary Research 28:1091–1105
    [Google Scholar]
  2. Bowne J. G., Jones R. H. 1966; Observations on bluetongue virus in the salivary glands of an insect vector, Culicoides variipennis. Virology 30:127
    [Google Scholar]
  3. Brown M., Faulkner P. 1977; A plaque assay for nuclear polyhedrosis viruses using a solid overlay. Journal of General Virology 36:361–364
    [Google Scholar]
  4. Chattopadhyay D., Banerjee A. K. 1987; Phosphorylation within a specific domain of the phosphoprotein of vesicular stomatitis virus regulates transcription in vitro. Cell 49:407–414
    [Google Scholar]
  5. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. 1977; Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. Journal of Biological Chemistry 252:1102–1106
    [Google Scholar]
  6. Clinton G. M., Burge B. W., Huang A. S. 1978; Effects of phosphorylation and pH on the association of NS protein with vesicular stomatitis virus cores. Journal of Virology 27:340–346
    [Google Scholar]
  7. Cooper J. A., Sefton B. M., Hunter T. 1983; Deletion and quantification of phosphotyrosine in proteins. Methods in Enzymology 99:387–402
    [Google Scholar]
  8. Cromack A. S., Blue J. L., Gratzek J. B. 1971; A quantitative ultrastructural study of the development of bluetongue virus in Madin-Darby bovine kidney cells. Journal of General Virology 13:229–244
    [Google Scholar]
  9. Cross R. K., Fields B. N. 1972; Temperature-sensitive mutants of reovirus type 3: studies on the synthesis of viral RNA. Journal of Virology 50:799–809
    [Google Scholar]
  10. Devaney M. A., Kendall J. M., Grubman M. J. 1988; Characterization of a non-structural phosphoprotein of two orbi-viruses. Virus Research 11:151–164
    [Google Scholar]
  11. Eaton B. T., Hyatt A. D. 1989; Association of bluetongue virus with the cytoskeleton. Subcellular Biochemistry 15:229–269
    [Google Scholar]
  12. Eaton B. T., Hyatt A. D., Brookes S. M. 1990; The replication of bluetongue virus. In Current Topics in Microbiology and Immunology, ‘Bluetongue and Related Viruses’ pp. 89–118 Roy P., Gorman B. M. Edited by Heidelberg: Springer-Verlag;
    [Google Scholar]
  13. French T. J., Inumaru S., Roy P. 1989; Expression of two related non-structural proteins of BTV-10 in insect cells by a recombinant baculovirus: production of polyclonal ascitic fluid and characteriza-tion of the gene product in BTV-infected BHK cells. Journal of Virology 63:3270–3278
    [Google Scholar]
  14. Fukusho A., Yu Y., Yamaguchi S., Roy P. 1989; Completion of the sequence of bluetongue virus serotype 10 by the characterization of a structural protein, VP6, and a non-structural protein, NS2. Journal of General Virology 70:1677–1689
    [Google Scholar]
  15. Gentsch J. R., Bishop D. H. L. 1978; Small viral RNA segment of bunyaviruses codes for viral nucleocapsid protein. Journal of Virology 28:417–419
    [Google Scholar]
  16. Gomatos P. J., Tamm I., Dales S., Franklin R. M. 1962; Reovirus type-3: physical characteristics and interactions with L cells. Virology 17:441–454
    [Google Scholar]
  17. Gould A. R., Hyatt A. D., Eaton B. T. 1988; Morphogenesis of a bluetongue virus variant with an amino acid alteration at a neutralization site in the outer coat protein, VP2. Virology 165:23–32
    [Google Scholar]
  18. Hooft van Iddenkinge B. J. L., Smith G. E., Summers M. D. 1983; Nucleotide sequence of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus. Virology 131:561–565
    [Google Scholar]
  19. Huismans H. 1979; Protein synthesis in bluetongue virus-infected cells. Virology 92:385–396
    [Google Scholar]
  20. Huismans H., Els H. J. 1979; Characterization of the microtubules associated with the replication of three different orbiviruses. Virology 92:397–405
    [Google Scholar]
  21. Huismans H., Joklik W. K. 1976; Reovirus-coded polypeptides in infected cells: isolation of two native monomeric polypeptides with affinity for single-stranded and double-stranded RNA, respectively. Virology 70:411
    [Google Scholar]
  22. Huismans H., van Dijk A. A., Bauskin A. R. 1987; In vitro phosphorylation and purification of a non-structural protein of bluetongue virus with affinity for single-stranded RNA. Journal of Virology 61:3589–3595
    [Google Scholar]
  23. Hyatt A. D., Eaton B. T. 1988; Ultrastructural distribution of the major capsid proteins within bluetongue virus and infected cells. Journal of General Virology 69:805–815
    [Google Scholar]
  24. Inumaru S., Roy P. 1987; Production and characterization of the neutralization antigen VP2 of bluetongue virus serotype 10 using a baculovirus expression vector. Virology 157:472–479
    [Google Scholar]
  25. Inumaru S., Ghiasa H., Roy P. 1987; Expression of bluetongue virus group-specific antigen VP3 in insect cells by a baculovirus vector: its use for the detection of bluetongue virus antibodies. Journal of General Virology 68:1627–1635
    [Google Scholar]
  26. Ito Y., Joklik W. K. 1972; Temperature-sensitive mutants of reovirus. III. Evidence that mutants of group D (‘RNA-negative’) are structural polypeptide mutants. Virology 50:282–286
    [Google Scholar]
  27. Kingsford L., Emerson S. U. 1980; Transcriptional activities of different phosphorylated species of NS protein purified from vesicular stomatitis virions and cytoplasm of infected cells. Journal of Virology 33:1097–1105
    [Google Scholar]
  28. Konarska M. M., Sharp P. A. 1987; Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell 49:763–774
    [Google Scholar]
  29. Laemmli UK. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 221680–685
    [Google Scholar]
  30. Leader D. P., Katan M. 1988; Viral aspects of protein phosphorylation. Journal of General Virology 69:1441–1464
    [Google Scholar]
  31. Lecatsas G. 1968; Electron microscopic study of the formation of bluetongue virus. Onderstepoort Journal of Veterinary Research 35:139
    [Google Scholar]
  32. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: A Laboratory Manual New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  33. Matsuura Y., Possee R. D., Overton H. A., Bishop D. H. L. 1987; Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins. Journal of General Virology 68:1233–1250
    [Google Scholar]
  34. Maxam A. M., Gilbert W. 1980; Sequencing end-labeled DNA with base-specific chemical cleavages. Methods in Enzymology 65:499–560
    [Google Scholar]
  35. Mertens P. P. C., Brown F., Sanger D. V. 1984; Assignment of the genome segments of bluetongue virus type 1 to the proteins which they encode. Virology 135:207–217
    [Google Scholar]
  36. Ramig R. F., Mustoe T. A., Sharpe A. H., Fields B. N. 1978; A genetic map of reovirus. II. Assignment of the double-stranded RNA-negative mutant groups C, D and E genome segments. Virology 85:531–534
    [Google Scholar]
  37. Roy P., Adachi A., Urakawa T., Booth T. F., Thomas C. P. 1990; Identification of bluetongue virus VP6 protein as a nucleic acid-binding protein and the localization of VP6 in virus-infected vertebrate cells. Journal of Virology 64:1–8
    [Google Scholar]
  38. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, U.S.A 74:5463–5467
    [Google Scholar]
  39. Scheidtmann K.-H., Hardung M., Echle B., Walter G. 1984; DNA-binding activity of simian virus 40 large T antigen correlates with a distinct phosphorylation state. Journal of Virology 50:1–12
    [Google Scholar]
  40. Schiff L. A., Fields B. N. 1990; Reoviruses and their replication. In Virology, 2nd edn. pp. 1275–1306 Fields B. N., Knipe D. M. Edited by New York: Raven Press;
    [Google Scholar]
  41. Silverstein S. C., Schur P. H. 1970; Immunofluorescent localization of double-stranded RNA in reovirus-infected cells. Virology 41:564–566
    [Google Scholar]
  42. Southern E. M. 1975; Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98:503–517
    [Google Scholar]
  43. Urakawa T., Roy P. 1988; Bluetongue virus tubules made in insect cells by recombinant baculoviruses: expression of the NS1 gene of bluetongue virus serotype 10. Journal of Virology 62:3919–3927
    [Google Scholar]
  44. Verwoerd D. W., Els H. J., De Villiers E. M., Huismans H. 1972; Structure of the bluetongue virus capsid. Journal of Virology 10:783–794
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-71-9-2073
Loading
/content/journal/jgv/10.1099/0022-1317-71-9-2073
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error