1887

Journal of General Virology header logo

Volume 75, Issue 12

Comparison of the expression and phosphorylation of the non-structural protein NS2 of three different orbiviruses: evidence for the involvement of an ubiquitous cellular kinase Free

Abstract

The non-structural protein NS2 of epizootic haemorrhagic disease (EHD), bluetongue (BT) and African horsesickness (AHS) viruses has each been expressed to high levels using a baculovirus vector gene expression system. It was found that the recombinant baculovirus- expressed EHDV NS2 protein was resolved as a doublet following PAGE. Peptide mapping of these protein bands indicated that they were identical. The difference in the sizes of the NS2 protein bands could not be attributed to the phosphorylation of NS2 or other posttranslational modification such as -glycosylation and remains obscure. The EHDV, BTV and AHSV baculo- virus-expressed NS2 proteins were all phosphorylated without the addition of an exogenous kinase. An unphosphorylated form of EHDV NS2, obtained by expressing the NS2 gene as a fusion protein in cells, could be phosphorylated by a protein kinase associated with the cytoplasm of insect cells. The phosphorylated version of this protein was found to be significantly less efficient in binding ssRNA, compared to the unphosphorylated version.

  • Received:
  • Accepted:
  • Published Online:
© The Journal of General Virology 1994
Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-75-12-3401
1994-12-01
2023-03-22
Loading full text...

Full text loading...

/deliver/fulltext/jgv/75/12/JV0750123401.html?itemId=/content/journal/jgv/10.1099/0022-1317-75-12-3401&mimeType=html&fmt=ahah

References

  1. Barik S., Banerjee A. K. 1991; Cloning and expression of the vesicular stomatitis virus phosphoprotein gene in Escherichia coli; analysis of phosphorylation status versus transcriptional activity. Journal of Virology 65:1719–1726
     [Google Scholar]
  2. Bogerd H. P., Huckaby G. L., Ahmed Y. F., Hanly S. M., Greene W. C. 1991; The type I human T-cell leukemia virus (HTLV-I) Rex trans-activator bind directly to the HTLV-I Rex and the type 1 human immunodeficiency virus Rev response elements. Proceedings of the National Academy of Sciences U.S.A: 885704–5708
     [Google Scholar]
  3. Boyle J. F., Fiolmes K. V. 1986; RNA-binding proteins of bovine rotavirus. Journal of Virology 58:561–568
     [Google Scholar]
  4. Bremer C. W. 1976; A gel electrophoretic study of the protein and nucleic acid components of African horsesickness virus. Onderste- poort Journal of Veterinary Research 43:193–200
     [Google Scholar]
  5. Brookes S. M., Hyatt A. D., Eaton B. T. 1993; Characterization of virus inclusion bodies in bluetongue virus-infected cells. Journal of General Virology 74:525–530
     [Google Scholar]
  6. 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]
  7. Cochrane A. W., Golub E., Volsky D., Ruben S., Rosen C. A. 1989; Functional significance of phosphorylation to the human immunodeficiency virus Rev protein. Journal of Virology 63:4438–4440
     [Google Scholar]
  8. Devaney M. A., Kendall J., Grubmann M. J. 1988; Characterization of a nonstructural protein of two orbiviruses. Virus Research 11:151–164
     [Google Scholar]
  9. Eaton B. T., Hyatt R. O., White J. R. 1987; Association of bluetongue virus with the cytoskeleton. Virology 157:107–116
     [Google Scholar]
  10. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. 1987; Lipofection: a highly efficient, lipid-mediated DNA-trans- fection procedure. Proceedings of the National Academy of Sciences U.S.A: 847413–7417
     [Google Scholar]
  11. Gorman B. M. 1992; An overview of the orbiviruses. In Bluetongue, African Horse Sickness and Related Orbiviruses Proceedings of the Second International Symposium pp 335–347 Walton T. E., Osborn B. I. Edited by Florida: CRC Press;
     [Google Scholar]
  12. Grässer F. A., König S. 1992; Phosphorylation of SV40 large T antigen at threonine residues results in conversion to a lower apparent molecular weight form. Archives of Virology 126:313–320
     [Google Scholar]
  13. Green P. L., Yip M. T., Xie Y., Chen I. S. Y. 1992; Phosphorylation regulates RNA binding by the human T-cell leukemia virus Rex protein. Journal of Virology 66:4325–4330
     [Google Scholar]
  14. Grubman M. J., Lewis S. A. 1992; Identification and characterization of the structural and nonstructural proteins of African horsesickness virus and determination of the genome coding assignments. Virology 186:444–451
     [Google Scholar]
  15. Huismans H. 1979; Protein synthesis in bluetongue virus-infected cells. Virology 92:385–396
     [Google Scholar]
  16. Huismans H., Basson H. M. 1983; The characterization of a phosphorylated nonstructural polypeptide of bluetongue virus with affinity for RNA. In: Double-stranded RNA Viruses pp 173–181 Compans W., Bishop D. H. L. Edited by Amsterdam: Elsevier Science Publishing;
     [Google Scholar]
  17. Huismans H., Els H. J. 1979; Characterization of the tubules associated with the replication of three different orbiviruses. Virology 92:397–306
     [Google Scholar]
  18. 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 70:411–124
     [Google Scholar]
  19. Huismans H., Bremer C. W., Barber T. L. 1979; The nucleic acid and proteins of epizootic haemorrhagic disease virus. Onderstepoort Journal of Veterinary Research 46:95–104
     [Google Scholar]
  20. 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]
  21. Hyatt A. D., Gould A. R., Coupar B., Eaton B. T. 1991; Localization of the non-structural protein NS3 in bluetongue virus- infected cells. Journal of General Virology 72:2263–2267
     [Google Scholar]
  22. Hyatt A. D., Zhao Y., Roy P. 1993; Release of bluetongue viruslike particles from insect cells is mediated by BTV nonstructural protein NS3/NS3A. Virology 157:472–479
     [Google Scholar]
  23. Kattoura M. D., Clapp L. L., Patton J. T. 1992; The Rotavirus nonstructural protein NS35 possesses RNA-binding activity in vitro and in vivo. Virology 191:698–708
     [Google Scholar]
  24. Köchel H. G., Kann M., Thomssen R. 1991; Identification of a binding site in the hepatitis B virus RNA pregenome for the viral Pol gene product. Virology 182:94–101
     [Google Scholar]
  25. Klausing K., Scheidtmann K., Baumann E. A., Knippers R. 1988; Effects of in vitro dephosphorylation on DNA-binding and DNA helicase activities of simian virus 40 large tumor antigen. Journal of Virology 62:1258–1265
     [Google Scholar]
  26. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 227680–685
     [Google Scholar]
  27. Livingstone G., Jones I. 1991; Baculovirus expression vectors with single strand capability. Nucleic Acids Research 17:2366
     [Google Scholar]
  28. Marston F. A. O. 1988; The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochemical Journal 240:1–12
     [Google Scholar]
  29. Mecham J. O., Dean V. C. 1988; Protein coding assignment for the genome of epizootic haemorrhagic disease virus. Journal of General Virology 69:1255–1262
     [Google Scholar]
  30. Miller L. K. 1988; Baculoviruses as gene expression vectors. Annual Reviews in Microbiology 42:177–199
     [Google Scholar]
  31. Nel L. H., Huismans H. 1990; A comparison of different cloned genome segments of epizootic haemorrhagic disease virus as serogroup-specific probes. Archives of Virology 110:103–112
     [Google Scholar]
  32. Nel L. H., Huismans H. 1991; Synthesis of the virus specified tubules of epizootic haemorrhagic disease virus using a baculovirus expression system. Virus Research 19:139–152
     [Google Scholar]
  33. 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]
  34. Simmons D. T., Chou W., Rodgers K. 1986; Phosphorylation downregulates the DNA-binding activity of simian virus 40 T antigen. Journal of Virology 60:888–894
     [Google Scholar]
  35. Smith D. B., Johnson K. S. 1989; Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione 5-transferase. Gene 67:31–39
     [Google Scholar]
  36. Thomas C. P., Booth T. F., Roy P. 1990; Synthesis of bluetongue virus-encoded phosphoprotein and formation of inclusion bodies by recombinant baculovirus in insect cells: it binds the single-stranded RNA species. Journal of General Virology 71:2073–2083
     [Google Scholar]
  37. Urakawa T., Roy P. 1988; Bluetongue virus tubules made in insect cells by recombinant baculoviruses: expression of the NS I gene of bluetongue virus serotype 10. Journal of Virology 62:3919–3927
     [Google Scholar]
  38. Van Duk A. A., Huismans H. 1988; In vitro transcription and translation of bluetongue virus RNA. Journal of General Virology 69:573–581
     [Google Scholar]
  39. Van Staden V., Huismans H. 1991; A comparison of the genes which encode non-structural protein NS3 of different orbiviruses. Journal of General Virology 72:1073–1079
     [Google Scholar]
  40. Van Staden V., Theron J., Greyling B. J., Huismans H., Nel L. H. 1991; A comparison of the nucleotide sequences of cognate NS2 genes of three different orbiviruses. Virology 185:500–504
     [Google Scholar]
  41. Verwoerd D. W., Huismans H., Erasmus B. J. 1979; Orbiviruses. In: Comprehensive Virology 14 pp 285–345 Fraenkel-Conrat H., Wagner R. R. Edited by New York: Plenum Press;
     [Google Scholar]
  42. Wu X., Chen S. -Y., Iwata H., Compans R. W., Roy P. 1992; Multiple glycoproteins synthesized by the smallest RNA segment (S10) of bluetongue virus. Journal of Virology 66:7104–7112
     [Google Scholar]
  43. Zuidema D., Schouten A., Usmany M., Maule A. J., Belsham G. J., Roosien J., Klinge-Roode E. C., Van Lent J. W. M., Vlak J. M. 1990; Expression of cauliflower mosaic virus gene I in insect cells using a novel polyhedrin-based baculovirus expression vector. Journal of General Virology 71:2201–2209
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-75-12-3401
Loading
Comparison of the expression and phosphorylation of the non-structural protein NS2 of three different orbiviruses: evidence for the involvement of an ubiquitous cellular kinase
J. Gen. Virol. 75, 3401 (1994); https://doi.org/10.1099/0022-1317-75-12-3401
/content/journal/jgv/10.1099/0022-1317-75-12-3401
/content/journal/jgv/10.1099/0022-1317-75-12-3401
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

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