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. 1987 Nov;61(11):3589–3595. doi: 10.1128/jvi.61.11.3589-3595.1987

In vitro phosphorylation and purification of a nonstructural protein of bluetongue virus with affinity for single-stranded RNA.

H Huismans 1, A A van Dijk 1, A R Bauskin 1
PMCID: PMC255959  PMID: 2822964

Abstract

A phosphorylated, nonstructural protein of bluetongue virus, NS2, is synthesized throughout the replication cycle in comparatively large amounts. The protein was detected in both the soluble and particulate fraction of the cytoplasm of infected cells. The particulate NS2 could be solubilized in 0.5 M NaCl. It was found that NS2 in the particulate fraction and immunoprecipitates of NS2 from the soluble protein fraction could be phosphorylated in vitro. It is not known whether the kinase involved is of cellular or viral origin, but after purification of NS2 by affinity chromatography on poly(U)-Sepharose it could still by phosphorylated in vitro without the addition of exogenous protein kinase. The affinity of NS2 for nucleic acid was also investigated. The protein was found to bind to single-stranded RNA. In the presence of purified bluetongue virus mRNA, NS2 formed a complex with an estimated S value of about 22S.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bell J. C., Brown E. G., Takayesu D., Prevec L. Protein kinase activity associated with immunoprecipitates of the vesicular stomatitis virus phosphoprotein NS. Virology. 1984 Jan 30;132(2):229–238. doi: 10.1016/0042-6822(84)90030-8. [DOI] [PubMed] [Google Scholar]
  2. Boyle J. F., Holmes K. V. RNA-binding proteins of bovine rotavirus. J Virol. 1986 May;58(2):561–568. doi: 10.1128/jvi.58.2.561-568.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Clinton G. M., Burge B. W., Huang A. S. Effects of phosphorylation and pH on the association of NS protein with vesicular stomatitis virus cores. J Virol. 1978 Aug;27(2):340–346. doi: 10.1128/jvi.27.2.340-346.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cooper P. C., Burgess A. W. Simultaneous detection of 35S- and 32P-labeled proteins on electrophoretic gels. Anal Biochem. 1982 Nov 1;126(2):301–305. doi: 10.1016/0003-2697(82)90519-x. [DOI] [PubMed] [Google Scholar]
  5. Gomatos P. J., Prakash O., Stamatos N. M. Small reovirus particle composed solely of sigma NS with specificity for binding different nucleic acids. J Virol. 1981 Jul;39(1):115–124. doi: 10.1128/jvi.39.1.115-124.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Harmon S. A., Marnell L. L., Summers D. F. The major ribonucleoprotein-associated protein kinase of vesicular stomatitis virus is a host cell protein. J Biol Chem. 1983 Dec 25;258(24):15283–15290. [PubMed] [Google Scholar]
  7. Huismans H., Cloete M. A comparison of different cloned bluetongue virus genome segments as probes for the detection of virus-specified RNA. Virology. 1987 Jun;158(2):373–380. doi: 10.1016/0042-6822(87)90209-1. [DOI] [PubMed] [Google Scholar]
  8. Huismans H., Els H. J. Characterization of the tubules associated with the replication of three different orbiviruses. Virology. 1979 Jan 30;92(2):397–406. doi: 10.1016/0042-6822(79)90144-2. [DOI] [PubMed] [Google Scholar]
  9. Huismans H., Joklik W. K. Reovirus-coded polypeptides in infected cells: isolation of two native monomeric polypeptides with affinity for single-stranded and double-stranded RNA, respectively. Virology. 1976 Apr;70(2):411–424. doi: 10.1016/0042-6822(76)90282-8. [DOI] [PubMed] [Google Scholar]
  10. Huismans H. Protein synthesis in bluetongue virus-infected cells. Virology. 1979 Jan 30;92(2):385–396. doi: 10.1016/0042-6822(79)90143-0. [DOI] [PubMed] [Google Scholar]
  11. Huismans H., Verwoerd D. W. Control of transcription during the expression of the bluetongue virus genome. Virology. 1973 Mar;52(1):81–88. doi: 10.1016/0042-6822(73)90400-5. [DOI] [PubMed] [Google Scholar]
  12. Huismans H., van der Walt N. T., Cloete M., Erasmus B. J. Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep. Virology. 1987 Mar;157(1):172–179. doi: 10.1016/0042-6822(87)90326-6. [DOI] [PubMed] [Google Scholar]
  13. Ito Y., Joklik W. K. Temperature-sensitive mutants of reovirus. 3. Evidence that mutants of group D ("RNA-negative") are structural polypeptide mutants. Virology. 1972 Oct;50(1):282–286. doi: 10.1016/0042-6822(72)90373-x. [DOI] [PubMed] [Google Scholar]
  14. Mertens P. P., Brown F., Sangar D. V. Assignment of the genome segments of bluetongue virus type 1 to the proteins which they encode. Virology. 1984 May;135(1):207–217. doi: 10.1016/0042-6822(84)90131-4. [DOI] [PubMed] [Google Scholar]
  15. Moyer S. A., Summers D. F. Phosphorylation of vesicular stomatitis virus in vivo and in vitro. J Virol. 1974 Feb;13(2):455–465. doi: 10.1128/jvi.13.2.455-465.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Scheidtmann K. H., Hardung M., Echle B., Walter G. DNA-binding activity of simian virus 40 large T antigen correlates with a distinct phosphorylation state. J Virol. 1984 Apr;50(1):1–12. doi: 10.1128/jvi.50.1.1-12.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Stamatos N. M., Gomatos P. J. Binding to selected regions of reovirus mRNAs by a nonstructural reovirus protein. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3457–3461. doi: 10.1073/pnas.79.11.3457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Van Dijk A. A., Huismans H. The in vitro activation and further characterization of the bluetongue virus-associated transcriptase. Virology. 1980 Jul 30;104(2):347–356. doi: 10.1016/0042-6822(80)90339-6. [DOI] [PubMed] [Google Scholar]
  19. Verwoerd D. W., Els H. J., De Villiers E. M., Huismans H. Structure of the bluetongue virus capsid. J Virol. 1972 Oct;10(4):783–794. doi: 10.1128/jvi.10.4.783-794.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]

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