1887

Abstract

The induction of proteolysis by expression of the influenza virus PA polymerase subunit is the only biochemical activity ascribed to this protein. In the course of studying viral protein synthesis by twodimensional gel electrophoresis, we observed the existence of several PA isoforms with different isoelectric points. These isoforms were also present when the PA gene was singly expressed in three different expression systems, indicating that a cellular activity is responsible for its post-translational modification. labelling with [P]ortho- phosphate, followed by two-dimensional gel electrophoresis, clearly demonstrated the incorporation of phosphate into the PA molecule. Phosphoserine and phosphothreonine epitopes were present in PA, while phosphotyrosine residues were absent, as tested by immunoblotting with specific antibodies. These facts, as well as the presence of multiple consensus sites for casein kinase II (CKII) phosphorylation, prompted us to test the involvement of this kinase in PA covalent modification. PA protein purified by immunoprecipitation could be specifically labelled by the catalytic subunit of human CKII, which was expressed and purified from bacteria. Collectively, these data demonstrate that the PAsubunit of the influenza virus RNA polymerase is a phosphoprotein.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-79-3-471
1998-03-01
2021-10-18
Loading full text...

Full text loading...

/deliver/fulltext/jgv/79/3/9519825.html?itemId=/content/journal/jgv/10.1099/0022-1317-79-3-471&mimeType=html&fmt=ahah

References

  1. Almond J. W., Felsenreich V. 1982; Phosphorylation of the nucleoprotein of an avian influenza virus. Journal of General Virology 60:295–305
    [Google Scholar]
  2. Arrese M., Portela A. 1996; Serine 3 is critical for phosphorylation at the N-terminal end ofthe nucleoprotein of influenza virus A/Victoria/ 3/75. Journal of Virology 70:3385–3391
    [Google Scholar]
  3. Barcena J., de la Luna S., Ochoa M., Melero J. A., Nieto A., Ortín J., Portela A. 1994; Monoclonal antibodies against the influenza virus PB2 and NP polypeptides interfere with the initiation step of viral mRNA synthesis in vitro. Journal of Virology 68:6900–6909
    [Google Scholar]
  4. Beloso A., Martínez C., Valcárcel J., Fernández-Santarén J., Ortín J. 1992; Degradation ofcellularmRNA during influenza virus infection: its possible role in protein synthesis shutoff. Journal of General Virology 73:575–581
    [Google Scholar]
  5. Biswas S. K., Nayak D. P. 1994; Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein 1. Journal of Virology 68:1819–1826
    [Google Scholar]
  6. Blaas D., Patzelt E., Keuchler E. 1982; Identification of the cap binding protein of influenza virus. Nucleic Acids Research 10:4803–4812
    [Google Scholar]
  7. Braam J., Ulmanen I., Krug R. M. 1983; Molecular model of a eucaryotic transcription complex: functions and movements of influenza P proteins during capped RNA-primed transcription. Cell 34:609–618
    [Google Scholar]
  8. de la Luna S., Martín J., Portela A., Ortín J. 1993; Influenza virus naked RNA can be expressed upon transfection into cells co-expressing the three subunits of the polymerase and the nucleoprotein from SV40 recombinant viruses. Journal of General Virology 74:535–539
    [Google Scholar]
  9. Edelman A. M., Blumenthal D. K., Krebs E. G. 1987; Protein serine/threonine kinases. Annual Review of Biochemistry 56:567–613
    [Google Scholar]
  10. Fuerst T. R., Earl P. L., Moss B. 1987; Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes. Molecular and Cellular Biology 7:2538–2544
    [Google Scholar]
  11. Gluzman Y. 1981; SV40 transformed simian cells support the replication or early SV40 mutants. Cell 23:175–182
    [Google Scholar]
  12. Gonzalez S., Zurcher T., Ortín J. 1996; Identification of two separate domains in the influenza virus PB1 protein involved in the interaction with the PB2 and PA subunits : a model for the viral RNA polymerase structure. Nucleic Acids Research 24:4456–4463
    [Google Scholar]
  13. Grankowski N., Boldyreff B., Issinger O.-G. 1991; Isolation and characterization of recombinant human casein kinase II subunits α and β from bacteria. European Journal of Biochemistry 198:25–30
    [Google Scholar]
  14. Gregoriades A., Christie T., Markarian K. 1984; The membrane (M1) protein of influenza virus occurs in two forms and is a phosphoprotein. Journal of Virology 49:229–235
    [Google Scholar]
  15. Hagen M., Chung T. D., Butcher J. A., Krystal M. 1994; Recombinant influenza virus polymerase: requirement of both 5ʹ and 3ʹ viral ends for endonuclease activity. Journal of Virology 68:1509–1515
    [Google Scholar]
  16. Horisberger M. A. 1980; The large P proteins of influenza A viruses are composed of one acidic and two basic polypeptides. Virology 107:302–305
    [Google Scholar]
  17. Kemp B. E., Pearson R. B. 1990; Protein kinase recognition sequence motifs. Trends in Biochemical Sciences 15:342–346
    [Google Scholar]
  18. Kistner O., Müller H., Becht H., Scholtissek C. 1985; Phospho- peptide fingerprints of nucleoprotein of various influenza A virus strains grown in different host cells. Journal of General Virology 66:465–472
    [Google Scholar]
  19. Krug R. M. 1989 The Influenza Viruses New York: Plenum Press;
    [Google Scholar]
  20. Lamb R. A. 1989; The genes and proteins of influenza viruses. In The Influenza Viruses pp 1–87 Krug R. M. Edited by New York: Plenum Press;
    [Google Scholar]
  21. Laskey R. A., Mills A. D. 1975; Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. European Journal of Biochemistry 56:335–341
    [Google Scholar]
  22. Licheng S., Summers D. F., Peng Q., Galarza J. M. 1995; Influenza A virus polymerase subunit PB2 is the endonuclease which cleaves host cell mRNA and functions only as the trimeric enzyme. Virology 208:38–47
    [Google Scholar]
  23. Mahy B. W. J. 1983; Mutants of influenza virus. In Genetics of Influenza Viruses pp 192–253 Palese P., Kingsbury D. W. Edited by Wien: Springer Verlag;
    [Google Scholar]
  24. Marión R.Ma , Zürcher T., de la Luna S., Ortín J. 1997; Influenza virus NS1 protein interacts with viral transcription-replication complexes in vivo . Journal of General Virology 78:2447–2451
    [Google Scholar]
  25. Mena I., de la Luna S., Albo C., Martín J., Nieto A., Ortín J., Portela A. 1994; Synthesis of biologically active influenza virus core proteins using a vaccinia-T7 RNA polymerase expression system. Journal of General Virology 75:2109–2114
    [Google Scholar]
  26. Nieto A., de la Luna S., Bárcena J., Portela A., Valcárcel J., Melero J. A., Ortín J. 1992; Nuclear transport of influenza virus polymerase PA protein. Virus Research 24:65–75
    [Google Scholar]
  27. O’Farrell P. H. 1975; High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemistry 250:4007–4021
    [Google Scholar]
  28. Ortín J., Nájera R., López C., Dávila M., Domingo E. 1980; Genetic variability of Hong Kong (H3N2) influenza viruses: spontaneous mutations and their location in the viral genome. Gene 11:319–331
    [Google Scholar]
  29. Perlmutter R. M., Marth J. D., Ziegler S. F., Garvin A. M., Pawar S., Cooke M. P., Abraham K. M. 1988; Specialized protein tyrosine kinase proto-oncogen in hematopoietic cells. Biochimica et Biophysica Acta 948:245–262
    [Google Scholar]
  30. Poch O., Sauvaget I., Delarue M., Tordo N. 1990; Identification of four conserved motifs among the RNA-dependent polymerases encoding elements. EMBO Journal 8:3867–3874
    [Google Scholar]
  31. Privalsky M. L., Penhoet E. E. 1978; Influenza virus proteins: identity, synthesis and modification analyzed by two-dimensional gel electrophoresis. Proceedings of the National Academy of Sciences, USA 75:3625–3629
    [Google Scholar]
  32. Privalsky M. L., Penhoet E. E. 1981; The structure and synthesis of influenza virus phosphoproteins. Journal of Biological Chemistry 256:5368–5376
    [Google Scholar]
  33. Richardson J. C., Akkina R. K. 1991; NS2 protein of influenza virus is found in purified virus and phosphorylated in infected cells. Archives of Virology 116:69–80
    [Google Scholar]
  34. Santarén R. J., Bravo R. 1987; Immediate induction of a 45K secreted glycoprotein by serum and growth factors in quiescent mouse 3T3 cells. Two-dimensional gel analysis. Experimental Cell Research 168:494–506
    [Google Scholar]
  35. Sanz-Ezquerro J. J., de la Luna S., Ortín J., Nieto A. 1995; Individual expression of influenza virus PA protein induces degradation of coexpressed proteins. Journal of Virology 69:2420–2426
    [Google Scholar]
  36. Sanz-Ezquerro J. J., Zürcher T., de la Luna S., Ortín J., Nieto A. 1996; The induction of proteolysis mediated by influenza virus PA protein maps to the amino-terminal third of the protein. Journal of Virology 70:1905–1911
    [Google Scholar]
  37. Towbin H., Staehlin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets : procedure and some applications. Proceedings of the National Academy of Sciences, USA 76:4350–4354
    [Google Scholar]
  38. Tucker S. P., Penn R. P., McCauley J. W. 1990; Characterization of the influenza virus associated protein kinase and its resemblance to casein kinase II. Virus Research 18:243–262
    [Google Scholar]
  39. Ulmanen I., Broni B. A., Krug R. M. 1981; Role of two of the influenza virus core P proteins in recognizing cap 1 structures (m7GpppNm) on RNAs and in initiating viral RNA transcription. Proceedings of the National Academy of Sciences, USA 78:7355–7359
    [Google Scholar]
  40. Vogel U., Kunerl M., Scholtissek C. 1994; Influenza A virus late mRNAs are specifically retained in the nucleus in the presence of a methyltransferase or a protein kinase inhibitor. Virology 198:227–233
    [Google Scholar]
  41. Whittaker G., Kemler I., Helenius A. 1995; Hyperphosphorylation of mutants influenza virus matrix protein, M1, causes its retention in the nucleus. Journal of Virology 69:439–445
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-79-3-471
Loading
/content/journal/jgv/10.1099/0022-1317-79-3-471
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