1887

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Volume 69, Issue 9

Semliki Forest Virus-specific Non-structural Protein nsP3 Is a Phosphoprotein Free

Abstract

Summary

Antisera were raised in rabbits against fusion proteins consisting of β-galactosidase and partial amino acid sequences of Semliki Forest virus (SFV)-specific non-structural proteins nsP1, nsP2, nsP3 and nsP4. The antisera were specific since each of them precipitated only one labelled protein of a size expected for nsP1, nsP2, nsP3 or nsP4 from lysates of [S]methionine-labelled SFV-infected BHK-21 cells. The specific antisera also precipitated p220 (with sequences of nsP1, nsP2 and nsP3), p155 (nsP1 and nsP2) and p135 (nsP3 and nsP4) which have been previously shown to be cleavage products of the polyprotein precursor of the non-structural proteins. nsP1, nsP4 and most of nsP3, together with the virus-specific RNA polymerase activity, were in the mitochondrial pellet (P15) fraction of infected BHK-21 cells whereas nsP2 was evenly distributed between P15 and the supernatant fraction (S15). Only antisera directed against nsP3 sequences precipitated a labelled protein from cells incubated with [P]orthophosphate during SFV infection. Treatment of the immunoprecipitate with calf alkaline intestinal phosphatase reduced the amount of labelled nsP3 considerably. Immunoprecipitated P-labelled nsP3, isolated by SDS-PAGE, was subjected to acid hydrolysis. Both phosphoserine and phosphothreonine but not phosphotyrosine could be identified in the hydrolysate. Approximately twice as much [P]serine as [P]threonine was detected in nsP3. P15 and S15 fractions were prepared from [S]methionine- and P-labelled SFV-infected cells and the S/P ratio of nsP3 was determined after immunoprecipitation and SDS-PAGE. The nsP3 in S15 was less heavily phosphorylated (about 50%) than P15-associated nsP3. Anti-nsP3 serum revealed large cytoplasmic vesicles in SFV-infected cells in indirect immunofluorescence microscopy.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): phosphoprotein , RNA polymerase and Semliki Forest virus
© Society for General Microbiology 1988
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1988-09-01
2024-04-20
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References

  1. ACHESON N. H., TAMM I. 1967; Replication of Semliki Forest virus: an electron microscopic study. Virology 32:123–143
     [Google Scholar]
  2. ALMOND J. W., FELSENREICH V. 1982; Phosphorylation of the nucleoprotein of an avian influenza virus. Journal of General Virology 60:295–305
     [Google Scholar]
  3. BANERJEE A. K. 1987; Transcription and replication of rhabdoviruses. Microbiological Reviews 51:66–87
     [Google Scholar]
  4. BELL J. C., PREVEC L. 1985; Phosphorylation sites on phosphoprotein NS of vesicular stomatitis virus. Journal of Virology 54:697–702
     [Google Scholar]
  5. BISHOP J. M. 1983; Cellular oncogenes and retroviruses. Annual Review of Biochemistry 52:301–354
     [Google Scholar]
  6. BISHOP J. M. 1985; Viral oncogenes. Cell 42:23–38
     [Google Scholar]
  7. BONNER W. M., LASKEY R. A. 1974; A film detection method for tritium-labeled proteins and nucleic acids in Polyacrylamide gels. European Journal of Biochemistry 46:83–88
     [Google Scholar]
  8. CHANG J. J. 1984; Analysis of phospho-amino acids and amino acid amides at the picomole level using 4′-dimethylaminoazobenzene-4-sulphonyl chloride. Journal of Chromatography 295:193–200
     [Google Scholar]
  9. CHATTOPADHYAY D., BANERJEE A. K. 1987; Phosphorylation within a specific domain of the phosphoprotein of vesicular stomatitis virus regulates transcription in vitro. Cell 49:407–417
     [Google Scholar]
  10. CHOCK P. B., RHEE S. G., STADTMAN E. R. 1980; Interconvertible enzyme cascades in cellular regulation. Annual Review of Biochemistry 49:813–843
     [Google Scholar]
  11. CLEWLEY J. P., KENNEDY S. I. T. 1976; Purification and polypeptide composition of Semliki Forest virus RNA polymerase. Journal of General Virology 32:395–411
     [Google Scholar]
  12. COHEN P. 1982; The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature, London 296:613–620
     [Google Scholar]
  13. CROSS R. K. 1983; Identification of a unique guanine-7-methyltransferase in Semliki Forest virus (SFV) infected cell extracts. Virology 130:452–463
     [Google Scholar]
  14. CROSS R. K., GOMATOS P. J. 1981; Concomitant methylation and synthesis in vitro of Semliki Forest virus (SFV) ss RNAs by a fraction from infected cells. Virology 114:542–554
     [Google Scholar]
  15. ERIKSON E., BRUGGE J., ERIKSON R. 1977; Phosphorylated and non-phosphorylated forms of avian sarcoma virus polypeptide pl9. Virology 80:177–185
     [Google Scholar]
  16. FRIEDMAN R. M., BEREZESKY I. K. 1967; Cytoplasmic fractions associated with Semliki Forest virus ribonucleic acid replication. Journal of Virology 1:274–383
     [Google Scholar]
  17. GAROFF H. 1985; Using recombinant DNA techniques to study protein targeting in the eucaryotic cell. Annual Review of Cell Biochemistry 1:403–445
     [Google Scholar]
  18. GAROFF H., FRISCHAUF A.-M., SIMONS K., LEHRACH H., DELIUS H. 1980a; The capsid protein of Semliki Forest virus has clusters of basic amino acids and prolines in its amino terminal region. Proceedings of the National Academy of Sciences, U.S.A 77:6376–6380
     [Google Scholar]
  19. GAROFF H., FRISCHAUF A.-M., SIMONS K., LEHRACH H., DELIUS H. 1980b; Nucleotide sequence of cDNA coding for Semliki Forest virus membrane glycoproteins. Nature, London 288:236–241
     [Google Scholar]
  20. GILL D. S., CHATTOPADHYAY D., BANERJEE A. K. 1986; Identification of a domain within the phosphoprotein of vesicular stomatitis virus that is essential for transcription in vitro. Proceedings of the National Academy of Sciences, U.S.A 83:8873–8877
     [Google Scholar]
  21. GLANVILLE N., RANKI M., MORSER J., KÄÄRIÄINEN L., SMITH A. E. 1976; Initiation of translation directed by 42S and 26S RNAs from Semliki Forest virus in vitro. Proceedings of the National Academy of Sciences, U.S.A 73:3059–3063
     [Google Scholar]
  22. GOMATOS P. J., KÄÄRIÄINEN L., KERÄNEN S., RANKI M., SAWICKI D. L. 1980; Semliki Forest virus replication complex capable of synthesizing 42S and 26S nascent RNA chains. Journal of General Virology 49:61–69
     [Google Scholar]
  23. GREEN M. 1985 Transformation and oncogenesis: DNA viruses. Virology183–234 Edited by Fields B. N. New York: Raven Press;
     [Google Scholar]
  24. GREGORIADES A., CHRISTIE T., MARKARIAN K. 1984; The membrane (M) protein of influenza virus occurs in two forms and is a phosphoprotein. Journal of Virology 49:229–235
     [Google Scholar]
  25. GRIMLEY P. M., BEREZESKY I. K., FRIEDMAN R. M. 1968; Cytoplasmic structures associated with an arbovirus infection: loci of viral ribonucleic acid synthesis. Journal of Virology 2:1326–1338
     [Google Scholar]
  26. GRIMLEY P. M., LEVIN J. G., BEREZESKY I. K., FRIEDMAN R. M. 1972; Specific membranous structures associated with the replication of group A arboviruses. Journal of Virology 10:492–503
     [Google Scholar]
  27. HARDY R. W. A, STRAUSS J. H. 1988; Processing the nonstructural polyproteins of Sindbis virus: study of the kinetics in vivo by using monospecific antibodies. Journal of Virology 62:998–1007
     [Google Scholar]
  28. HUNTER T., SEFTON B. M. 1980; Transforming gene of Rous sarcoma virus phosphorylates tyrosine. Proceedings of the National Academy of Sciences, U.S.A 77:1311–1315
     [Google Scholar]
  29. IKUTA K., LUFTIG R. B. 1988; Detection of phosphorylated forms of Moloney murine leukemia virus major capsid protein p30 by immunoprecipitation and two-dimensional gel electrophoresis. Journal of Virology 62:40–46
     [Google Scholar]
  30. JALANKO A., SÖDERLUND H. 1986; The repeated regions of Semliki Forest virus defective-interfering RNA interfere with the encapsidation process of the standard virus. Virology 141:257–266
     [Google Scholar]
  31. JEPPESEN P. G. N. 1980; Separation of DNA fragments using linear Polyacrylamide gradient gel electrophoresis. Methods in Enzymology 65:305–319
     [Google Scholar]
  32. KÄÄRIÄINEN L., SAWICKI D., GOMATOS P. J. 1978; Cleavage defect in the non-structural polyprotein of Semliki Forest virus has two separate effects on virus RNA synthesis. Journal of General Virology 39:463–473
     [Google Scholar]
  33. KÄÄRIÄINEN L., TAKKINEN K., KERÄNEN S., SÖDERLUND H. 1987; Replication of the genome of alphaviruses. Journal of Cell Science 7:231–250
     [Google Scholar]
  34. KALKKINEN N. 1986 Radio-sequence analysis; an ultra-sensitive method to align proteins and nucleotide sequences. Advanced Methods in Protein Microsequence Analysis194–206 Edited by Wittmann-Liebold B., Salnikow J., Erdman V. A. Wien & New York: Springer-Verlag;
     [Google Scholar]
  35. KALKKINEN N., LAAKSONEN M., SÖDERLUND H., JÖRNVALL H. 1981; Radio-sequence analysis of in vivo multilabeled nonstructural protein ns86 of Semliki Forest virus. Virology 113:188–195
     [Google Scholar]
  36. KERÄNEN S., KÄÄRIÄINEN L. 1974; Isolation and basic characterization of temperature-sensitive mutants from Semliki Forest virus. Acta pathologica et microbiologica scandinavica B 82:810–820
     [Google Scholar]
  37. KERÄNEN S., KÄÄRIÄINEN L. 1979; Functional defects of RNA-negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses. Journal of Virology 32:19–29
     [Google Scholar]
  38. KERÄNEN S., RUOHONEN L. 1983; Nonstructural proteins of Semliki Forest virus: synthesis, processing and stability in infected cells. Journal of Virology 47:505–515
     [Google Scholar]
  39. KREBS E. G., BEAVO J. A. 1979; Phosphorylation -dephosphorylation of enzymes. Annual Review of Biochemistry 48:923–959
     [Google Scholar]
  40. KONDOR-KOCH C, BURKE B., GAROFF H. 1983; Expression of Semliki Forest virus proteins from cloned complementary DNA. I. The fusion activity of the spike glycoprotein. Journal of Cell Biology 97:644–651
     [Google Scholar]
  41. KUISMANEN E., HEDMAN K., SARASTE J., PETTERSSON R. F. 1982; Uukuniemi virus maturation: accumulation of virus particles and viral antigens in the Golgi complex. Molecular and Cellular Biology 2:1444–1458
     [Google Scholar]
  42. KUISMANEN E., BANG B., HURME M., PETTERSSON R. F. 1984; Uukuniemi virus maturation: immunofluorescence microscopy with monoclonal glycoprotein-specific antibodies. Journal of Virology 51:137–146
     [Google Scholar]
  43. LACHMI B., KÄÄRIÄINEN L. 1976; Sequential translation of nonstructural proteins in cells infected with a Semliki Forest virus mutant. Proceedings of the National Academy of Sciences, U.S.A 73:1936–1940
     [Google Scholar]
  44. LACHMI B., KÄÄRIÄINEN L. 1977; Control of protein synthesis in Semliki Forest virus infected cells. Journal of Virology 22:142–149
     [Google Scholar]
  45. LAEMMLI U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227:680–685
     [Google Scholar]
  46. LAI M. M. C. 1976; Phosphoproteins of Rous sarcoma virus. Virology 74:287–301
     [Google Scholar]
  47. LAMB R. A., CHOPPIN P. W. 1977; The synthesis of Sendai virus polypeptides in infected cells. III. Phosphorylation of polypeptides. Virology 81:382–397
     [Google Scholar]
  48. LEHTOVAARA P., ULMANEN I., KÄÄRIÄINEN L., KERÄNEN S., PHILIPSON L. 1980; Synthesis and processing of Semliki Forest virus-specific nonstructural proteins in vivo and in vitro. European Journal of Biochemistry 112:461–468
     [Google Scholar]
  49. LEIS J., JENTOFT J. 1983; Characteristics and regulation of avian retrovirus ppl2 protein with viral RNA. Journal of Virology 48:361–369
     [Google Scholar]
  50. LEIS J., JOHNSON S., COLLINS L. S., TRAUGH J. A. 1984; Effects of phosphorylation of avian retrovirus nucleocapsid protein ppl2 on binding of viral RNA. Journal of Biological Chemistry 259:7726–7732
     [Google Scholar]
  51. LEVIS R., WEISS B. G., TSIANG M., HUANG H., SCHLESINGER S. 1986; Deletion mapping of Sindbis virus DI RNAs derived from cDNA defines the sequences essential for replication and packaging. Cell 44:137–145
     [Google Scholar]
  52. LEVIS R., HUANG H., SCHLESINGER S. 1987; Engineered defective interfering RNAs of Sindbis virus express bacterial chloramphenicol acetyltransferase in avian cells. Proceedings of the National Academy of Sciences, U.S.A 84:4811–4815
     [Google Scholar]
  53. MANIATIS T., FRITSCH E. F., SAMBROOK J. 1982 Molecular Cloning: A Laboratory Manual New York: Cold Spring Harbor Laboratory;
     [Google Scholar]
  54. MAXAM A. M., GILBERT W. 1980; Sequencing end-labeled DNA with base-specific chemical cleavages. Methods in Enzymology 65:499–560
     [Google Scholar]
  55. MELANCON P., GAROFF H. 1986; Reinitiation of translocation in the Semliki Forest virus structural polyprotein: identification of the signal sequence for the E1 glycoprotein. EMBO Journal 6:1543–1550
     [Google Scholar]
  56. MELANCON P., GAROFF H. 1987; Processing of the Semliki Forest virus structural polyprotein: role of the capsid protease. Journal of Virology 61:1301–1309
     [Google Scholar]
  57. MOHR I. J., STILLMAN B., GLUZMAN Y. 1987; Regulation of SV40 DNA replication by phosphorylation of T-antigen. EMBO Journal 7:153–160
     [Google Scholar]
  58. PAL B. K., ROY-BURMAN P. 1975; Phosphoproteins: structural components of oncornaviruses. Journal of Virology 15:540–549
     [Google Scholar]
  59. PAL B. K., MCALLISTER R. M., GARDNER M. B., ROY-BURMAN P. 1975; Comparative studies of the structural phosphoproteins of mammalian type C viruses. Journal of Virology 16:123–131
     [Google Scholar]
  60. PRIVALSKY M. L., PENHOET E. E. 1981; The structure and synthesis of influenza virus phosphoproteins. Journal of Biological Chemistry 256:5368–5376
     [Google Scholar]
  61. RANKI M., KÄÄRIÄINEN L. 1979; Solubilized RNA replication complex from Semliki Forest virus-infected cells. Virology 98:298–307
     [Google Scholar]
  62. RANKI M., ULMANEN I., KÄÄRIÄINEN L. 1979; Semliki Forest virus-specific nonstructural protein is associated with ribosomes. FEBS Letters 108:299–302
     [Google Scholar]
  63. RICE C. M., FRANKE C. A., STRAUSS J. H., HRUBY D. E. 1985; Expression of Sindbis virus structural proteins via recombinant vaccinia virus: synthesis, processing and incorporation into mature Sindbis virions. Journal of Virology 56:227–239
     [Google Scholar]
  64. RICE C. M., LEVIS R., STRAUSS J. H., HUANG H. V. 1987; Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. Journal of Virology 61:3809–3819
     [Google Scholar]
  65. SARASTE J., KÄÄRIÄINEN L., SÖDERLUND H., KERÄNEN S. 1977; RNA synthesis directed by a temperature-sensitive mutant of Semliki Forest virus. Journal of General Virology 37:399–406
     [Google Scholar]
  66. SAWICKI D. L., KÄÄRIÄINEN L., LAMBEK C., GOMATOS P. J. 1978; Mechanism for control of synthesis of Semliki Forest virus 26S and 42S RNA. Journal of Virology 25:19–27
     [Google Scholar]
  67. SAWICKI D. L., SAWICKI S. G., KERÄNEN S., KÄÄRIÄINEN L. 1981a; Specific Sindbis virus-coded function for minus-strand RNA synthesis. Journal of Virology 39:348–358
     [Google Scholar]
  68. SAWICKI S. G., SAWICKI D. L., KÄÄRIÄINEN L., KERÄNEN S. 1981b; A Sindbis virus mutant temperature-sensitive in the regulation of minus-strand RNA synthesis. Virology 115:161–172
     [Google Scholar]
  69. SCHLESINGER M. J., SCHLESINGER S. (editors) 1986 The Togaviridae and Flaviviridae New York & London: Plenum Press;
     [Google Scholar]
  70. SOKOL F., CLARK H F. 1973; Phosphoproteins, structural components of rhabdoviruses. Virology 52:246–263
     [Google Scholar]
  71. STANLEY K. K., LUZIO J. P. 1984; Construction of a new family of high-efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins. EMBO Journal 3:1429–1434
     [Google Scholar]
  72. STRAUSS E. G., STRAUSS J. H. 1983; Replication strategies of the single stranded RNA viruses of eukaryotes. Current Topics in Microbiology and Immunology 105:1–98
     [Google Scholar]
  73. STRAUSS E. G., STRAUSS J. H. 1986 Structure and replication of the alphavirus genome. The Togaviridae and Flaviviridae35–90 Edited by Schlesinger S., Schlesinger M. J. New York & London: Plenum Press;
     [Google Scholar]
  74. STRAUSS E. G., RICE C M., STRAUSS J. H. 1984; Complete nucleotide sequence of the genomic RNA of Sindbis virus. Virology 133:92–110
     [Google Scholar]
  75. TAKKINEN K. 1986; Complete nucleotide sequence of the nonstructural protein genes of Semliki Forest virus. Nucleic Acids Research 14:5667–5682
     [Google Scholar]
  76. TEGTMEYER P., RANDELL K., COLLINS S. K. 1977; Modification of simian virus 40 protein A. Journal of Virology 21:647–657
     [Google Scholar]
  77. TSIANG M., MONROE S. S., SCHLESINGER S. 1985; Studies of defective interfering RNAs of Sindbis virus with and without tRNAAsP sequences at their 5′ termini. Journal of Virology 54:38–44
     [Google Scholar]
  78. TSIANG M., WEISS B. G., SCHLESINGER S. 1988; Effects of 5′-terminal modifications on biological activity of defective interfering RNAs of Sindbis virus. Journal of Virology 62:47–53
     [Google Scholar]
  79. WAITE M. R. F., LUBIN M., JONES K., BOSE H. R. 1974; Phosphorylated proteins of Sindbis virus. Journal of Virology 13:244–246
     [Google Scholar]
  80. WEN D., SCHLESINGER M. J. 1986; Regulated expression of Sindbis and vesicular stomatitis virus glycoproteins in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences, U.S.A 83:3639–3643
     [Google Scholar]
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Semliki Forest Virus-specific Non-structural Protein nsP3 Is a Phosphoprotein
J. Gen. Virol. 69, 2165 (1988); https://doi.org/10.1099/0022-1317-69-9-2165
/content/journal/jgv/10.1099/0022-1317-69-9-2165
/content/journal/jgv/10.1099/0022-1317-69-9-2165
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