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Volume 64, Issue 10

Herpes Simplex Virus Non-structural Proteins. IV. Purification of the Virus-induced Deoxyribonuclease and Characterization of the Enzyme Using Monoclonal Antibodies Free

Abstract

SUMMARY

The alkaline nucleases induced by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) have been purified from high salt extracts of virus-infected cells. The purification used three types of column chromatography and resulted in apparently homogeneous DNase preparations with good recovery. The enzyme from HSV-2-infected cells has been characterized. It had both exonuclease and endonuclease activity, each with an unusually high pH optimum. The enzyme had an absolute requirement for magnesium which could not be replaced by other divalent cations. Analysis of the sedimentation characteristics and electrophoretic properties of the purified enzyme indicated that it was composed of a single subunit of mol. wt. 85000. The purified HSV-2 enzyme was used as an immunogen to prime BALB/c mice which were used to prepare monoclonal antibodies. Three monoclonal antibodies were shown by several criteria to react with the enzyme. Thus, we were able to confirm that the 85K polypeptide did indeed have nuclease activity. This polypeptide was designated ICSP 22 in earlier studies and is a major polypeptide of virus-infected cells.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): HSV-1 and -2 , induced enzyme , monoclonal antibodies and nuclease
© Journal of General Virology 1983
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1983-10-01
2024-04-18
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References

  1. 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]
  2. Fahey J. L., Horbett A. P. 1959; Human gamma globulin fractionation on anion exchange cellulose columns. Journal of Biological Chemistry 234:2645–2651
     [Google Scholar]
  3. Francke B., Garrett B. 1982; The effect of a temperature-sensitive lesion in the alkaline DNase of herpes simplex virus type 2 on the synthesis of viral DNA. Virology 116:116–127
     [Google Scholar]
  4. Francke B., Moss H., Timbury M. C., Hay J. 1978; Alkaline DNase activity in cells infected with a temperature-sensitive mutant of herpes simplex virus type 2. Journal of Virology 26:209–213
     [Google Scholar]
  5. Hoffman P. J., Cheng Y. 1978; The deoxyribonuclease induced after infection of KB cells by herpes simplex virus type 1 or type 2. Journal of Biological Chemistry 253:3557–3562
     [Google Scholar]
  6. Hoffman P. J., Cheng Y. 1979; DNase induced after infection of KB cells by herpes simplex virus type 1 or type 2. Journal of Virology 32:449–457
     [Google Scholar]
  7. Honess R. W., Roizman B. 1973; Proteins specified by herpes simplex virus. XI. Identification and relative molar rates of synthesis of structural and non-structural herpes virus polypeptides in the infected cell. Journal of Virology 12:1347–1365
     [Google Scholar]
  8. Honess R. W., Watson D. H. 1974; Absence of a requirement for host polypeptides in the herpes virus thymidine kinase. Journal of General Virology 24:215–220
     [Google Scholar]
  9. Keir H. M. 1968; Virus-induced enzymes in mammalian cells infected with DNA-viruses. In The Molecular Biology of Viruses pp 67–99 Edited by Crawford L. V., Stoker M. G. P. Cambridge: Cambridge University Press;
     [Google Scholar]
  10. Keir H. M., Gold E. 1963; Deoxyribonucleic acid nucleotidyltransferase and deoxyribonuclease from cultured cells infected with herpes simplex virus. Biochimica et biophysica acta 72:263–276
     [Google Scholar]
  11. Kennett R. H., Denis K. A., Tung A. S., Klinman N. R. 1978; Hybrid plasmacytoma production: fusion with adult spleen cells, monoclonal spleen fragments, neonatal spleen cells and human spleen cells. Current Topics in Microbiology and Immunology 81:77–91
     [Google Scholar]
  12. Killington R. A., Newhook L., Balachandran N., Rawls W. E., Bacchetti S. 1981; Production of hybrid cell lines secreting antibodies to herpes simplex virus type 2. Journal of Virological Methods 2:223–236
     [Google Scholar]
  13. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. 1951; Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193:265–275
     [Google Scholar]
  14. Morrison J. M., Keir H. M. 1968; A new DNA-exonuclease in cells infected with herpes virus: partial purification and properties of the enzyme. Journal of General Virology 3:337–347
     [Google Scholar]
  15. Moss H., Chartrand P., Timbury M. C., Hay J. 1979; Mutant of herpes simplex virus type 2 with temperature-sensitive lesions affecting virion thermostability and DNase activity: identification of the lethal mutation and physical mapping of the nuc− lesion. Journal of Virology 32:140–146
     [Google Scholar]
  16. Powell K. L., Courtney R. J. 1975; Polypeptides synthesised in herpes simplex virus type 2 infected Hep-2 cells. Virology 66:217–228
     [Google Scholar]
  17. Powell K. L., Purifoy D. J. M. 1977; Nonstructural proteins of herpes simplex virus. I. Purification of the induced DNA polymerase. Journal of Virology 24:618–626
     [Google Scholar]
  18. Preston C. M., Cordingley M. G. 1982; mRNA and DNA-directed synthesis of herpes simplex virus-coded exonuclease in Xenopus laevis oocytes. Journal of Virology 43:386–394
     [Google Scholar]
  19. Purifoy D. J. M., Benyesh-Melnick M. 1975; DNA polymerase induction by DNA negative temperature-sensitive mutants of herpes simplex virus type 2. Virology 68:374–386
     [Google Scholar]
  20. Purifoy D. J. M., Powell K. L. 1976; DNA binding proteins induced by herpes simplex virus type 2 in Hep-2 cells. Journal of Virology 19:717–731
     [Google Scholar]
  21. Rawls W. E., Laurel D., Melnick J. L., Glicksman J. M., Kaufman R. H. 1968; A search for viruses in smegma, premalignant and early malignant cervical tissues. The isolation of herpesviruses with distinct antigenic properties. American Journal of Epidemiology 87:647–655
     [Google Scholar]
  22. Schulman M., Wilde C. D., Köhler G. 1978; A better cell line for making hybridomas secreting specific antibodies. Nature, London 276:269–270
     [Google Scholar]
  23. Strobel-Fidler M., Francke B. 1980; Alkaline deoxyribonuclease induced by herpes simplex virus type 1: composition and properties of the purified enzyme. Virology 103:493–501
     [Google Scholar]
  24. Watson D. H., Sheddon W. I. H., Elliott A., Tetsuka T., Wildy P., Bourgaux-Ramoisy D., Gold E. 1966; Virus specific antigens in mammalian cells infected with herpes simplex virus. Immunology 11:399–408
     [Google Scholar]
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Herpes Simplex Virus Non-structural Proteins. IV. Purification of the Virus-induced Deoxyribonuclease and Characterization of the Enzyme Using Monoclonal Antibodies
J. Gen. Virol. 64, 2249 (1983); https://doi.org/10.1099/0022-1317-64-10-2249
/content/journal/jgv/10.1099/0022-1317-64-10-2249
/content/journal/jgv/10.1099/0022-1317-64-10-2249
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