Herpes Simplex Virus-induced Ribonucleotide Reductase: Development of Antibodies Specific for the Enzyme Free

Abstract

SUMMARY

We have previously reported on the characterization of a novel ribonucleotide reductase induced in herpes simplex virus type 2 (HSV-2)-infected mammalian cells. The virus-induced enzyme was partially purified, free of the constitutive cell isozyme, by fractionation of infected cell extracts with ammonium sulphate. In this report we describe a further purification of the virus-induced enzyme and the development of a rabbit antiserum capable of specifically inhibiting its activity. Enzymically active salt fractions from infected cell extracts were sedimented through glycerol gradients; the virus-induced enzyme was found to sediment approximately 2.5 times faster than the constitutive, or control, enzyme and was separated from the majority of the protein in the sample. Immunization of rabbits with the partially purified enzyme preparation recovered from gradients resulted in the synthesis of antibodies which completely and specifically inhibited the virus-induced reductase, and precipitated one major polypeptide and a few minor species from both HSV-1- and HSV-2-infected cells. The antibodies, however, exhibited much greater affinity for the HSV-2 than the HSV-1 antigen. These results demonstrate that the virus-induced enzyme differs antigenically, as well as biochemically, from the constitutive cell isozyme and lend further support to the hypothesis that the enzyme is virus-coded.

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/content/journal/jgv/10.1099/0022-1317-64-6-1327
1983-06-01
2024-03-29
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References

  1. Allen G. P., Cohen J. C., Randall C. C., O’Callaghan D. J. 1978; Replication of equine herpesvirus type 1 and type 2: resistance to hydroxyurea and thymidine. Intervirology 9:276–285
    [Google Scholar]
  2. Balachandran N., Harnish D., Killington R. A., Bacchetti S., Rawls W. E. 1981; Monoclonal antibodies to two glycoproteins of herpes simplex virus type 2. Journal of Virology 39:438–446
    [Google Scholar]
  3. Banerjee A. K., Rhodes D. P. 1973; In vitro synthesis of RNA that contains polyadenylate by virion-associated RNA polymerase of vesicular stomatitis virus. Proceedings of the National Academy of Sciences, U,. S,. A 70:3566–3570
    [Google Scholar]
  4. Blue W. T., Stobbs D. G. 1981; Isolation of a protein kinase induced by herpes simplex virus type 1. Journal of Virology 38:383–388
    [Google Scholar]
  5. Chang C-H., Cheng Y. C. 1979; Demonstration of two components and association of adenosine diphosphate-cytidine diphosphate reductase from cultured human lymphoblast cells (molt-4F). Cancer Research 39:436–442
    [Google Scholar]
  6. Cohen J. C., Henry B. E., Randall C. C., O’Callaghan D. J. 1977; Ribonucleotide reductase activity in hydroxyurea resistant herpesvirus replication. Proceedings of the Society for Experimental Biology and Medicine 155:395–399
    [Google Scholar]
  7. Engstrom Y., Eriksson S., Thelander L., Akerman M. 1979; Ribonucleotide reductase from calf thymus. Purification and properties. Biochemistry 18:2941–2948
    [Google Scholar]
  8. Henry B. E., Glaser R., Hewetson J., O’Callaghan D. J. 1978; Expression of altered ribonucleotide reductase activity associated with the replication of the Epstein-Barr virus. Virology 89:262–271
    [Google Scholar]
  9. Holmgren A. 1981; Regulation of ribonucleotide reductase. Current Topics in Cellular Regulation 19:47–76
    [Google Scholar]
  10. Huszar D., Bacchetti S. 1981; Partial purification and characterization of the ribonucleotide reductase induced by herpes simplex virus infection of mammalian cells. Journal of Virology 37:580–588
    [Google Scholar]
  11. Keir H. M., Subak-Sharpe H., Shedden W. I. H., Watson D. H., Wildy P. 1966; Immunological evidence for a specific DNA polymerase produced after infection by herpes simplex virus. Virology 30:154–157
    [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. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227:680–685
    [Google Scholar]
  14. Langelier Y., Buttin G. 1981; Characterization of ribonucleotide reductase induction in BHK-21/C13 Syrian hamster cell line upon infection by herpes simplex virus (HSV). Journal of General Virology 57:21–31
    [Google Scholar]
  15. Langelier Y., Dechamps M., Buttin G. 1978; Analysis of dCMP deaminase and CDP reductase levels in hamster cells infected with herpes simplex virus. Journal of Virology 26:547–553
    [Google Scholar]
  16. Lankinen H., Graslund A., Thelander L. 1982; Induction of a new ribonucleotide reductase after infection of mouse L cells with pseudorabies virus. Journal of Virology 41:893–900
    [Google Scholar]
  17. 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 3:337–374
    [Google Scholar]
  18. Munyon W., Buchsbaum R., Paoletti E., Mann I., Kraiselburd E., Davis D. 1972; Electrophoresis of thymidine kinase activity synthesized by cells transformed by herpes simplex virus. Virology 49:683–689
    [Google Scholar]
  19. Ponce De Leon M., Eisenberg R. J., Cohen G. H. 1977; Ribonucleotide reductase from herpes simplex virus (types 1 and 2) infected and uninfected KB cells: properties of the partially purified enzymes. Journal of General Virology 36:163–173
    [Google Scholar]
  20. Reichard P. 1958; Chromatographic separations of cytosine containing compounds. Acta chemica scandinavica 12:2048
    [Google Scholar]
  21. Seth P., Rawls W. E., Duff R., Rapp F., Adam E., Melnick J. L. 1974; Antigenic differences between isolates of herpesvirus type 2. Intervirology 3:1–14
    [Google Scholar]
  22. Thelander L., Reichard P. 1979; Reduction of ribonucleotides. Annual Review of Biochemistry 48:133–158
    [Google Scholar]
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