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Volume 72, Issue 6

Purification and characterization of the herpes simplex virus type 1 ribonucleotide reductase small subunit following expression in Free

Abstract

The herpes simplex virus type 1 (HSV-1) gene encoding the ribonucleotide reductase (RR) small subunit (R2) was cloned as an unfused and intact open reading frame into a T7 RNA polymerase expression system in . The expressed product was recovered from bacteria in soluble form and constituted 7% of the soluble protein. Protein purification yielded 3.5 mg of 95% pure R2 per litre of bacterial culture. The correct composition of the purified protein was verified by amino acid analysis and N-terminal sequencing. The isoelectric point of the protein was 5.3. Atomic emission spectroscopy indicated that the iron content of the -expressed R2 was 0.2 to 0.5 atoms of iron per R2 protomer as compared with a theoretical maximum value of 2. The -expressed HSV-1 R2 existed as a combination of a stable dimer and monomer. Combination of the -expressed R2 with the -expressed large subunit (R1) gave an active holoenzyme. Thus, the T7 expression system provides a rich source of enzymically active HSV-1 RR.

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© Journal of General Virology 1991
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1991-06-01
2024-04-24
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References

  1. Atkin C. L., Thelander L., Reichard P., Lang G. 1973; Iron and free radical in ribonucleotide reductase. Journal of Biological Chemistry 248:7464–7472
     [Google Scholar]
  2. Bacchetti S., Evelegh M. J., Muirhead B. 1986; Identification and separation of the two subunits of the herpes simplex virus ribonucleotide reductase. Journal of Virology 57:1177–1181
     [Google Scholar]
  3. Baer R., Bankier A. T., Biggin M. D., Deininger P. L., Farrell P. J., Gibson T. J., Hatfull G., Hudson G. S., Satchwell S. C., Seguin C., Tuffnell P. S., Barrell B. G. 1984; DNA sequence and expression of the B95-8 Epstein–Barr virus genome. Nature, London 310:207–211
     [Google Scholar]
  4. Cameron J. M., McDougall I., Marsden H. S., Preston V. G., Ryan D. M., Subak-Sharpe J. H. 1988; Ribonucleotide reductase encoded by herpes simplex virus is a determinant of the pathogenicity of the virus in mice and a valid antiviral target. Journal of General Virology 69:2607–2612
     [Google Scholar]
  5. Cohen E. A., Charron J., Perret J., Langelier Y. 1985; Herpes simplex virus ribonucleotide reductase induced in infected BHK-21/C13 cells: biochemical evidence for the existence of two non-identical subunits, H1 and H2. Journal of General Virology 66:733–745
     [Google Scholar]
  6. Cohen E. A., Gaudreau P., Brazeau P., Langelier Y. 1986a; Specific inhibition of herpesvirus ribonucleotide reductase by a nonapeptide derived from the carboxy terminus of subunit 2. Nature, London 321:441–443
     [Google Scholar]
  7. Cohen E. A., Gaudreau P., Brazeau P., Langelier Y. 1986b; Neutralisation of herpes simplex virus ribonucleotide reductase activity by an oligopeptide-induced antiserum directed against subunit H2. Journal of Virology 60:1130–1133
     [Google Scholar]
  8. Cohen E. A., Paradis H., Gaudreau P., Brazeau P., Langelier Y. 1987; Identification of viral polypeptides involved in pseudorabies virus ribonucleotide reductase activity. Journal of Virology 61:2046–2049
     [Google Scholar]
  9. 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]
  10. Darling A. J., Dutia B. M., Marsden H. S. 1987; Improved method for the measurement of ribonucleotide reductase activity. Journal of Virological Methods 180:281–290
     [Google Scholar]
  11. Darling A. J., MacKay E. M., Ingemarson R., Booth B. 1990; Herpes simplex virus encoded ribonucleotide reductase: evidence for the dissociation/reassociation of the holoenzyme. Virus Genes 3:4:367–372
     [Google Scholar]
  12. Davison A. J., Scott J. E. 1986; The complete DNA sequence of varicella-zoster virus. Journal of General Virology 67:1759–1816
     [Google Scholar]
  13. Dutia B. M. 1983; Ribonucleotide reductase induced by herpes simplex virus has a virus-specified constituent. Journal of General Virology 64:513–521
     [Google Scholar]
  14. Dutia B. M., Frame M. C., Subak-Sharpe J. H., Clark W. N., Marsden H. S. 1986; Specific inhibition of herpesvirus ribonucleotide reductase by synthetic peptides. Nature, London 321:439–441
     [Google Scholar]
  15. Eriksson S., Sjöberg B.-M. 1988; Ribonucleotide reductase. In Allosteric Enzymes pp 189–217 Edited by Herve G. Boca Raton: CRC Press;
     [Google Scholar]
  16. Frame M. C., Marsden H. S., Dutia B. M. 1985; The ribonucleotide reductase induced by herpes simplex virus type 1 involves minimally a complex of two polypeptides (136K and 38K). Journal of General Virology 66:1581–1587
     [Google Scholar]
  17. Furlong J., Conner J., McLauchlan J., Lankinen H., Galt C., Marsden H. S., Clements J. B. 1991; The large subunit of herpes simplex virus type 1 ribonucleotide reductase: expression in Escherichia coli and purification. Virology 182: (in press)
    [Google Scholar]
  18. Gaudreau P., Michaud J., Cohen E. A., Langelier Y., Brazeau P. 1987; Structure-activity studies on synthetic peptides inhibiting herpes simplex virus ribonucleotide reductase. Journal of Biological Chemistry 262:12413–12416
     [Google Scholar]
  19. Gibson T., Stockwell P., Ginsberg M., Barrell B. 1984; Homology between two EBV early genes and HSV ribonucleotide reductase and 38K. genes. Nucleic Acids Research 12:5087–5099
     [Google Scholar]
  20. Goldstein D. J., Weller S. K. 1988; Herpes simplex virus type 1-induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis: isolation and characterization of an ICP6 lacZ insertion mutant. Journal of Virology 62:196–205
     [Google Scholar]
  21. 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]
  22. Huszar D., Bacchetti S. 1981; Partial purification and characterisation of the ribonucleotide reductase induced by herpes simplex virus infection of mammalian cells. Journal of Virology 37:580–588
     [Google Scholar]
  23. Ingemarson R., Lankinen H. 1987; The herpes simplex virus type 1 ribonucleotide reductase is a tight complex of the type α 2 β 2 composed of 40K and 140K proteins, of which the latter shows multiple forms due to proteolysis. Journal of Virology 156:417–422
     [Google Scholar]
  24. Ingemarson R., Gräslund A., Darling A., Thelander L. 1989; Herpes simplex virus ribonucleotide reductase: expression in Escherichia coli and purification to homogeneity of a tyrosyl free radical-containing, enzymatically active form of the 38-kilodalton subunit. Journal of Virology 63:3769–3776
     [Google Scholar]
  25. Jacobson J. G., Leib D. A., Goldstein D. J., Bogard C. L., Schaffer P. A., Weller S. K., Coen D. M. 1989; A herpes simplex virus ribonucleotide reductase deletion mutant is defective for productive acute and reactivatable latent infections of mice and for replication in mouse cells. Virology 173:276–283
     [Google Scholar]
  26. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227:680–685
     [Google Scholar]
  27. Lamarche N., Massie B., Richer M., Paradis H., Langelier Y. 1990; High level expression in 293 cells of the herpes simplex virus type 2 ribonucleotide reductase subunit 2 using an adenovirus vector. Journal of General Virology 71:1785–1792
     [Google Scholar]
  28. Lammers M., Follman H. 1983; The ribonucleotide reductases – a unique group of metalloenzymes essential for cell proliferation. Structure and Bonding 54:27–91
     [Google Scholar]
  29. Lankinen H., Gräslund 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]
  30. Lankinen H., Telford E., MacDonald D., Marsden H. 1989; The unique N-terminal domain of the large subunit of herpes simplex virus ribonucleotide reductase is preferentially sensitive to proteolysis. Journal of General Virology 70:3159–3169
     [Google Scholar]
  31. Lynch J. B., Juarez-Garcia C., Münck E., Que L. J. R. 1989; Mossbauer and EPR studies of the binuclear iron center in ribonucleotide reductase from Escherichia coli . Journal of Biological Chemistry 264:8091–8096
     [Google Scholar]
  32. McClements W., Yamanaka G., Garsky V., Perry H., Bacchetti S., Colonno R., Stein R. B. 1988; Oligopeptides inhibit the ribonucleotide reductase of herpes simplex virus by causing subunit separation. Virology 162:270–273
     [Google Scholar]
  33. McGeoch D. J., Dalrymple M. A., Davison A. J., Dolan A., Frame M. C., McNab D., Perry L. J., Scott J. E., Taylor P. 1988; The complete DNA sequence of the long unique region in the genome of herpes simplex virus type I. Journal of General Virology 69:1531–1574
     [Google Scholar]
  34. McLauchlan J., Clements J. B. 1983; Organization of the herpes simplex virus type 1 transcription unit encoding two early proteins with molecular weights of 140000 and 40000. Journal of General Virology 64:997–1006
     [Google Scholar]
  35. Marsden H. S., Stow N. D., Preston V. G., Timbury M. C., Wilkie N. M. 1978; Physical mapping of herpes simplex virus-induced polypeptides. Journal of Virology 28:624–642
     [Google Scholar]
  36. Nikas I., Darling A. J., Lankinen H. M., Cross A. M., Marsden H. S., Clements J. B. 1990; A single amino acid substitution in the large subunit of herpes simplex virus type 1 ribonucleotide reductase which prevents subunit association. Journal of General Virology 71:2369–2376
     [Google Scholar]
  37. Nordlund P., Sjӧberg B. M., Eklund H. 1990; Threedimensional structure of the free radical protein of ribonucleotide reductase. Nature, London 345:593–598
     [Google Scholar]
  38. Paradis H., Gaudreau P., Brazeau P., Langelier Y. 1988; Mechanism of inhibition of herpes simplex virus (HSV) ribonucleotide reductase by a nonapeptide corresponding to the carboxyl terminus of its subunit 2. Journal of Biological Chemistry 263:16045–16050
     [Google Scholar]
  39. Pereira L., Wolff M. H., Fenwick M., Roizman B. 1977; Regulation of herpesvirus macromolecular synthesis. Virology 77:733–749
     [Google Scholar]
  40. Preston V. G., Palfreyman J. W., Dutia B. M. 1984; Identification of a herpes simplex virus type 1 polypeptide which is a component of the virus-induced ribonucleotide reductase. Journal of General Virology 65:1457–1466
     [Google Scholar]
  41. Preston V. G., Darling A. J., McDougall I. M. 1988; The herpes simplex type 1 temperature-sensitive mutant ts 1222 has a single base pair deletion in the small subunit of ribonucleotide reductase. Virology 167:458–467
     [Google Scholar]
  42. Reichard P. 1988; Interactions between deoxyribonucleotide and DNA synthesis. Annual Review of Biochemistry 57:349–374
     [Google Scholar]
  43. Sjӧberg B. M., Grӓslund A. 1983; Ribonucleotide reductase. In Advances in Inorganic Biochemistry vol 5 Iron Binding Proteins without Cofactors or Sulfur Clusters pp 87–110 Edited by Theil E. C. New York: Elsevier;
     [Google Scholar]
  44. Sjӧberg B. M., Karlsson M., Jӧrnvall H. 1987; Half-site reactivity of the tyrosyl radical of ribonucleotide reductase from Escherichia coli . Journal of Biological Chemistry 262:9736–9743
     [Google Scholar]
  45. Spector T., Stonehuerner J. G., Biron K. K., Averett E. R. 1987; Ribonucleotide reductase induced by varicella zoster virus. Characterisation and potentiation of its inhibition by acyclovir. Biochemical Pharmacology 36:4341–4346
     [Google Scholar]
  46. Stubbe J. 1989; Protein radical involvement in biological catalysis?. Annual Review of Biochemistry 58:257–285
     [Google Scholar]
  47. Stubbe J. 1990; Ribonucleotide reductases: amazing and confusing. Journal of Biological Chemistry 265:5329–5332
     [Google Scholar]
  48. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. 1990; Use of T7 RNA polymerase to direct expression of cloned genes. Methods in Enzymology 185:60–89
     [Google Scholar]
  49. Telford E., Lankinen H., Marsden H. 1990; Inhibition of equine herpesvirus type 1 subtype 1-induced ribonucleotide reductase by the nonapeptide YAGAVVNDL. Journal of General Virology 71:1373–1378
     [Google Scholar]
  50. Thelander L., Reichard P. 1979; Reduction of ribonucleotides. Annual Review of Biochemistry 48:133–158
     [Google Scholar]
  51. Thelander L., Eriksson S., Akerman M. 1980; Ribonucleotide reductase from calf thymus. Separation of the enzyme into two non-identical subunits, proteins Ml and M2. Journal of Biological Chemistry 255:7426–7432
     [Google Scholar]
  52. Wilcox K. W., Kohn A., Sklyanskaya E., Roizman B. 1980; Herpes simplex virus phosphoproteins. I. Phosphate cycles on and off some viral polypeptides and can alter their affinity for DNA. Journal of Virology 33:167–182
     [Google Scholar]
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Purification and characterization of the herpes simplex virus type 1 ribonucleotide reductase small subunit following expression in Escherichia coli
J. Gen. Virol. 72, 1383 (1991); https://doi.org/10.1099/0022-1317-72-6-1383
/content/journal/jgv/10.1099/0022-1317-72-6-1383
/content/journal/jgv/10.1099/0022-1317-72-6-1383
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