1887

Abstract

The large subunit (R1) of herpes simplex virus (HSV) ribonucleotide reductase is a bifunctional protein consisting of a unique N-terminal protein kinase domain and a ribonucleotide reductase domain. Previous studies showed that the two functional domains are linked by a protease sensitive site. Here we provide evidence for two subdomains, of 30K and 53K, within the reductase domain. The two fragments, which were produced by limited proteolysis and were resistant to further degradation, remained tightly associated in a complex containing two molecules of each. They were capable of binding the R2 subunit of HSV ribonucleotide reductase with approximately the same affinity as the intact protein but the complex did not complement the small subunit (R2) to give an active enzyme. At low concentrations (0·4 μg/ml) of trypsin or V8 protease, cleavage between the subdomains was prevented by the presence of the N-terminal protein kinase domain. At higher protease concentrations (1 μg/ml) the N-terminal domain is extensively proteolysed and the 30K and 53K domains were generated. Identical results were obtained using purified R1 isolated from infected cell extracts or following expression in . The origin of the two domains was investigated by N-terminal sequencing of the 53K fragment and by examining their reactivity with a panel of R1-specific monoclonal antibodies which we isolated and epitope mapped for that purpose. The trypsin cleavage site was found to lie between arginine 575 and asparagine 576, and proteolysis in this region was not prevented by the presence of R2 or the nonapep-tide YAGAWNDL. We propose that the ribonucleotide reductase region of HSV R1 exists in a two domain structure, and that the interdomain linking region is protected by the unique N terminus.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-75-12-3327
1994-12-01
2021-10-25
Loading full text...

Full text loading...

/deliver/fulltext/jgv/75/12/JV0750123327.html?itemId=/content/journal/jgv/10.1099/0022-1317-75-12-3327&mimeType=html&fmt=ahah

References

  1. Aberg A., Hahn S., Karlsson M., Larsson A., Ormo M., Ahgren A., Sjöberg B. -M. 1989; Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli . Journal of Biological Chemistry 264:12249–12252
    [Google Scholar]
  2. Bio-Mega inc 1990; Antiherpestetrapeptide derivatives having a substituted aspartic acid side chain. European Patent Application no EP/0/411/333/A1.
  3. 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 and a valid antiviral target. Journal of General Virology 69:2607–2612
    [Google Scholar]
  4. Chung T. D., Wymer J. P., Smith C. C., Kulka M., Aurelian L. 1989; Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP0). Journal of Virology 63:3389–3398
    [Google Scholar]
  5. Climent I., Sjöberg B. -M., Huang Y. C. 1991; Carboxy-terminal peptides as probes for Escherichia coli ribonucleotide reductase subunit interaction: kinetic analysis of inhibition studies. Biochemistry 30:5164–5171
    [Google Scholar]
  6. Cohen E. A., Gaudreau P., Brazeau P., Langelier Y. 1986; Specific inhibition of herpes simplex virus ribonucleotide reductase by a nonapeptide derived from the carboxy terminus of subunit 2. Nature; London: 321:441–443
    [Google Scholar]
  7. 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]
  8. Conner J., Cooper J., Furlong J., Clements J. B. 1992a; An autophosphorylating but not transphosphorylating activity is associated with the unique N-terminus of the herpes simplex virus type 1 ribonucleotide reductase large subunit. Journal of Virology 66:7511–7516
    [Google Scholar]
  9. Conner J., MacFarlane J., Lankinen H., Marsden H. 1992b; The unique N terminus of the herpes simplex virus type 1 large subunit is not required for ribonucleotide reductase activity. Journal of General Virology 73:103–112
    [Google Scholar]
  10. Conner J., Furlong J., Murray J., Meighan M., Cross A., Marsden H., Clements J. B. 1993; Herpes simplex virus type 1 ribonucleotide reductase large subunit: regions of the protein essential for subunit interaction and dimerisation. Biochemistry 32:13673–13680
    [Google Scholar]
  11. Conner J., Marsden H., Clements J. B. 1994; Ribonucleotide reductase of herpesviruses. Reviews in Medical Virology 4:25–34
    [Google Scholar]
  12. Consentino G., Lavalee P., Rakhit S., Plante R., Gaudette Y., Lawetz C., Whitehead P. W., Duceppe J. S., Lepine-Frenette C., Dansereau N., Guilbaut C., Langelier Y., Gaudreau P., Thelander L., Guidon Y. 1991; Specific inhibition of ribonucleotide reductase by peptides corresponding to the C-terminal of their second subunit. Journal of Biochemistry and Cellular Biology 69:79–83
    [Google Scholar]
  13. Cross A. M., Hope R. G., Marsden H. S. 1987; Generation and properties of the glycoprotein E-related 32K/34K/35K and 55K/57K polypeptide encoded by herpes simplex virus type 1. Journal of General Virology 68:2093–2104
    [Google Scholar]
  14. Darling A. J., Dutia B. M., Marsden H. S. 1987; Improved method for the measurement of ribonucleotide reductase activity. Journal of Virological Methods 180:280–290
    [Google Scholar]
  15. Darling A. J., MacKay E. M., Ingemarson R. 1990; Herpes simplex virus encoded ribonucleotide reductase: evidence for the dissociation/reassociation of the holoenzyme. Virus Genes 3:367–372
    [Google Scholar]
  16. De Wind N., Berns A., Gielkens A., Kimman T. 1993; Ribonucleotide reductase-deficient mutants of pseudorabies virus are avirulent for pigs and induce partial protective immunity. Journal of General Virology 74:351–359
    [Google Scholar]
  17. Dutia B. M., Frame M. C., Subak-Sharpe J. H., Clark W. N., Marsden H. S. 1986; Specific inhibition of herpes virus ribonucleotide reductase by synthetic peptides. Nature; London: 321439–441
    [Google Scholar]
  18. Filatov D., Ingemarson R., Graslund A., Thelander L. 1992; The role of herpes simplex virus ribonucleotide reductase small subunit carboxyl terminus in subunit interaction and formation of iron-tyrosyl center structure. Journal of Biological Chemistry 267:15816–15822
    [Google Scholar]
  19. Furlong J., Conner J., McLaughlan 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 182:846–851
    [Google Scholar]
  20. 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. Virology 156:417–422
    [Google Scholar]
  21. 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 and reactivatable latent infections of mice and for replication in mouse cells. Virology 173:276–283
    [Google Scholar]
  22. 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]
  23. Lankinen H., McLauchlan J., Weir M., Furlong J., Conner J., McGarrity A., Mistry A., Clements J. B., Marsden H. S. 1991; Purification and characterization of the herpes simplex virus type 1 ribonucleotide reductase small subunit following expression in Escherichia coli . Journal of General Virology 72:1383–1392
    [Google Scholar]
  24. Lankinen H., Everett R., Cross A., Conner J., Marsden H. S. 1993; Epitope mapping identifies an exposed loop between the unique amino- and conserved carboxy-domains of the large subunit of herpes simplex virus type 1 ribonucleotide reductase. Journal of General Virology 74:1871–1877
    [Google Scholar]
  25. Luo J. -H., Aurelian L. 1992; The transmembrane helical segment but not the invariant lysine is required for the kinase activity of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10). Journal of Biological Chemistry 267:9645–9653
    [Google Scholar]
  26. 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]
  27. Mao S. S., Johnston M. I., Bollinger J. M., Stubbe J. 1989; >Mechanism-based inhibition of a mutant Escherichia coli ribonucleotide reductase (cysteine-255 → serine) by its substrate CDP. Proceedings of the National Academy of Sciences, U.S.A 86:1485–1489
    [Google Scholar]
  28. Nikas I., McLauchlan J., Davison A. J., Taylor W. R., Clements J. B. 1986; Structural features of ribonucleotide reductase. Protein Structure Function and Genetics 1:376–384
    [Google Scholar]
  29. Nordlund P., Sjöberg B. -M., Eklund H. 1990; Threedimensional structure of the free radical protein of ribonucleotide reductase. Nature; London: 345593–598
    [Google Scholar]
  30. 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 carboxy-terminus of its subunit 2. Journal of Biological Chemistry 263:16045–16050
    [Google Scholar]
  31. Paradis H., Gaudreau P., Massie B., Lamarche N., Guilbault C., Gravel S., Langelier Y. 1991; Affinity purification of active subunit 1 of herpes simplex virus type 1 ribonucleotide reductase exhibiting a protein kinase activity. Journal of Biological Chemistry 266:9647–9651
    [Google Scholar]
  32. Price N., Johnson C. M. 1989; Proteinases as probes of conformation of soluble proteins. In Proteolytic Enzymes: A Practical Approach pp. 163–180 Benyon A. J., Bond J. S. Edited by Oxford, U.K: IRL Press;
    [Google Scholar]
  33. Reichard P. 1988; Interactions between deoxyribonucleotide and DNA synthesis. Annual Review of Biochemistry 57:349–374
    [Google Scholar]
  34. Slabaugh M. B., Davis R. E., Roseman N. A., Matthews C. K. 1993; Vaccinia virus ribonucleotide reductase expression and isolation of the recombinant large subunit. Journal of Biological Chemistry 268:17803–17810
    [Google Scholar]
  35. Stubbe J. 1990; Ribonucleotide reductases: amazing and confusing. Journal of Biological Chemistry 256:5329–5332
    [Google Scholar]
  36. Telford E., Lankinen H., Marsden H. S. 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]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-75-12-3327
Loading
/content/journal/jgv/10.1099/0022-1317-75-12-3327
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