Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1993 Jun 15;292(Pt 3):883–889. doi: 10.1042/bj2920883

Herpes simplex virus type 1 uracil-DNA glycosylase: isolation and selective inhibition by novel uracil derivatives.

F Focher 1, A Verri 1, S Spadari 1, R Manservigi 1, J Gambino 1, G E Wright 1
PMCID: PMC1134197  PMID: 8391260

Abstract

We have purified Herpes simplex type 1 (HSV1) uracil-DNA glycosylase from the nuclei of HSV1-infected HeLa cells harvested 8 h post-infection, at which time the induction of the enzyme is a maximum. The enzyme has been shown to be distinct from the host enzyme, isolated from HeLa cells, by its lack of sensitivity to a monoclonal antibody to human uracil-DNA glycosylase. Furthermore, several uracil analogues were synthesized and screened for their capacity to discriminate between the viral and human uracil-DNA glycosylases. Both enzymes were inhibited by 6-(p-alkylanilino)uracils, but the viral enzyme was significantly more sensitive than the HeLa enzyme to most analogues. Substituents providing the best inhibitors of HSV1 uracil-DNA glycosylase were found to be in the order: p-n-butyl < p-n-pentl = p-n-hexyl < p-n-heptyl < p-n-octyl. The most potent HSV1 enzyme inhibitor, 6-(p-n-octylanilino)uracil (OctAU), with an IC50 of 8 microM, was highly selective for the viral enzyme. Short-term [3H]thymidine incorporation into the DNA of HeLa cells in culture was partially inhibited by OctAU, whereas it was unchanged when 6-(p-n-hexylanilino)uracil was present at concentrations that completely inhibited HSV1 uracil-DNA glycosylase activity. These compounds represent the first class of inhibitors that inhibit HSV1 uracil-DNA glycosylase at concentrations in the micromolar range. The results suggest their possible use to evaluate the functional role of HSV1 uracil-DNA glycosylase in viral infections and re-activation in nerve cells.

Full text

PDF
883

Images in this article

886Figure 2
on p.886
886Figure 3
on p.886

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arenaz P., Sirover M. A. Isolation and characterization of monoclonal antibodies directed against the DNA repair enzyme uracil DNA glycosylase from human placenta. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5822–5826. doi: 10.1073/pnas.80.19.5822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baker B. R., Rzeszotarski W. Irreversible enzyme inhibitors. CXXI. Thymidine phosphorylase. IX. On the nature and dimensions of the hydrophobic bonding region. J Med Chem. 1968 Jul;11(4):639–644. doi: 10.1021/jm00310a001. [DOI] [PubMed] [Google Scholar]
  3. Brown N. C., Dudycz L. W., Wright G. E. Rational design of substrate analogues targeted to selectively inhibit replication-specific DNA polymerases. Drugs Exp Clin Res. 1986;12(6-7):555–564. [PubMed] [Google Scholar]
  4. Caradonna S. J., Cheng Y. C. Induction of uracil-DNA glycosylase and dUTP nucleotidohydrolase activity in herpes simplex virus-infected human cells. J Biol Chem. 1981 Oct 10;256(19):9834–9837. [PubMed] [Google Scholar]
  5. Caradonna S. J., Cheng Y. C. Uracil DNA-glycosylase. Purification and properties of this enzyme isolated from blast cells of acute myelocytic leukemia patients. J Biol Chem. 1980 Mar 25;255(6):2293–2300. [PubMed] [Google Scholar]
  6. Caradonna S., Worrad D., Lirette R. Isolation of a herpes simplex virus cDNA encoding the DNA repair enzyme uracil-DNA glycosylase. J Virol. 1987 Oct;61(10):3040–3047. doi: 10.1128/jvi.61.10.3040-3047.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Challberg M. D., Kelly T. J. Animal virus DNA replication. Annu Rev Biochem. 1989;58:671–717. doi: 10.1146/annurev.bi.58.070189.003323. [DOI] [PubMed] [Google Scholar]
  8. Ejercito P. M., Kieff E. D., Roizman B. Characterization of herpes simplex virus strains differing in their effects on social behaviour of infected cells. J Gen Virol. 1968 May;2(3):357–364. doi: 10.1099/0022-1317-2-3-357. [DOI] [PubMed] [Google Scholar]
  9. Focher F., Hildebrand C., Freese S., Ciarrocchi G., Noonan T., Sangalli S., Brown N., Spadari S., Wright G. N2-phenyldeoxyguanosine: a novel selective inhibitor of herpes simplex thymidine kinase. J Med Chem. 1988 Aug;31(8):1496–1500. doi: 10.1021/jm00403a004. [DOI] [PubMed] [Google Scholar]
  10. Focher F., Mazzarello P., Verri A., Hübscher U., Spadari S. Activity profiles of enzymes that control the uracil incorporation into DNA during neuronal development. Mutat Res. 1990 Mar;237(2):65–73. doi: 10.1016/0921-8734(90)90012-g. [DOI] [PubMed] [Google Scholar]
  11. Focher F., Verri A., Verzeletti S., Mazzarello P., Spadari S. Uracil in OriS of herpes simplex 1 alters its specific recognition by origin binding protein (OBP): does virus induced uracil-DNA glycosylase play a key role in viral reactivation and replication? Chromosoma. 1992;102(1 Suppl):S67–S71. doi: 10.1007/BF02451788. [DOI] [PubMed] [Google Scholar]
  12. Hübscher U., Kuenzle C. C., Limacher W., Scherrer P., Spadari S. Functions of DNA polymerases alpha, beta, and gamma in neurons during development. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):625–629. doi: 10.1101/sqb.1979.043.01.069. [DOI] [PubMed] [Google Scholar]
  13. Hübscher U., Kuenzle C. C., Spadari S. Variation of DNA polymerases-alpha, -beta. and -gamma during perinatal tissue growth and differentiation. Nucleic Acids Res. 1977 Aug;4(8):2917–2929. doi: 10.1093/nar/4.8.2917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Krokan H., Wittwer C. U. Uracil DNa-glycosylase from HeLa cells: general properties, substrate specificity and effect of uracil analogs. Nucleic Acids Res. 1981 Jun 11;9(11):2599–2613. doi: 10.1093/nar/9.11.2599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Leib D. A., Ruffner K. L., Hildebrand C., Schaffer P. A., Wright G. E., Coen D. M. Specific inhibitors of herpes simplex virus thymidine kinase diminish reactivation of latent virus from explanted murine ganglia. Antimicrob Agents Chemother. 1990 Jun;34(6):1285–1286. doi: 10.1128/aac.34.6.1285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mazzarello P., Focher F., Verri A., Spadari S. Misincorporation of uracil into DNA as possible contributor to neuronal aging and abiotrophy. Int J Neurosci. 1990 Feb;50(3-4):169–174. doi: 10.3109/00207459008987169. [DOI] [PubMed] [Google Scholar]
  17. Mullaney J., Moss H. W., McGeoch D. J. Gene UL2 of herpes simplex virus type 1 encodes a uracil-DNA glycosylase. J Gen Virol. 1989 Feb;70(Pt 2):449–454. doi: 10.1099/0022-1317-70-2-449. [DOI] [PubMed] [Google Scholar]
  18. Seal G., Arenaz P., Sirover M. A. Purification and properties of the human placental uracil DNA glycosylase. Biochim Biophys Acta. 1987 Aug 13;925(2):226–233. doi: 10.1016/0304-4165(87)90113-9. [DOI] [PubMed] [Google Scholar]
  19. Spadari S., Focher F., Hübscher U. Developmental activity profile of DNA polymerases delta and alpha in rat neurons suggests a coordinated in vivo function. In Vivo. 1988 Sep-Oct;2(5):317–320. [PubMed] [Google Scholar]
  20. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Verri A., Mazzarello P., Biamonti G., Spadari S., Focher F. The specific binding of nuclear protein(s) to the cAMP responsive element (CRE) sequence (TGACGTCA) is reduced by the misincorporation of U and increased by the deamination of C. Nucleic Acids Res. 1990 Oct 11;18(19):5775–5780. doi: 10.1093/nar/18.19.5775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Weiser T., Gassmann M., Thömmes P., Ferrari E., Hafkemeyer P., Hübscher U. Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus. J Biol Chem. 1991 Jun 5;266(16):10420–10428. [PubMed] [Google Scholar]
  23. Worrad D. M., Caradonna S. Identification of the coding sequence for herpes simplex virus uracil-DNA glycosylase. J Virol. 1988 Dec;62(12):4774–4777. doi: 10.1128/jvi.62.12.4774-4777.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wright G. E., Baril E. F., Brown N. C. Butylanilinouracil: a selective inhibitor of HeLa cell DNA synthesis and HeLa cell DNA polymerase alpha. Nucleic Acids Res. 1980 Jan 11;8(1):99–109. doi: 10.1093/nar/8.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wright G. E., Brown N. C. Inhibitors of Bacillus subtilis DNA polymerase III. 6-Anilinouracils and 6-(alkylamino)uracils. J Med Chem. 1980 Jan;23(1):34–38. doi: 10.1021/jm00175a007. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES