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. 1988 Sep;85(18):6672–6676. doi: 10.1073/pnas.85.18.6672

Identification of amino acids in herpes simplex virus DNA polymerase involved in substrate and drug recognition.

J S Gibbs 1, H C Chiou 1, K F Bastow 1, Y C Cheng 1, D M Coen 1
PMCID: PMC282039  PMID: 2842788

Abstract

Herpes simplex virus (HSV) encodes a DNA polymerase that is similar in several respects to the replicative mammalian DNA polymerase alpha. Recently, these and other DNA polymerases have been shown to share several regions of protein sequence similarity. Despite these similarities, antiviral drugs that mimic natural polymerase substrates specifically inhibit herpesvirus DNA polymerases. To study amino acids involved in substrate and drug recognition, we have characterized and mapped altered drug sensitivity markers of nine HSV pol mutants and sequenced the relevant portions of these mutants. The mutations were found to occur within four relatively small regions. One such region, which we designate region A, has sequence similarity only to DNA polymerases that are sensitive to certain antiviral drugs. The other three regions contain sequences that are similar among various DNA polymerases. The multiple mutations occurring within two of these regions make it likely that the regions interact directly with drugs and substrates. Our results lead us to favor a model in which protein folding allows interactions among the four regions to form the substrate and drug binding sites.

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Selected References

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

  1. Argos P., Tucker A. D., Philipson L. Primary structural relationships may reflect similar DNA replication strategies. Virology. 1986 Mar;149(2):208–216. doi: 10.1016/0042-6822(86)90122-4. [DOI] [PubMed] [Google Scholar]
  2. Aron G. M., Purifoy D. J., Schaffer P. A. DNA synthesis and DNA polymerase activity of herpes simplex virus type 1 temperature-sensitive mutants. J Virol. 1975 Sep;16(3):498–507. doi: 10.1128/jvi.16.3.498-507.1975. [DOI] [PMC free article] [PubMed] [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., Séguin C. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature. 1984 Jul 19;310(5974):207–211. doi: 10.1038/310207a0. [DOI] [PubMed] [Google Scholar]
  4. Bastow K. F., Derse D. D., Cheng Y. C. Susceptibility of phosphonoformic acid-resistant herpes simplex virus variants to arabinosylnucleosides and aphidicolin. Antimicrob Agents Chemother. 1983 Jun;23(6):914–917. doi: 10.1128/aac.23.6.914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blanco L., Salas M. Effect of aphidicolin and nucleotide analogs on the phage phi 29 DNA polymerase. Virology. 1986 Sep;153(2):179–187. doi: 10.1016/0042-6822(86)90021-8. [DOI] [PubMed] [Google Scholar]
  6. Bolden A., Aucker J., Weissbach A. Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid. J Virol. 1975 Dec;16(6):1584–1592. doi: 10.1128/jvi.16.6.1584-1592.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chiou H. C., Kerns K. M., Coen D. M. Mutation within the herpes simplex virus DNA polymerase gene conferring resistance to (R)-9-(3,4-dihydroxybutyl)guanine. Antimicrob Agents Chemother. 1986 Sep;30(3):502–504. doi: 10.1128/aac.30.3.502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Coen D. M., Aschman D. P., Gelep P. T., Retondo M. J., Weller S. K., Schaffer P. A. Fine mapping and molecular cloning of mutations in the herpes simplex virus DNA polymerase locus. J Virol. 1984 Jan;49(1):236–247. doi: 10.1128/jvi.49.1.236-247.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Coen D. M., Fleming H. E., Jr, Leslie L. K., Retondo M. J. Sensitivity of arabinosyladenine-resistant mutants of herpes simplex virus to other antiviral drugs and mapping of drug hypersensitivity mutations to the DNA polymerase locus. J Virol. 1985 Feb;53(2):477–488. doi: 10.1128/jvi.53.2.477-488.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Coen D. M., Furman P. A., Aschman D. P., Schaffer P. A. Mutations in the herpes simplex virus DNA polymerase gene conferring hypersensitivity to aphidicolin. Nucleic Acids Res. 1983 Aug 11;11(15):5287–5297. doi: 10.1093/nar/11.15.5287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Coen D. M., Furman P. A., Gelep P. T., Schaffer P. A. Mutations in the herpes simplex virus DNA polymerase gene can confer resistance to 9-beta-D-arabinofuranosyladenine. J Virol. 1982 Mar;41(3):909–918. doi: 10.1128/jvi.41.3.909-918.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Darby G., Larder B. A., Bastow K. F., Field H. J. Sensitivity of viruses to phosphorylated 9-(2-hydroxyethoxymethyl)guanine revealed in TK-transformed cells. J Gen Virol. 1980 Jun;48(Pt 2):451–454. doi: 10.1099/0022-1317-48-2-451. [DOI] [PubMed] [Google Scholar]
  13. Davison A. J., Scott J. E. The complete DNA sequence of varicella-zoster virus. J Gen Virol. 1986 Sep;67(Pt 9):1759–1816. doi: 10.1099/0022-1317-67-9-1759. [DOI] [PubMed] [Google Scholar]
  14. Derse D., Bastow K. F., Cheng Y. Characterization of the DNA polymerases induced by a group of herpes simplex virus type I variants selected for growth in the presence of phosphonoformic acid. J Biol Chem. 1982 Sep 10;257(17):10251–10260. [PubMed] [Google Scholar]
  15. Dicioccio R. A., Chadha K., Sahai Srivastava B. I. Inhibition of herpes simplex virus-induced DNA polymerase, cellular DNA polymerase alpha, and virus production by aphidicolin. Biochim Biophys Acta. 1980 Sep 19;609(2):224–231. doi: 10.1016/0005-2787(80)90233-6. [DOI] [PubMed] [Google Scholar]
  16. Earl P. L., Jones E. V., Moss B. Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3659–3663. doi: 10.1073/pnas.83.11.3659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Frank K. B., Cheng Y. C. Mutually exclusive inhibition of herpesvirus DNA polymerase by aphidicolin, phosphonoformate, and acyclic nucleoside triphosphates. Antimicrob Agents Chemother. 1985 Apr;27(4):445–448. doi: 10.1128/aac.27.4.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Frank K. B., Derse D. D., Bastow K. F., Cheng Y. C. Novel interaction of aphidicolin with herpes simplex virus DNA polymerase and polymerase-associated exonuclease. J Biol Chem. 1984 Nov 10;259(21):13282–13286. [PubMed] [Google Scholar]
  19. Freitas V. R., Smee D. F., Chernow M., Boehme R., Matthews T. R. Activity of 9-(1,3-dihydroxy-2-propoxymethyl)guanine compared with that of acyclovir against human, monkey, and rodent cytomegaloviruses. Antimicrob Agents Chemother. 1985 Aug;28(2):240–245. doi: 10.1128/aac.28.2.240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Furman P. A., Coen D. M., St Clair M. H., Schaffer P. A. Acyclovir-resistant mutants of herpes simplex virus type 1 express altered DNA polymerase or reduced acyclovir phosphorylating activities. J Virol. 1981 Dec;40(3):936–941. doi: 10.1128/jvi.40.3.936-941.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Furman P. A., St Clair M. H., Fyfe J. A., Rideout J. L., Keller P. M., Elion G. B. Inhibition of herpes simplex virus-induced DNA polymerase activity and viral DNA replication by 9-(2-hydroxyethoxymethyl)guanine and its triphosphate. J Virol. 1979 Oct;32(1):72–77. doi: 10.1128/jvi.32.1.72-77.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Germershausen J., Bostedor R., Field A. K., Perry H., Liou R., Bull H., Tolman R. L., Karkas J. D. A comparison of the antiviral agents 2'-nor-2'-deoxyguanosine and acyclovir: uptake and phosphorylation in tissue culture and kinetics of in vitro inhibition of viral and cellular DNA polymerases by their respective triphosphates. Biochem Biophys Res Commun. 1983 Oct 31;116(2):360–367. doi: 10.1016/0006-291x(83)90530-2. [DOI] [PubMed] [Google Scholar]
  23. Gibbs J. S., Chiou H. C., Hall J. D., Mount D. W., Retondo M. J., Weller S. K., Coen D. M. Sequence and mapping analyses of the herpes simplex virus DNA polymerase gene predict a C-terminal substrate binding domain. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7969–7973. doi: 10.1073/pnas.82.23.7969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gingeras T. R., Sciaky D., Gelinas R. E., Bing-Dong J., Yen C. E., Kelly M. M., Bullock P. A., Parsons B. L., O'Neill K. E., Roberts R. J. Nucleotide sequences from the adenovirus-2 genome. J Biol Chem. 1982 Nov 25;257(22):13475–13491. [PubMed] [Google Scholar]
  25. Hall J. D. Modeling functional sites in DNA polymerases. Trends Genet. 1988 Feb;4(2):42–46. doi: 10.1016/0168-9525(88)90065-0. [DOI] [PubMed] [Google Scholar]
  26. Jung G. H., Leavitt M. C., Hsieh J. C., Ito J. Bacteriophage PRD1 DNA polymerase: evolution of DNA polymerases. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8287–8291. doi: 10.1073/pnas.84.23.8287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kallin B., Sternås L., Saemundssen A. K., Luka J., Jörnvall H., Eriksson B., Tao P. Z., Nilsson M. T., Klein G. Purification of Epstein-Barr virus DNA polymerase from P3HR-1 cells. J Virol. 1985 May;54(2):561–568. doi: 10.1128/jvi.54.2.561-568.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Knopf C. W. Nucleotide sequence of the DNA polymerase gene of herpes simplex virus type 1 strain Angelotti. Nucleic Acids Res. 1986 Oct 24;14(20):8225–8226. doi: 10.1093/nar/14.20.8225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kouzarides T., Bankier A. T., Satchwell S. C., Weston K., Tomlinson P., Barrell B. G. Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene. J Virol. 1987 Jan;61(1):125–133. doi: 10.1128/jvi.61.1.125-133.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Krokan H., Schaffer P., DePamphilis M. L. Involvement of eucaryotic deoxyribonucleic acid polymerases alpha and gamma in the replication of cellular and viral deoxyribonucleic acid. Biochemistry. 1979 Oct 2;18(20):4431–4443. doi: 10.1021/bi00587a025. [DOI] [PubMed] [Google Scholar]
  31. Larder B. A., Darby G. Susceptibility to other antiherpes drugs of pathogenic variants of herpes simplex virus selected for resistance to acyclovir. Antimicrob Agents Chemother. 1986 May;29(5):894–898. doi: 10.1128/aac.29.5.894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Larder B. A., Kemp S. D., Darby G. Related functional domains in virus DNA polymerases. EMBO J. 1987 Jan;6(1):169–175. doi: 10.1002/j.1460-2075.1987.tb04735.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Larder B. A., Lisle J. J., Darby G. Restoration of wild-type pathogenicity to an attenuated DNA polymerase mutant of herpes simplex virus type 1. J Gen Virol. 1986 Nov;67(Pt 11):2501–2506. doi: 10.1099/0022-1317-67-11-2501. [DOI] [PubMed] [Google Scholar]
  34. Mao J. C., Robishaw E. E., Overby L. R. Inhibition of DNA polymerase from herpes simplex virus-infected wi-38 cells by phosphonoacetic Acid. J Virol. 1975 May;15(5):1281–1283. doi: 10.1128/jvi.15.5.1281-1283.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Overby L. R., Duff R. G., Mao J. C. Antiviral potential of phosphonoacetic acid. Ann N Y Acad Sci. 1977 Mar 4;284:310–320. doi: 10.1111/j.1749-6632.1977.tb21966.x. [DOI] [PubMed] [Google Scholar]
  36. Owen L. J., Field H. J. Genomic localization and sequence analysis of the putative bovine herpesvirus-1 DNA polymerase gene. Arch Virol. 1988;98(1-2):27–38. doi: 10.1007/BF01321003. [DOI] [PubMed] [Google Scholar]
  37. Pedrali-Noy G., Spadari S. Mechanism of inhibition of herpes simplex virus and vaccinia virus DNA polymerases by aphidicolin, a highly specific inhibitor of DNA replication in eucaryotes. J Virol. 1980 Nov;36(2):457–464. doi: 10.1128/jvi.36.2.457-464.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Quinn J. P., McGeoch D. J. DNA sequence of the region in the genome of herpes simplex virus type 1 containing the genes for DNA polymerase and the major DNA binding protein. Nucleic Acids Res. 1985 Nov 25;13(22):8143–8163. doi: 10.1093/nar/13.22.8143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Reno J. M., Lee L. F., Boezi J. A. Inhibition of herpesvirus replication and herpesvirus-induced deoxyribonucleic acid polymerase by phosphonoformate. Antimicrob Agents Chemother. 1978 Feb;13(2):188–192. doi: 10.1128/aac.13.2.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ruth J. L., Cheng Y. C. Nucleoside analogues with clinical potential in antivirus chemotherapy. The effect of several thymidine and 2'-deoxycytidine analogue 5'-triphosphates on purified human (alpha, beta) and herpes simplex virus (types 1, 2) DNA polymerases. Mol Pharmacol. 1981 Sep;20(2):415–422. [PubMed] [Google Scholar]
  41. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. St Clair M. H., Miller W. H., Miller R. L., Lambe C. U., Furman P. A. Inhibition of cellular alpha DNA polymerase and herpes simplex virus-induced DNA polymerases by the triphosphate of BW759U. Antimicrob Agents Chemother. 1984 Feb;25(2):191–194. doi: 10.1128/aac.25.2.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tsurumi T., Maeno K., Nishiyama Y. A single-base change within the DNA polymerase locus of herpes simplex virus type 2 can confer resistance to aphidicolin. J Virol. 1987 Feb;61(2):388–394. doi: 10.1128/jvi.61.2.388-394.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tsurumi T., Maeno K., Nishiyama Y. Nucleotide sequence of the DNA polymerase gene of herpes simplex virus type 2 and comparison with the type 1 counterpart. Gene. 1987;52(2-3):129–137. doi: 10.1016/0378-1119(87)90039-4. [DOI] [PubMed] [Google Scholar]
  46. Wahren B., Rudén U., Gadler H., Oberg B., Eriksson B. Activity of the cytomegalovirus genome in the presence of PPi analogs. J Virol. 1985 Dec;56(3):996–1001. doi: 10.1128/jvi.56.3.996-1001.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Weissbach A., Hong S. C., Aucker J., Muller R. Characterization of herpes simplex virus-induced deoxyribonucleic acid polymerase. J Biol Chem. 1973 Sep 25;248(18):6270–6277. [PubMed] [Google Scholar]
  48. Wong S. W., Wahl A. F., Yuan P. M., Arai N., Pearson B. E., Arai K., Korn D., Hunkapiller M. W., Wang T. S. Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases. EMBO J. 1988 Jan;7(1):37–47. doi: 10.1002/j.1460-2075.1988.tb02781.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yager D. R., Coen D. M. Analysis of the transcript of the herpes simplex virus DNA polymerase gene provides evidence that polymerase expression is inefficient at the level of translation. J Virol. 1988 Jun;62(6):2007–2015. doi: 10.1128/jvi.62.6.2007-2015.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Yoshikawa H., Ito J. Nucleotide sequence of the major early region of bacteriophage phi 29. Gene. 1982 Mar;17(3):323–335. doi: 10.1016/0378-1119(82)90149-4. [DOI] [PubMed] [Google Scholar]

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