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. 1989 Nov;63(11):4913–4918. doi: 10.1128/jvi.63.11.4913-4918.1989

In vitro mutagenesis of the herpes simplex virus type 1 DNA polymerase gene results in altered drug sensitivity of the enzyme.

J T Matthews 1, R D Carroll 1, J T Stevens 1, M L Haffey 1
PMCID: PMC251137  PMID: 2552170

Abstract

A mutation (asparagine 815 to serine 815) was introduced into the herpes simplex virus type 1 (HSV-1) DNA polymerase (pol). The HSV-1 pol enzyme in lysates of Saccharomyces cerevisiae cells expressing the mutant protein showed increased resistance to acyclovir triphosphate and increased sensitivity to phosphonoacetate but was not substantially altered with respect to sensitivity to phosphonoformate or aphidicolin. These results directly demonstrate that both resistance to acyclovir triphosphate and sensitivity to phosphonoacetate can be conferred by this mutation in the absence of other viral factors and that the yeast expression system can be used for structure-function studies on HSV-1 pol.

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Selected References

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

  1. Beggs J. D. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978 Sep 14;275(5676):104–109. doi: 10.1038/275104a0. [DOI] [PubMed] [Google Scholar]
  2. Brugge J. S., Jarosik G., Andersen J., Queral-Lustig A., Fedor-Chaiken M., Broach J. R. Expression of Rous sarcoma virus transforming protein pp60v-src in Saccharomyces cerevisiae cells. Mol Cell Biol. 1987 Jun;7(6):2180–2187. doi: 10.1128/mcb.7.6.2180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Challberg M. D. A method for identifying the viral genes required for herpesvirus DNA replication. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9094–9098. doi: 10.1073/pnas.83.23.9094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chartrand P., Crumpacker C. S., Schaffer P. A., Wilkie N. M. Physical and genetic analysis of the herpes simplex virus DNA polymerase locus. Virology. 1980 Jun;103(2):311–326. doi: 10.1016/0042-6822(80)90190-7. [DOI] [PubMed] [Google Scholar]
  5. Chiou H. C., Weller S. K., Coen D. M. Mutations in the herpes simplex virus major DNA-binding protein gene leading to altered sensitivity to DNA polymerase inhibitors. Virology. 1985 Sep;145(2):213–226. doi: 10.1016/0042-6822(85)90155-2. [DOI] [PubMed] [Google Scholar]
  6. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Coen D. M., Schaffer P. A. Two distinct loci confer resistance to acycloguanosine in herpes simplex virus type 1. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2265–2269. doi: 10.1073/pnas.77.4.2265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Gibbs J. S., Chiou H. C., Bastow K. F., Cheng Y. C., Coen D. M. Identification of amino acids in herpes simplex virus DNA polymerase involved in substrate and drug recognition. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6672–6676. doi: 10.1073/pnas.85.18.6672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Haffey M. L., Stevens J. T., Terry B. J., Dorsky D. I., Crumpacker C. S., Wietstock S. M., Ruyechan W. T., Field A. K. Expression of herpes simplex virus type 1 DNA polymerase in Saccharomyces cerevisiae and detection of virus-specific enzyme activity in cell-free lysates. J Virol. 1988 Dec;62(12):4493–4498. doi: 10.1128/jvi.62.12.4493-4498.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Johnston M., Davis R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. doi: 10.1128/mcb.4.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Knopf C. W. The herpes simplex virus type 1 DNA polymerase gene: site of phosphonoacetic acid resistance mutation in strain Angelotti is highly conserved. J Gen Virol. 1987 May;68(Pt 5):1429–1433. doi: 10.1099/0022-1317-68-5-1429. [DOI] [PubMed] [Google Scholar]
  17. Knopf C. W., Weisshart K. The herpes simplex virus DNA polymerase: analysis of the functional domains. Biochim Biophys Acta. 1988 Dec 20;951(2-3):298–314. doi: 10.1016/0167-4781(88)90100-5. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Larder B. A., Darby G. Selection and characterisation of acyclovir-resistant herpes simplex virus type 1 mutants inducing altered DNA polymerase activities. Virology. 1985 Oct 30;146(2):262–271. doi: 10.1016/0042-6822(85)90009-1. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Novotný J., Auffray C. A program for prediction of protein secondary structure from nucleotide sequence data: application to histocompatibility antigens. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):243–255. doi: 10.1093/nar/12.1part1.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ostrander M., Cheng Y. C. Properties of herpes simplex virus type 1 and type 2 DNA polymerase. Biochim Biophys Acta. 1980 Sep 19;609(2):232–245. doi: 10.1016/0005-2787(80)90234-8. [DOI] [PubMed] [Google Scholar]
  25. Pizzagalli A., Valsasnini P., Plevani P., Lucchini G. DNA polymerase I gene of Saccharomyces cerevisiae: nucleotide sequence, mapping of a temperature-sensitive mutation, and protein homology with other DNA polymerases. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3772–3776. doi: 10.1073/pnas.85.11.3772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Powell K. L., Purifoy D. J. Nonstructural proteins of herpes simplex virus. I. Purification of the induced DNA polymerase. J Virol. 1977 Nov;24(2):618–626. doi: 10.1128/jvi.24.2.618-626.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. 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]
  31. Wang T. S., Wong S. W., Korn D. Human DNA polymerase alpha: predicted functional domains and relationships with viral DNA polymerases. FASEB J. 1989 Jan;3(1):14–21. doi: 10.1096/fasebj.3.1.2642867. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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