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. 1986 Nov;83(22):8769–8773. doi: 10.1073/pnas.83.22.8769

A human cytomegalovirus mutant resistant to the nucleoside analog 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U) induces reduced levels of BW B759U triphosphate.

K K Biron, J A Fyfe, S C Stanat, L K Leslie, J B Sorrell, C U Lambe, D M Coen
PMCID: PMC387013  PMID: 3022304

Abstract

We have isolated a human cytomegalovirus mutant that is resistant to the antiviral drug 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U), yet exhibits wild-type sensitivity to inhibitors of herpesvirus DNA polymerases such as phosphonoformic acid and aphidicolin. Cells infected with the mutant accumulate approximately equal to 1/10th the amount of drug triphosphate as do those infected with the wild-type parent. This reduction in drug triphosphate could not be attributed to altered drug uptake or to reduced stability of the triphosphate, once formed. The induction of normal nucleoside and deoxynucleoside triphosphates and certain cellular nucleoside kinases was comparable in mutant and wild-type virus infections. These results provide strong evidence for the importance of phosphorylation in the selectivity of this antiviral compound and raise the possibility that human cytomegalovirus encodes a nucleoside kinase. The mutant may identify the existence of a cytomegalovirus function whose properties could facilitate genetic analysis of this important pathogen.

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Selected References

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

  1. Ashton W. T., Karkas J. D., Field A. K., Tolman R. L. Activation by thymidine kinase and potent antiherpetic activity of 2'-nor-2'-deoxyguanosine (2'NDG). Biochem Biophys Res Commun. 1982 Oct 29;108(4):1716–1721. doi: 10.1016/s0006-291x(82)80109-5. [DOI] [PubMed] [Google Scholar]
  2. Bach M. C., Bagwell S. P., Knapp N. P., Davis K. M., Hedstrom P. S. 9-(1,3-Dihydroxy-2-propoxymethyl)guanine for cytomegalovirus infections in patients with the acquired immunodeficiency syndrome. Ann Intern Med. 1985 Sep;103(3):381–382. doi: 10.7326/0003-4819-103-3-381. [DOI] [PubMed] [Google Scholar]
  3. Biron K. K., Stanat S. C., Sorrell J. B., Fyfe J. A., Keller P. M., Lambe C. U., Nelson D. J. Metabolic activation of the nucleoside analog 9-[( 2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine in human diploid fibroblasts infected with human cytomegalovirus. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2473–2477. doi: 10.1073/pnas.82.8.2473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cheng Y. C., Huang E. S., Lin J. C., Mar E. C., Pagano J. S., Dutschman G. E., Grill S. P. Unique spectrum of activity of 9-[(1,3-dihydroxy-2-propoxy)methyl]-guanine against herpesviruses in vitro and its mode of action against herpes simplex virus type 1. Proc Natl Acad Sci U S A. 1983 May;80(9):2767–2770. doi: 10.1073/pnas.80.9.2767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. DUBBS D. R., KIT S. MUTANT STRAINS OF HERPES SIMPLEX DEFICIENT IN THYMIDINE KINASE-INDUCING ACTIVITY. Virology. 1964 Apr;22:493–502. doi: 10.1016/0042-6822(64)90070-4. [DOI] [PubMed] [Google Scholar]
  9. Estes J. E., Huang E. S. Stimulation of cellular thymidine kinases by human cytomegalovirus. J Virol. 1977 Oct;24(1):13–21. doi: 10.1128/jvi.24.1.13-21.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Felsenstein D., D'Amico D. J., Hirsch M. S., Neumeyer D. A., Cederberg D. M., de Miranda P., Schooley R. T. Treatment of cytomegalovirus retinitis with 9-[2-hydroxy-1-(hydroxymethyl)ethoxymethyl]guanine. Ann Intern Med. 1985 Sep;103(3):377–380. doi: 10.7326/0003-4819-103-3-377. [DOI] [PubMed] [Google Scholar]
  11. Field A. K., Davies M. E., DeWitt C., Perry H. C., Liou R., Germershausen J., Karkas J. D., Ashton W. T., Johnston D. B., Tolman R. L. 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine: a selective inhibitor of herpes group virus replication. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4139–4143. doi: 10.1073/pnas.80.13.4139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Fyfe J. A., Keller P. M., Furman P. A., Miller R. L., Elion G. B. Thymidine kinase from herpes simplex virus phosphorylates the new antiviral compound, 9-(2-hydroxyethoxymethyl)guanine. J Biol Chem. 1978 Dec 25;253(24):8721–8727. [PubMed] [Google Scholar]
  15. Fyfe J. A., McKee S. A., Keller P. M. Altered thymidine-thymidylate kinases from strains of herpes simplex virus with modified drug sensitivities to acyclovir and (E)-5-(2-bromovinyl)-2'-deoxyuridine. Mol Pharmacol. 1983 Sep;24(2):316–323. [PubMed] [Google Scholar]
  16. Gadler H. Nucleic acid hybridization for measurement of effects of antiviral compounds on human cytomegalovirus DNA replication. Antimicrob Agents Chemother. 1983 Sep;24(3):370–374. doi: 10.1128/aac.24.3.370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Garrett C., Santi D. V. A rapid and sensitive high pressure liquid chromatography assay for deoxyribonucleoside triphosphates in cell extracts. Anal Biochem. 1979 Nov 1;99(2):268–273. doi: 10.1016/s0003-2697(79)80005-6. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. HOGGAN M. D., ROIZMAN B. The isolation and properties of a variant of Herpes simplex producing multinucleated giant cells in monolayer cultures in the presence of antibody. Am J Hyg. 1959 Sep;70:208–219. doi: 10.1093/oxfordjournals.aje.a120071. [DOI] [PubMed] [Google Scholar]
  20. Hall J. D., Coen D. M., Fisher B. L., Weisslitz M., Randall S., Almy R. E., Gelep P. T., Schaffer P. A. Generation of genetic diversity in herpes simplex virus: an antimutator phenotype maps to the DNA polymerase locus. Virology. 1984 Jan 15;132(1):26–37. doi: 10.1016/0042-6822(84)90088-6. [DOI] [PubMed] [Google Scholar]
  21. Halpern M. E., Smiley J. R. Effects of deletions on expression of the herpes simplex virus thymidine kinase gene from the intact viral genome: the amino terminus of the enzyme is dispensable for catalytic activity. J Virol. 1984 Jun;50(3):733–738. doi: 10.1128/jvi.50.3.733-738.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hay J., Subak-Sharpe J. H. Mutants of herpes simplex virus types 1 and 2 that are resistant to phosphonoacetic acid induce altered DNA polymerase activities in infected cells. J Gen Virol. 1976 Apr;31(1):145–148. doi: 10.1099/0022-1317-31-1-145. [DOI] [PubMed] [Google Scholar]
  23. Lewis R. A., Watkins L., St Jeor S. Enhancement of deoxyguanosine kinase activity in human lung fibroblast cells infected with human cytomegalovirus. Mol Cell Biochem. 1984 Nov;65(1):67–71. doi: 10.1007/BF00226020. [DOI] [PubMed] [Google Scholar]
  24. Mar E. C., Chiou J. F., Cheng Y. C., Huang E. S. Inhibition of cellular DNA polymerase alpha and human cytomegalovirus-induced DNA polymerase by the triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 9-(1,3-dihydroxy-2-propoxymethyl)guanine. J Virol. 1985 Mar;53(3):776–780. doi: 10.1128/jvi.53.3.776-780.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mocarski E. S., Post L. E., Roizman B. Molecular engineering of the herpes simplex virus genome: insertion of a second L-S junction into the genome causes additional genome inversions. Cell. 1980 Nov;22(1 Pt 1):243–255. doi: 10.1016/0092-8674(80)90172-5. [DOI] [PubMed] [Google Scholar]
  26. Post L. E., Mackem S., Roizman B. Regulation of alpha genes of herpes simplex virus: expression of chimeric genes produced by fusion of thymidine kinase with alpha gene promoters. Cell. 1981 May;24(2):555–565. doi: 10.1016/0092-8674(81)90346-9. [DOI] [PubMed] [Google Scholar]
  27. ROWE W. P., HARTLEY J. W., WATERMAN S., TURNER H. C., HUEBNER R. J. Cytopathogenic agent resembling human salivary gland virus recovered from tissue cultures of human adenoids. Proc Soc Exp Biol Med. 1956 Jun;92(2):418–424. [PubMed] [Google Scholar]
  28. Schaffer P. A., Carter V. C., Timbury M. C. Collaborative complementation study of temperature-sensitive mutants of herpes simplex virus types 1 and 2. J Virol. 1978 Sep;27(3):490–504. doi: 10.1128/jvi.27.3.490-504.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shepp D. H., Dandliker P. S., de Miranda P., Burnette T. C., Cederberg D. M., Kirk L. E., Meyers J. D. Activity of 9-[2-hydroxy-1-(hydroxymethyl)ethoxymethyl]guanine in the treatment of cytomegalovirus pneumonia. Ann Intern Med. 1985 Sep;103(3):368–373. doi: 10.7326/0003-4819-103-3-368. [DOI] [PubMed] [Google Scholar]
  30. Smee D. F. Interaction of 9-(1,3-dihydroxy-2-propoxymethyl)guanine with cytosol and mitochondrial deoxyguanosine kinases: possible role in anti-cytomegalovirus activity. Mol Cell Biochem. 1985 Nov;69(1):75–81. doi: 10.1007/BF00225929. [DOI] [PubMed] [Google Scholar]
  31. Suzuki S., Saneyoshi M., Nakayama C., Nishiyama Y., Yoshida S. Mechanism of selective inhibition of human cytomegalovirus replication by 1-beta-D-arabinofuranosyl-5-fluorouracil. Antimicrob Agents Chemother. 1985 Aug;28(2):326–330. doi: 10.1128/aac.28.2.326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Varmuza S. L., Smiley J. R. Signals for site-specific cleavage of HSV DNA: maturation involves two separate cleavage events at sites distal to the recognition sequences. Cell. 1985 Jul;41(3):793–802. doi: 10.1016/s0092-8674(85)80060-x. [DOI] [PubMed] [Google Scholar]
  33. Yamanishi K., Rapp F. Induction of host DNA synthesis and DNA polymerase by DNA-negative temperature-sensitive mutants of human cytomegalovirus. Virology. 1979 Apr 15;94(1):237–241. doi: 10.1016/0042-6822(79)90457-4. [DOI] [PubMed] [Google Scholar]
  34. Yamanishi K., Rapp F. Temperature-sensitive mutants of human cytomegalovirus. J Virol. 1977 Oct;24(1):416–418. doi: 10.1128/jvi.24.1.416-418.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Závada V., Erban V., Rezácová D., Vonka V. Thymidine-kinase in cytomegalovirus infected cells. Arch Virol. 1976;52(4):333–339. doi: 10.1007/BF01315622. [DOI] [PubMed] [Google Scholar]

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