Drug-resistant strains of herpes simplex virus type 1 (HSV-1) were selected under the pressure of ()-3-hydroxy-2-phosphonylmethoxypropyl (HPMP) derivatives of cytosine (HPMPC, cidofovir) and adenine (HPMPA) and 2-phosphonylmethoxyethyl (PME) derivatives of adenine (PMEA, adefovir) and 2,6-diaminopurine (PMEDAP). HPMPC-resistant (HPMPC) and HPMPA strains were cross-resistant to one another, but they remained sensitive to foscarnet (PFA), acyclovir (ACV) and the PME derivatives, while the PMEA and PMEDAP strains showed cross-resistance to PFA and ACV. The PMEA, PMEDAP and PFA mutants all revealed a single nucleotide change resulting in a Ser-724 to Asn mutation within the conserved region II of the DNA polymerase. Two HPMPA clones and one HPMPC clone possessed single amino acid changes in the DNA polymerase (HPMPA clone D1, Leu-1007 to Met; HPMPA clone B5, Ile-1028 to Thr; HPMPC clone C3, Val-573 to Met). The HPMPC clone A4 contained two mutations, Ala-136 to Thr and Arg-700 to Met. The mutation at position 136, located outside the catalytic domain of the enzyme, was not detected in other HPMPC clones, suggesting that this mutation may not be responsible for the resistant phenotype. Residue 573 is located within the 3′→5′ exonuclease editing domain close to the catalytically important residues Tyr-577 and Asp-581. Similarly, residue 700 is located in the palm subdomain of the catalytic domain, adjacent to the Asp residues 717, 886 and 888 that are vital for polymerase activity. The HPMPA mutations at residues 1007 and 1028, beyond the last conserved region, still fall within the thumb subdomain of the catalytic domain. The different drug-resistant mutants varied in neurovirulent behaviour, the HPMPC strains showing reduced neurovirulence compared with the wild-type.


Article metrics loading...

Loading full text...

Full text loading...



  1. Andrei, G., Snoeck, R. & De Clercq, E. (1995). Susceptibilities of several drug-resistant herpes simplex virus type 1 strains to alternative antiviral compounds. Antimicrobial Agents and Chemotherapy 39, 1632-1635.[CrossRef] [Google Scholar]
  2. Chatis, P. A. & Crumpacker, C. S. (1991). Analysis of the thymidine kinase gene from clinically isolated acyclovir-resistant herpes simplex viruses. Virology 180, 793-797.[CrossRef] [Google Scholar]
  3. Chen, E. Y. & Seeburg, P. H. (1985). Supercoil sequencing: a fast and simple method for sequencing plasmid DNA.DNA 4, 165-170.[CrossRef] [Google Scholar]
  4. Chiou, H. C., Weller, S. K. & Coen, D. M. (1985). Mutations in the herpes simplex virus major DNA-binding protein gene leading to altered sensitivity to DNA polymerase inhibitors. Virology 145, 213-226.[CrossRef] [Google Scholar]
  5. Chiou, H. C., Kumura, K., Hu, A., Kerns, K. M. & Coen, D. M. (1995). Penciclovir-resistance mutations in the herpes simplex virus DNA polymerase gene. Antiviral Chemistry & Chemotherapy 6, 281-288.[CrossRef] [Google Scholar]
  6. Chou, J., Kern, E. R., Whitley, R. J. & Roizman, B. (1990). Mapping of herpes simplex virus-1 neurovirulence to gamma-134.5, a gene nonessential for growth in culture. Science 250, 1262-1266.[CrossRef] [Google Scholar]
  7. Coen, D. M. (1996a). Nucleosides and foscarnet – mechanisms. In Antiviral Drug Resistance, pp. 81-102. Edited by D. D. Richman. Chichester: John Wiley.
  8. Coen, D. M. (1996b). Viral DNA polymerases. In DNA Replication in Eukaryotic Cells, pp. 495-523. Edited by M. De Pamphilis. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  9. Collins, P. & Darby, G. (1991). Laboratory studies of herpes simplex virus strains resistant to acyclovir. Reviews in Medical Virology 1, 19-28.[CrossRef] [Google Scholar]
  10. Collins, P., Larder, B. A., Oliver, N. M., Kemp, S., Smith, I. W. & Darby, G. (1989). Characterization of a DNA polymerase mutant of herpes simplex virus from a severely immunocompromised patient receiving acyclovir. Journal of General Virology 70, 375-382.[CrossRef] [Google Scholar]
  11. De Clercq, E., Holy, A., Rosenberg, I., Sakuma, T., Balzarini, J. & Maudgal, P. C. (1986). A novel selective broad-spectrum anti-DNA virus agent. Nature 323, 464-467.[CrossRef] [Google Scholar]
  12. De Clercq, E., Sakuma, T., Baba, M., Pauwels, R., Balzarini, J., Rosenberg, I. & Holy, A. (1987). Antiviral activity of phosphonylmethoxyalkyl derivatives of purine and pyrimidines. Antiviral Research 8, 261-272.[CrossRef] [Google Scholar]
  13. Digard, P., Bebrin, W. R., Weisshart, K. & Coen, D. M. (1993). The extreme C terminus of herpes simplex virus DNA polymerase is crucial for functional interaction with processivity factor UL42 and for viral replication. Journal of Virology 67, 398-406. [Google Scholar]
  14. Earl, P. L., Jones, E. V. & Moss, B. (1986). Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene. Proceedings of the National Academy of Sciences, USA 83, 3659-3663.[CrossRef] [Google Scholar]
  15. Ellis, M. N., Keller, P. M., Fyfe, J. A., Martin, J. L., Rooney, J. F., Straus, S. E., Lehrman, S. N. & Barry, D. W. (1987). Clinical isolate of herpes simplex virus type 2 that induces a thymidine kinase with altered substrate specificity. Antimicrobial Agents and Chemotherapy 31, 1117-1125.[CrossRef] [Google Scholar]
  16. Field, H. J. & Coen, D. M. (1986). Pathogenicity of herpes simplex virus mutants containing drug resistance mutations in the viral DNA polymerase gene. Journal of Virology 60, 286-289. [Google Scholar]
  17. Field, H. J. & Darby, G. (1980). Pathogenicity in mice of strains of herpes simplex virus which are resistant to acyclovir in vitro and in vivo. Antimicrobial Agents and Chemotherapy 17, 209-216.[CrossRef] [Google Scholar]
  18. Foster, S. A., Cerny, J. & Cheng, Y. C. (1991). Herpes simplex virus-specified DNA polymerase is the target for the antiviral action of 9-(2-phosphonylmethoxyethyl)adenine. Journal of Biological Chemistry 266, 238-244. [Google Scholar]
  19. Gibbs, J. S., Chiou, H. C., Bastow, K. F., Cheng, Y. C. & Coen, D. M. (1988). Identification of amino acids in herpes simplex virus DNA polymerase involved in substrate and drug recognition. Proceedings of the National Academy of Sciences, USA 85, 6672-6676.[CrossRef] [Google Scholar]
  20. Hanahan, D. (1985). Techniques for transformation of E. coli. In DNA Cloning: A Practical Approach, pp. 109-138. Edited by D. M. Glover. Oxford: IRL Press.
  21. Hill, E. L., Hunter, G. A. & Ellis, M. N. (1991). In vitro and in vivo characterization of herpes simplex virus clinical isolates recovered from patients infected with human immunodeficiency virus. Antimicrobial Agents and Chemotherapy 35, 2322-2328.[CrossRef] [Google Scholar]
  22. Hitchcock, M. J. M., Jaffe, H. S., Martin, J. C. & Stagg, R. J. (1996). Cidofovir, a new agent with potent anti-herpesvirus activity. Antiviral Chemistry & Chemotherapy 7, 115-127.[CrossRef] [Google Scholar]
  23. Ho, H.-T., Woods, K. L., Bronson, J. J., De Boeck, H., Martin, J. C. & Hitchcock, M. J. M. (1992). Intracellular metabolism of the antiherpes agent (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine. Molecular Pharmacology 41, 197-202. [Google Scholar]
  24. Hwang, C. B. C., Ruffner, K. L. & Coen, D. M. (1992). A point mutation within a distinct conserved region of the herpes simplex virus DNA polymerase gene confers drug resistance. Journal of Virology 66, 1774-1776. [Google Scholar]
  25. Knopf, C. W. & Weisshart, K. (1988). The herpes simplex virus DNA polymerase: analysis of the functional domains. Biochimica et Biophysica Acta 951, 298-314.[CrossRef] [Google Scholar]
  26. Lalezari, J., Schacker, T., Feinberg, J., Gathe, J., Lee, S., Cheung, T., Kramer, F., Kessler, H., Corey, L., Drew, W. L., Boggs, J., McGuire, B., Jaffe, H. S. & Safrin, S. (1997). A randomized, double-blind, placebo-controlled trial of cidofovir gel for the treatment of acyclovir-unresponsive mucocutaneous herpes simplex virus infection in patients with AIDS. Journal of Infectious Diseases 176, 892-898.[CrossRef] [Google Scholar]
  27. Larder, B. A., Kemp, S. D. & Darby, G. (1987). Related functional domains in virus DNA polymerases. EMBO Journal 6, 169-175. [Google Scholar]
  28. McLaren, C., Chen, M. S., Ghazzouli, I., Saral, R. & Burns, W. H. (1985). Drug resistance patterns of herpes simplex virus isolates from patients treated with acyclovir. Antimicrobial Agents and Chemotherapy 28, 740-744.[CrossRef] [Google Scholar]
  29. Marcy, A. I., Hwang, C. B. C., Ruffner, K. L. & Coen, D. M. (1990). Engineered herpes simplex virus DNA polymerase point mutants: the most highly conserved region shared among α-like DNA polymerases is involved in substrate recognition.Journal of Virology 64, 5883-5890. [Google Scholar]
  30. Mendel, D. B., Barkhimer, D. B. & Chen, M. S. (1995). Biochemical basis for increased susceptibility to Cidofovir of herpes simplex viruses with altered or deficient thymidine kinase activity. Antimicrobial Agents and Chemotherapy 39, 2120-2122.[CrossRef] [Google Scholar]
  31. Mendel, D. B., Tai, C. Y., Barkhimer, D. B., Kern, E. R. & Chen, M. S. (1997). Characterization of an in vitro selected herpes simplex virus type 2 (HSV-2) strain with decreased susceptibility to cidofovir. Fourth Conference on Retroviruses and Opportunistic Infections, Washington, DC, USA (abstract 681).
  32. Merta, A., Votruba, I., Rosenberg, I., Otmar, M., Hrebabecký, H., Bernaerts, R. & Holý, A. (1990). Inhibition of herpes simplex virus DNA polymerase by diphosphates of acyclic phosphonylmethoxyalkyl nucleotide analogues. Antiviral Research 13, 209-218.[CrossRef] [Google Scholar]
  33. Naesens, L., Snoeck, R., Andrei, G., Balzarini, J., Neyts, J. & De Clercq, E. (1997). HPMPC (cidofovir), PMEA (adefovir) and related acyclic nucleoside phosphonate analogues: a review of their pharmacology and clinical potential in the treatment of viral infections. Antiviral Chemistry & Chemotherapy 8, 1-23. [Google Scholar]
  34. Nugier, F., Collins, P., Larder, B. A., Langlois, M., Aymard, M. & Darby, G. (1991). Herpes simplex virus isolates from an immunocompromised patient who failed to respond to acyclovir treatment express thymidine kinase with altered substrate specificity. Antiviral Chemistry & Chemotherapy 2, 295-302.[CrossRef] [Google Scholar]
  35. Parker, A. C., Craig, J. I. O., Collins, P., Oliver, N. & Smith, I. (1987). Acyclovir-resistant herpes simplex virus infection due to altered DNA polymerase. Lancet ii, 1461.
  36. Pelosi, E., Mulamba, G. B. & Coen, D. M. (1998a). Penciclovir and pathogenesis phenotypes of drug-resistant herpes simplex virus mutants. Antiviral Research 37, 17-28.[CrossRef] [Google Scholar]
  37. Pelosi, E., Rozenberg, F., Coen, D. M. & Tyler, K. L. (1998b). A herpes simplex virus DNA polymerase mutation that specifically attenuates neurovirulence in mice. Virology 252, 364-372.[CrossRef] [Google Scholar]
  38. Pyles, R. B., Sawtell, N. M. & Thompson, R. L. (1992). Herpes simplex virus type 1 dUTPase mutants are attenuated for neurovirulence, neuroinvasiveness, and reactivation from latency. Journal of Virology 66, 6706-6713. [Google Scholar]
  39. Sacks, S. L., Wanklin, R. J., Reece, D. E., Hicks, K. A., Tyler, K. L. & Coen, D. M. (1989). Progressive esophagitis from acyclovir-resistant herpes simplex. Clinical roles for DNA polymerase mutants and viral heterogeneity? Annals of Internal Medicine 111, 893-899.[CrossRef] [Google Scholar]
  40. Safrin, S. (1996). Nucleosides and foscarnet – clinical aspects. In Antiviral Drug Resistance, pp. 103-122. Edited by D. D. Richman. Chichester: John Wiley.
  41. Safrin, S., Crumpacker, C., Chatis, P., Davis, R., Hafner, R., Rush, J., Kessler, H. A., Landry, B. & Mills, J. (1991). A controlled trial comparing foscarnet with vidarabine for acyclovir-resistant mucocutaneous herpes simplex in the acquired immunodeficiency syndrome. The AIDS Clinical Trials Group. New England Journal of Medicine 325, 551-555.[CrossRef] [Google Scholar]
  42. Safrin, S., Kemmerly, S., Plotkin, B., Smith, T., Weissbach, N., De Veranez, D., Phan, L. D. & Cohn, D. (1994). Foscarnet-resistant herpes simplex virus infection in patients with AIDS. Journal of Infectious Diseases 169, 193-196.[CrossRef] [Google Scholar]
  43. Safrin, S., Cherrington, J. & Jaffe, H. S. (1997). Clinical uses of cidofovir. Reviews in Medical Virology 7, 145-156.[CrossRef] [Google Scholar]
  44. Snoeck, R., Andrei, G., De Clercq, E., Gerard, M., Clumeck, N., Tricot, G. & Sadzot-Delvaux, C. (1993). A new topical treatment for resistant herpes simplex infections. New England Journal of Medicine 329, 968-969.[CrossRef] [Google Scholar]
  45. Snoeck, R., Andrei, G., Gérard, M., Silverman, A., Hedderman, A., Balzarini, J., Sadzot-Delvaux, C., Tricot, G., Clumeck, N. & De Clercq, E. (1994). Successful treatment of progressive mucocutaneous infection due to acyclovir- and foscarnet-resistant herpes simplex virus with (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC). Clinical Infectious Diseases 18, 570-578.[CrossRef] [Google Scholar]
  46. Tsurumi, T., Maeno, K. & Nishiyama, Y. (1987). A single-base change within the DNA polymerase locus of herpes simplex virus type 2 can confer resistance to aphidicolin. Journal of Virology 61, 388-394. [Google Scholar]
  47. Wang, J., Sattar, A. K., Wang, C. C., Karam, J. D., Konigsberg, W. H. & Steitz, T. A. (1997). Crystal structure of a pol α family replication DNA polymerase from bacteriophage RB69. Cell 89, 1087-1099.[CrossRef] [Google Scholar]
  48. Whitley, R. J. & Gnann, J. W.Jr (1992). Acyclovir: a decade later. New England Journal of Medicine 327, 782-789.[CrossRef] [Google Scholar]
  49. Xiong, X., Smith, J. L., Kim, C., Huang, E. S. & Chen, M. S. (1996). Kinetic analysis of the interaction of cidofovir diphosphate with human cytomegalovirus DNA polymerase. Biochemical Pharmacology 51, 1563-1567.[CrossRef] [Google Scholar]
  50. Xiong, X., Smith, J. L. & Chen, M. S. (1997). Effect of incorporation of cidofovir into DNA by human cytomegalovirus DNA polymerase on DNA elongation. Antimicrobial Agents and Chemotherapy 41, 594-599. [Google Scholar]

Data & Media loading...

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