Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Mar;70(3):1390–1395. doi: 10.1128/jvi.70.3.1390-1395.1996

Single amino acid changes in the DNA polymerase confer foscarnet resistance and slow-growth phenotype, while mutations in the UL97-encoded phosphotransferase confer ganciclovir resistance in three double-resistant human cytomegalovirus strains recovered from patients with AIDS.

F Baldanti 1, M R Underwood 1, S C Stanat 1, K K Biron 1, S Chou 1, A Sarasini 1, E Silini 1, G Gerna 1
PMCID: PMC189958  PMID: 8627655

Abstract

Three human cytomegalovirus (HCMV) strains (VR4760, VR4955, and VR5120) showing double resistance to ganciclovir (GCV) and foscarnet (PFA) were isolated from three patients with AIDS who underwent multiple sequential courses of therapy with GCV and PFA (A. Sarasini, F. Baldanti, M. Furione, E. Percivalle, R. Brerra, M. Barbi, and G. Gerna, J. Med. Virol., 47:237-244, 1995). We previously demonstrated that the three strains were genetically unrelated and that each of them was present as a single viral population in vivo. Thus, in each of the three cases, a single viral strain was resistant to both GCV and PFA. In the present paper, we report the characterization of the molecular bases of the double resistance and demonstrate that the PFA resistance is associated with a slower replication of HCMV strains in cell cultures. Sequencing of the UL97 and UL54 genes, GCV anabolism assays, and marker transfer experiments showed that GCV resistance was due to single amino acid changes in the UL97 gene product (VR4760, Met-460 --> Ile; VR4955, Ala-594 --> Val; VR5120, Leu595 --> Ser), while single amino acid changes in domain II of the DNA polymerase (VR4760 and VR5120, Val-715 --> Met; VR4955, Thr-700 --> Ala) were responsible for both the PFA resistance and the slow-growth phenotype. Thus, in these three cases, double resistance to GCV and PFA was not due to a single mutation conferring cross-resistance or to the presence of a mixture of strains with different drug susceptibilities. The HCMV DNA polymerase recombinant strains carrying the mutations conferring PFA resistance were sensitive to GCV and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC). In addition, the same UL54 mutations were responsible for the slow growth of the clinical isolates, since the recombinant strains showed a marked delay in immediate-early antigen plaque formation and a reduction of infectious virus yield compared with AD169, from which they were derived. These results may have some important implications for the successful isolation, propagation, and characterization of PFA-resistant strains from clinical samples containing mixed viral populations.

Full Text

The Full Text of this article is available as a PDF (375.7 KB).

Selected References

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

  1. Baldanti F., Silini E., Sarasini A., Talarico C. L., Stanat S. C., Biron K. K., Furione M., Bono F., Palù G., Gerna G. A three-nucleotide deletion in the UL97 open reading frame is responsible for the ganciclovir resistance of a human cytomegalovirus clinical isolate. J Virol. 1995 Feb;69(2):796–800. doi: 10.1128/jvi.69.2.796-800.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chee M. S., Lawrence G. L., Barrell B. G. Alpha-, beta- and gammaherpesviruses encode a putative phosphotransferase. J Gen Virol. 1989 May;70(Pt 5):1151–1160. doi: 10.1099/0022-1317-70-5-1151. [DOI] [PubMed] [Google Scholar]
  3. Chou S., Erice A., Jordan M. C., Vercellotti G. M., Michels K. R., Talarico C. L., Stanat S. C., Biron K. K. Analysis of the UL97 phosphotransferase coding sequence in clinical cytomegalovirus isolates and identification of mutations conferring ganciclovir resistance. J Infect Dis. 1995 Mar;171(3):576–583. doi: 10.1093/infdis/171.3.576. [DOI] [PubMed] [Google Scholar]
  4. Chrisp P., Clissold S. P. Foscarnet. A review of its antiviral activity, pharmacokinetic properties and therapeutic use in immunocompromised patients with cytomegalovirus retinitis. Drugs. 1991 Jan;41(1):104–129. doi: 10.2165/00003495-199141010-00009. [DOI] [PubMed] [Google Scholar]
  5. D'Aquila R. T., Summers W. C. Isolation and characterization of phosphonoacetic acid-resistant mutants of human cytomegalovirus. J Virol. 1987 Apr;61(4):1291–1295. doi: 10.1128/jvi.61.4.1291-1295.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Drew W. L., Miner R. C., Busch D. F., Follansbee S. E., Gullett J., Mehalko S. G., Gordon S. M., Owen W. F., Jr, Matthews T. R., Buhles W. C. Prevalence of resistance in patients receiving ganciclovir for serious cytomegalovirus infection. J Infect Dis. 1991 Apr;163(4):716–719. doi: 10.1093/infdis/163.4.716. [DOI] [PubMed] [Google Scholar]
  7. Drobyski W. R., Knox K. K., Carrigan D. R., Ash R. C. Foscarnet therapy of ganciclovir-resistant cytomegalovirus in marrow transplantation. Transplantation. 1991 Jul;52(1):155–157. [PubMed] [Google Scholar]
  8. Erice A., Chou S., Biron K. K., Stanat S. C., Balfour H. H., Jr, Jordan M. C. Progressive disease due to ganciclovir-resistant cytomegalovirus in immunocompromised patients. N Engl J Med. 1989 Feb 2;320(5):289–293. doi: 10.1056/NEJM198902023200505. [DOI] [PubMed] [Google Scholar]
  9. Field A. K., Biron K. K. "The end of innocence" revisited: resistance of herpesviruses to antiviral drugs. Clin Microbiol Rev. 1994 Jan;7(1):1–13. doi: 10.1128/cmr.7.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Gerna G., Baldanti F., Zavattoni M., Sarasini A., Percivalle E., Revello M. G. Monitoring of ganciclovir sensitivity of multiple human cytomegalovirus strains coinfecting blood of an AIDS patient by an immediate-early antigen plaque assay. Antiviral Res. 1992 Oct 1;19(4):333–345. doi: 10.1016/0166-3542(92)90014-v. [DOI] [PubMed] [Google Scholar]
  12. Gerna G., Sarasini A., Percivalle E., Zavattoni M., Baldanti F., Revello M. G. Rapid screening for resistance to ganciclovir and foscarnet of primary isolates of human cytomegalovirus from culture-positive blood samples. J Clin Microbiol. 1995 Mar;33(3):738–741. doi: 10.1128/jcm.33.3.738-741.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Jacobson M. A., Drew W. L., Feinberg J., O'Donnell J. J., Whitmore P. V., Miner R. D., Parenti D. Foscarnet therapy for ganciclovir-resistant cytomegalovirus retinitis in patients with AIDS. J Infect Dis. 1991 Jun;163(6):1348–1351. doi: 10.1093/infdis/163.6.1348. [DOI] [PubMed] [Google Scholar]
  15. Knox K. K., Drobyski W. R., Carrigan D. R. Cytomegalovirus isolate resistant to ganciclovir and foscarnet from a marrow transplant patient. Lancet. 1991 May 25;337(8752):1292–1293. doi: 10.1016/0140-6736(91)92965-5. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Littler E., Stuart A. D., Chee M. S. Human cytomegalovirus UL97 open reading frame encodes a protein that phosphorylates the antiviral nucleoside analogue ganciclovir. Nature. 1992 Jul 9;358(6382):160–162. doi: 10.1038/358160a0. [DOI] [PubMed] [Google Scholar]
  19. Lurain N. S., Spafford L. E., Thompson K. D. Mutation in the UL97 open reading frame of human cytomegalovirus strains resistant to ganciclovir. J Virol. 1994 Jul;68(7):4427–4431. doi: 10.1128/jvi.68.7.4427-4431.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lurain N. S., Thompson K. D., Holmes E. W., Read G. S. Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents. J Virol. 1992 Dec;66(12):7146–7152. doi: 10.1128/jvi.66.12.7146-7152.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Sarasini A., Baldanti F., Furione M., Percivalle E., Brerra R., Barbi M., Gerna G. Double resistance to ganciclovir and foscarnet of four human cytomegalovirus strains recovered from AIDS patients. J Med Virol. 1995 Nov;47(3):237–244. doi: 10.1002/jmv.1890470309. [DOI] [PubMed] [Google Scholar]
  23. Stanat S. C., Reardon J. E., Erice A., Jordan M. C., Drew W. L., Biron K. K. Ganciclovir-resistant cytomegalovirus clinical isolates: mode of resistance to ganciclovir. Antimicrob Agents Chemother. 1991 Nov;35(11):2191–2197. doi: 10.1128/aac.35.11.2191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sullivan V., Biron K. K., Talarico C., Stanat S. C., Davis M., Pozzi L. M., Coen D. M. A point mutation in the human cytomegalovirus DNA polymerase gene confers resistance to ganciclovir and phosphonylmethoxyalkyl derivatives. Antimicrob Agents Chemother. 1993 Jan;37(1):19–25. doi: 10.1128/aac.37.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sullivan V., Coen D. M. Isolation of foscarnet-resistant human cytomegalovirus patterns of resistance and sensitivity to other antiviral drugs. J Infect Dis. 1991 Oct;164(4):781–784. doi: 10.1093/infdis/164.4.781. [DOI] [PubMed] [Google Scholar]
  26. Sullivan V., Talarico C. L., Stanat S. C., Davis M., Coen D. M., Biron K. K. A protein kinase homologue controls phosphorylation of ganciclovir in human cytomegalovirus-infected cells. Nature. 1992 Jul 9;358(6382):162–164. doi: 10.1038/358162a0. [DOI] [PubMed] [Google Scholar]
  27. Tatarowicz W. A., Lurain N. S., Thompson K. D. A ganciclovir-resistant clinical isolate of human cytomegalovirus exhibiting cross-resistance to other DNA polymerase inhibitors. J Infect Dis. 1992 Oct;166(4):904–907. doi: 10.1093/infdis/166.4.904. [DOI] [PubMed] [Google Scholar]
  28. Teo I. A., Griffin B. E., Jones M. D. Characterization of the DNA polymerase gene of human herpesvirus 6. J Virol. 1991 Sep;65(9):4670–4680. doi: 10.1128/jvi.65.9.4670-4680.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zipeto D., Revello M. G., Silini E., Parea M., Percivalle E., Zavattoni M., Milanesi G., Gerna G. Development and clinical significance of a diagnostic assay based on the polymerase chain reaction for detection of human cytomegalovirus DNA in blood samples from immunocompromised patients. J Clin Microbiol. 1992 Feb;30(2):527–530. doi: 10.1128/jcm.30.2.527-530.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES