Skip to main content
PMC home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1993 Jul;37(7):1480–1487. doi: 10.1128/aac.37.7.1480

pol mutations conferring zidovudine and didanosine resistance with different effects in vitro yield multiply resistant human immunodeficiency virus type 1 isolates in vivo.

J J Eron 1, Y K Chow 1, A M Caliendo 1, J Videler 1, K M Devore 1, T P Cooley 1, H A Liebman 1, J C Kaplan 1, M S Hirsch 1, R T D'Aquila 1
PMCID: PMC187998  PMID: 7689822

Abstract

Specific mutations in the human immunodeficiency virus type 1 (HIV-1) pol gene that cause zidovudine (3'-azido-2',3'-dideoxythymidine; AZT) and didanosine (2',3'-dideoxyinosine; ddI) resistance were studied. The 50% inhibitory concentrations (IC50s) of nucleosides for cloned viruses containing these mutations were compared with the IC50s of the corresponding triphosphate analogs for mutant recombinant-expressed reverse transcriptases (RTs). Changes in ddATP inhibition of RNA-dependent DNA polymerase activity fully accounted for the ddI resistance of the virus caused by a Leu-74-->Val substitution in RT, including an augmentation by the AZT-selected substitutions Thr-215-->Tyr and Lys-219-->Gln in RT. In contrast, the AZT-selected substitutions studied did not cause as great a change in the IC50 of AZT-triphosphate (AZT-TP) for polymerase as they did in the IC50 of AZT for mutant virus. In addition, the mutation at codon 74 suppressed AZT resistance in the virus caused by the mutations at codons 215 and 219 but did not suppress the AZT-TP resistance of enzyme containing these same mutations in RT. The mutation at codon 74 was found in clinical isolates whether or not the patient had received AZT prior to starting ddI therapy. AZT resistance coexisted with ddI resistance following acquisition of Leu-74-->Val in three clinical isolates, indicating that the suppressive effect of Val-74 on the AZT resistance of the virus does not occur in all genetic contexts. When this suppression of AZT resistance was seen in the virus, Val-74 did not appear to cause mutually exclusive changes in AZT-TP and ddATP binding to RT in vitro. The results of the in vitro experiments and characterization of clinical isolates suggest that there are differences in the functional effects of these AZT and ddI resistance mutations.

Full text

PDF
1487

Selected References

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

  1. Casanova J. L., Pannetier C., Jaulin C., Kourilsky P. Optimal conditions for directly sequencing double-stranded PCR products with sequenase. Nucleic Acids Res. 1990 Jul 11;18(13):4028–4028. doi: 10.1093/nar/18.13.4028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chow Y. K., Hirsch M. S., Merrill D. P., Bechtel L. J., Eron J. J., Kaplan J. C., D'Aquila R. T. Use of evolutionary limitations of HIV-1 multidrug resistance to optimize therapy. Nature. 1993 Feb 18;361(6413):650–654. doi: 10.1038/361650a0. [DOI] [PubMed] [Google Scholar]
  3. Cooley T. P., Kunches L. M., Saunders C. A., Ritter J. K., Perkins C. J., McLaren C., McCaffrey R. P., Liebman H. A. Once-daily administration of 2',3'-dideoxyinosine (ddI) in patients with the acquired immunodeficiency syndrome or AIDS-related complex. Results of a Phase I trial. N Engl J Med. 1990 May 10;322(19):1340–1345. doi: 10.1056/NEJM199005103221902. [DOI] [PubMed] [Google Scholar]
  4. D'Aquila R. T., Summers W. C. HIV-1 reverse transcriptase/ribonuclease H: high level expression in Escherichia coli from a plasmid constructed using the polymerase chain reaction. J Acquir Immune Defic Syndr. 1989;2(6):579–587. [PubMed] [Google Scholar]
  5. Eron J. J., Gorczyca P., Kaplan J. C., D'Aquila R. T. Susceptibility testing by polymerase chain reaction DNA quantitation: a method to measure drug resistance of human immunodeficiency virus type 1 isolates. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3241–3245. doi: 10.1073/pnas.89.8.3241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eron J. J., Jr, Johnson V. A., Merrill D. P., Chou T. C., Hirsch M. S. Synergistic inhibition of replication of human immunodeficiency virus type 1, including that of a zidovudine-resistant isolate, by zidovudine and 2',3'-dideoxycytidine in vitro. Antimicrob Agents Chemother. 1992 Jul;36(7):1559–1562. doi: 10.1128/aac.36.7.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fischl M. A., Richman D. D., Hansen N., Collier A. C., Carey J. T., Para M. F., Hardy W. D., Dolin R., Powderly W. G., Allan J. D. The safety and efficacy of zidovudine (AZT) in the treatment of subjects with mildly symptomatic human immunodeficiency virus type 1 (HIV) infection. A double-blind, placebo-controlled trial. The AIDS Clinical Trials Group. Ann Intern Med. 1990 May 15;112(10):727–737. doi: 10.7326/0003-4819-112-10-727. [DOI] [PubMed] [Google Scholar]
  8. Fitzgibbon J. E., Howell R. M., Haberzettl C. A., Sperber S. J., Gocke D. J., Dubin D. T. Human immunodeficiency virus type 1 pol gene mutations which cause decreased susceptibility to 2',3'-dideoxycytidine. Antimicrob Agents Chemother. 1992 Jan;36(1):153–157. doi: 10.1128/aac.36.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gao Q., Gu Z. X., Parniak M. A., Li X. G., Wainberg M. A. In vitro selection of variants of human immunodeficiency virus type 1 resistant to 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine. J Virol. 1992 Jan;66(1):12–19. doi: 10.1128/jvi.66.1.12-19.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Harada S., Koyanagi Y., Yamamoto N. Infection of HTLV-III/LAV in HTLV-I-carrying cells MT-2 and MT-4 and application in a plaque assay. Science. 1985 Aug 9;229(4713):563–566. doi: 10.1126/science.2992081. [DOI] [PubMed] [Google Scholar]
  11. Johnson V. A., Merrill D. P., Videler J. A., Chou T. C., Byington R. E., Eron J. J., D'Aquila R. T., Hirsch M. S. Two-drug combinations of zidovudine, didanosine, and recombinant interferon-alpha A inhibit replication of zidovudine-resistant human immunodeficiency virus type 1 synergistically in vitro. J Infect Dis. 1991 Oct;164(4):646–655. doi: 10.1093/infdis/164.4.646. [DOI] [PubMed] [Google Scholar]
  12. Kahn J. O., Lagakos S. W., Richman D. D., Cross A., Pettinelli C., Liou S. H., Brown M., Volberding P. A., Crumpacker C. S., Beall G. A controlled trial comparing continued zidovudine with didanosine in human immunodeficiency virus infection. The NIAID AIDS Clinical Trials Group. N Engl J Med. 1992 Aug 27;327(9):581–587. doi: 10.1056/NEJM199208273270901. [DOI] [PubMed] [Google Scholar]
  13. Kellam P., Boucher C. A., Larder B. A. Fifth mutation in human immunodeficiency virus type 1 reverse transcriptase contributes to the development of high-level resistance to zidovudine. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1934–1938. doi: 10.1073/pnas.89.5.1934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kohlstaedt L. A., Steitz T. A. Reverse transcriptase of human immunodeficiency virus can use either human tRNA(3Lys) or Escherichia coli tRNA(2Gln) as a primer in an in vitro primer-utilization assay. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9652–9656. doi: 10.1073/pnas.89.20.9652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  16. Kornfeld H., Riedel N., Viglianti G. A., Hirsch V., Mullins J. I. Cloning of HTLV-4 and its relation to simian and human immunodeficiency viruses. Nature. 1987 Apr 9;326(6113):610–613. doi: 10.1038/326610a0. [DOI] [PubMed] [Google Scholar]
  17. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  18. Lacey S. F., Reardon J. E., Furfine E. S., Kunkel T. A., Bebenek K., Eckert K. A., Kemp S. D., Larder B. A. Biochemical studies on the reverse transcriptase and RNase H activities from human immunodeficiency virus strains resistant to 3'-azido-3'-deoxythymidine. J Biol Chem. 1992 Aug 5;267(22):15789–15794. [PubMed] [Google Scholar]
  19. Land S., McGavin C., Lucas R., Birch C. Incidence of zidovudine-resistant human immunodeficiency virus isolated from patients before, during, and after therapy. J Infect Dis. 1992 Nov;166(5):1139–1142. doi: 10.1093/infdis/166.5.1139. [DOI] [PubMed] [Google Scholar]
  20. Land S., Terloar G., McPhee D., Birch C., Doherty R., Cooper D., Gust I. Decreased in vitro susceptibility to zidovudine of HIV isolates obtained from patients with AIDS. J Infect Dis. 1990 Feb;161(2):326–329. doi: 10.1093/infdis/161.2.326. [DOI] [PubMed] [Google Scholar]
  21. Larder B. A. 3'-Azido-3'-deoxythymidine resistance suppressed by a mutation conferring human immunodeficiency virus type 1 resistance to nonnucleoside reverse transcriptase inhibitors. Antimicrob Agents Chemother. 1992 Dec;36(12):2664–2669. doi: 10.1128/aac.36.12.2664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Larder B. A., Darby G., Richman D. D. HIV with reduced sensitivity to zidovudine (AZT) isolated during prolonged therapy. Science. 1989 Mar 31;243(4899):1731–1734. doi: 10.1126/science.2467383. [DOI] [PubMed] [Google Scholar]
  23. Larder B. A., Kemp S. D. Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). Science. 1989 Dec 1;246(4934):1155–1158. doi: 10.1126/science.2479983. [DOI] [PubMed] [Google Scholar]
  24. McLeod G. X., McGrath J. M., Ladd E. A., Hammer S. M. Didanosine and zidovudine resistance patterns in clinical isolates of human immunodeficiency virus type 1 as determined by a replication endpoint concentration assay. Antimicrob Agents Chemother. 1992 May;36(5):920–925. doi: 10.1128/aac.36.5.920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mitsuya H., Yarchoan R., Broder S. Molecular targets for AIDS therapy. Science. 1990 Sep 28;249(4976):1533–1544. doi: 10.1126/science.1699273. [DOI] [PubMed] [Google Scholar]
  26. Ratner L., Fisher A., Jagodzinski L. L., Mitsuya H., Liou R. S., Gallo R. C., Wong-Staal F. Complete nucleotide sequences of functional clones of the AIDS virus. AIDS Res Hum Retroviruses. 1987 Spring;3(1):57–69. doi: 10.1089/aid.1987.3.57. [DOI] [PubMed] [Google Scholar]
  27. Richman D. D., Fischl M. A., Grieco M. H., Gottlieb M. S., Volberding P. A., Laskin O. L., Leedom J. M., Groopman J. E., Mildvan D., Hirsch M. S. The toxicity of azidothymidine (AZT) in the treatment of patients with AIDS and AIDS-related complex. A double-blind, placebo-controlled trial. N Engl J Med. 1987 Jul 23;317(4):192–197. doi: 10.1056/NEJM198707233170402. [DOI] [PubMed] [Google Scholar]
  28. Rooke R., Parniak M. A., Tremblay M., Soudeyns H., Li X. G., Gao Q., Yao X. J., Wainberg M. A. Biological comparison of wild-type and zidovudine-resistant isolates of human immunodeficiency virus type 1 from the same subjects: susceptibility and resistance to other drugs. Antimicrob Agents Chemother. 1991 May;35(5):988–991. doi: 10.1128/aac.35.5.988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rooke R., Tremblay M., Soudeyns H., DeStephano L., Yao X. J., Fanning M., Montaner J. S., O'Shaughnessy M., Gelmon K., Tsoukas C. Isolation of drug-resistant variants of HIV-1 from patients on long-term zidovudine therapy. Canadian Zidovudine Multi-Centre Study Group. AIDS. 1989 Jul;3(7):411–415. doi: 10.1097/00002030-198907000-00001. [DOI] [PubMed] [Google Scholar]
  30. St Clair M. H., Martin J. L., Tudor-Williams G., Bach M. C., Vavro C. L., King D. M., Kellam P., Kemp S. D., Larder B. A. Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase. Science. 1991 Sep 27;253(5027):1557–1559. doi: 10.1126/science.1716788. [DOI] [PubMed] [Google Scholar]
  31. Tudor-Williams G., St Clair M. H., McKinney R. E., Maha M., Walter E., Santacroce S., Mintz M., O'Donnell K., Rudoll T., Vavro C. L. HIV-1 sensitivity to zidovudine and clinical outcome in children. Lancet. 1992 Jan 4;339(8784):15–19. doi: 10.1016/0140-6736(92)90140-x. [DOI] [PubMed] [Google Scholar]
  32. Volberding P. A., Lagakos S. W., Koch M. A., Pettinelli C., Myers M. W., Booth D. K., Balfour H. H., Jr, Reichman R. C., Bartlett J. A., Hirsch M. S. Zidovudine in asymptomatic human immunodeficiency virus infection. A controlled trial in persons with fewer than 500 CD4-positive cells per cubic millimeter. The AIDS Clinical Trials Group of the National Institute of Allergy and Infectious Diseases. N Engl J Med. 1990 Apr 5;322(14):941–949. doi: 10.1056/NEJM199004053221401. [DOI] [PubMed] [Google Scholar]
  33. Wahlberg J., Albert J., Lundeberg J., Cox S., Wahren B., Uhlén M. Dynamic changes in HIV-1 quasispecies from azidothymidine (AZT)-treated patients. FASEB J. 1992 Jul;6(10):2843–2847. doi: 10.1096/fasebj.6.10.1634047. [DOI] [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES