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. 1997 Jun;71(6):4254–4263. doi: 10.1128/jvi.71.6.4254-4263.1997

The antiretrovirus drug 3'-azido-3'-deoxythymidine increases the retrovirus mutation rate.

J G Julias 1, T Kim 1, G Arnold 1, V K Pathak 1
PMCID: PMC191640  PMID: 9151812

Abstract

It was previously observed that the nucleoside analog 5-azacytidine increased the spleen necrosis virus (SNV) mutation rate 13-fold in one cycle of retrovirus replication (V. K. Pathak and H. M. Temin, J. Virol. 66:3093-3100, 1992). Based on this observation, we hypothesized that nucleoside analogs used as antiviral drugs may also increase retrovirus mutation rates. We sought to determine if 3'-azido-3'-deoxythymidine (AZT), the primary treatment for human immunodeficiency virus type 1 (HIV-1) infection, increases the retrovirus mutation rate. Two assays were used to determine the effects of AZT on retrovirus mutation rates. The strategy of the first assay involved measuring the in vivo rate of inactivation of the lacZ gene in one replication cycle of SNV- and murine leukemia virus-based retroviral vectors. We observed 7- and 10-fold increases in the SNV mutant frequency following treatment of target cells with 0.1 and 0.5 microM AZT, respectively. The murine leukemia virus mutant frequency increased two- and threefold following treatment of target cells with 0.5 and 1.0 microM AZT, respectively. The second assay used an SNV-based shuttle vector containing the lacZ alpha gene. Proviruses were recovered as plasmids in Escherichia coli, and the rate of inactivation of lacZ alpha was measured. The results indicated that treatment of target cells increased the overall mutation rate two- to threefold. DNA sequence analysis of mutant proviruses indicated that AZT increased both the deletion and substitution rates. These results suggest that AZT treatment of HIV-1 infection may increase the degree of viral variation and alter virus evolution or pathogenesis.

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Selected References

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  1. Bebenek K., Abbotts J., Roberts J. D., Wilson S. H., Kunkel T. A. Specificity and mechanism of error-prone replication by human immunodeficiency virus-1 reverse transcriptase. J Biol Chem. 1989 Oct 5;264(28):16948–16956. [PubMed] [Google Scholar]
  2. Coffin J. M. HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy. Science. 1995 Jan 27;267(5197):483–489. doi: 10.1126/science.7824947. [DOI] [PubMed] [Google Scholar]
  3. Dougherty J. P., Temin H. M. Determination of the rate of base-pair substitution and insertion mutations in retrovirus replication. J Virol. 1988 Aug;62(8):2817–2822. doi: 10.1128/jvi.62.8.2817-2822.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Essex M. Strategies of research for a vaccine against AIDS. Hist Philos Life Sci. 1995;17(1):141–149. [PubMed] [Google Scholar]
  5. 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]
  6. Frick L. W., Nelson D. J., St Clair M. H., Furman P. A., Krenitsky T. A. Effects of 3'-azido-3'-deoxythymidine on the deoxynucleotide triphosphate pools of cultured human cells. Biochem Biophys Res Commun. 1988 Jul 15;154(1):124–129. doi: 10.1016/0006-291x(88)90659-6. [DOI] [PubMed] [Google Scholar]
  7. Furman P. A., Fyfe J. A., St Clair M. H., Weinhold K., Rideout J. L., Freeman G. A., Lehrman S. N., Bolognesi D. P., Broder S., Mitsuya H. Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8333–8337. doi: 10.1073/pnas.83.21.8333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gao W. Y., Cara A., Gallo R. C., Lori F. Low levels of deoxynucleotides in peripheral blood lymphocytes: a strategy to inhibit human immunodeficiency virus type 1 replication. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8925–8928. doi: 10.1073/pnas.90.19.8925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Glickman B. W., Saddi V. A., Curry J. International Commission for Protection Against Environmental Mutagens and Carcinogens. Working paper no. 2. Spontaneous mutations in mammalian cells. Mutat Res. 1994 Jan;304(1):19–32. doi: 10.1016/0027-5107(94)90319-0. [DOI] [PubMed] [Google Scholar]
  10. Gritz L., Davies J. Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene. 1983 Nov;25(2-3):179–188. doi: 10.1016/0378-1119(83)90223-8. [DOI] [PubMed] [Google Scholar]
  11. Gu Z., Gao Q., Li X., Parniak M. A., Wainberg M. A. Novel mutation in the human immunodeficiency virus type 1 reverse transcriptase gene that encodes cross-resistance to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine. J Virol. 1992 Dec;66(12):7128–7135. doi: 10.1128/jvi.66.12.7128-7135.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ho D. D., Neumann A. U., Perelson A. S., Chen W., Leonard J. M., Markowitz M. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature. 1995 Jan 12;373(6510):123–126. doi: 10.1038/373123a0. [DOI] [PubMed] [Google Scholar]
  13. Hu W. S., Temin H. M. Genetic consequences of packaging two RNA genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1556–1560. doi: 10.1073/pnas.87.4.1556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Japour A. J., Welles S., D'Aquila R. T., Johnson V. A., Richman D. D., Coombs R. W., Reichelderfer P. S., Kahn J. O., Crumpacker C. S., Kuritzkes D. R. Prevalence and clinical significance of zidovudine resistance mutations in human immunodeficiency virus isolated from patients after long-term zidovudine treatment. AIDS Clinical Trials Group 116B/117 Study Team and the Virology Committee Resistance Working Group. J Infect Dis. 1995 May;171(5):1172–1179. doi: 10.1093/infdis/171.5.1172. [DOI] [PubMed] [Google Scholar]
  15. Ji J. P., Loeb L. A. Fidelity of HIV-1 reverse transcriptase copying RNA in vitro. Biochemistry. 1992 Feb 4;31(4):954–958. doi: 10.1021/bi00119a002. [DOI] [PubMed] [Google Scholar]
  16. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. doi: 10.1007/BF00267254. [DOI] [PubMed] [Google Scholar]
  17. Kawai S., Nishizawa M. New procedure for DNA transfection with polycation and dimethyl sulfoxide. Mol Cell Biol. 1984 Jun;4(6):1172–1174. doi: 10.1128/mcb.4.6.1172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kim T., Mudry R. A., Jr, Rexrode C. A., 2nd, Pathak V. K. Retroviral mutation rates and A-to-G hypermutations during different stages of retroviral replication. J Virol. 1996 Nov;70(11):7594–7602. doi: 10.1128/jvi.70.11.7594-7602.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kohalmi S. E., Kunz B. A. Mutational specificity of thymine nucleotide depletion in yeast. Mutat Res. 1993 Sep;289(1):73–81. doi: 10.1016/0027-5107(93)90132-y. [DOI] [PubMed] [Google Scholar]
  20. Kunkel T. A., Alexander P. S. The base substitution fidelity of eucaryotic DNA polymerases. Mispairing frequencies, site preferences, insertion preferences, and base substitution by dislocation. J Biol Chem. 1986 Jan 5;261(1):160–166. [PubMed] [Google Scholar]
  21. Kunz B. A., Kohalmi S. E. Modulation of mutagenesis by deoxyribonucleotide levels. Annu Rev Genet. 1991;25:339–359. doi: 10.1146/annurev.ge.25.120191.002011. [DOI] [PubMed] [Google Scholar]
  22. LaCasse R. A., Remington K. M., North T. W. The mutation frequency of feline immunodeficiency virus enhanced by 3'-azido-3'-deoxythymidine. J Acquir Immune Defic Syndr Hum Retrovirol. 1996 May 1;12(1):26–32. doi: 10.1097/00042560-199605010-00004. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Libby R. T., Gallant J. A. Phosphorolytic error correction during transcription. Mol Microbiol. 1994 Apr;12(1):121–129. doi: 10.1111/j.1365-2958.1994.tb01001.x. [DOI] [PubMed] [Google Scholar]
  26. Loeb L. A., Kunkel T. A. Fidelity of DNA synthesis. Annu Rev Biochem. 1982;51:429–457. doi: 10.1146/annurev.bi.51.070182.002241. [DOI] [PubMed] [Google Scholar]
  27. Lori F., Malykh A., Cara A., Sun D., Weinstein J. N., Lisziewicz J., Gallo R. C. Hydroxyurea as an inhibitor of human immunodeficiency virus-type 1 replication. Science. 1994 Nov 4;266(5186):801–805. doi: 10.1126/science.7973634. [DOI] [PubMed] [Google Scholar]
  28. Mansky L. M., Temin H. M. Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase. J Virol. 1995 Aug;69(8):5087–5094. doi: 10.1128/jvi.69.8.5087-5094.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mansky L. M., Temin H. M. Lower mutation rate of bovine leukemia virus relative to that of spleen necrosis virus. J Virol. 1994 Jan;68(1):494–499. doi: 10.1128/jvi.68.1.494-499.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Martinez M. A., Vartanian J. P., Wain-Hobson S. Hypermutagenesis of RNA using human immunodeficiency virus type 1 reverse transcriptase and biased dNTP concentrations. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11787–11791. doi: 10.1073/pnas.91.25.11787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Meuth M. The molecular basis of mutations induced by deoxyribonucleoside triphosphate pool imbalances in mammalian cells. Exp Cell Res. 1989 Apr;181(2):305–316. doi: 10.1016/0014-4827(89)90090-6. [DOI] [PubMed] [Google Scholar]
  32. Milich L., Margolin B., Swanstrom R. V3 loop of the human immunodeficiency virus type 1 Env protein: interpreting sequence variability. J Virol. 1993 Sep;67(9):5623–5634. doi: 10.1128/jvi.67.9.5623-5634.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Miller A. D., Garcia J. V., von Suhr N., Lynch C. M., Wilson C., Eiden M. V. Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J Virol. 1991 May;65(5):2220–2224. doi: 10.1128/jvi.65.5.2220-2224.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Moore K. H., Raasch R. H., Brouwer K. L., Opheim K., Cheeseman S. H., Eyster E., Lemon S. M., van der Horst C. M. Pharmacokinetics and bioavailability of zidovudine and its glucuronidated metabolite in patients with human immunodeficiency virus infection and hepatic disease (AIDS Clinical Trials Group protocol 062). Antimicrob Agents Chemother. 1995 Dec;39(12):2732–2737. doi: 10.1128/aac.39.12.2732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Parthasarathi S., Varela-Echavarría A., Ron Y., Preston B. D., Dougherty J. P. Genetic rearrangements occurring during a single cycle of murine leukemia virus vector replication: characterization and implications. J Virol. 1995 Dec;69(12):7991–8000. doi: 10.1128/jvi.69.12.7991-8000.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pathak V. K., Temin H. M. 5-Azacytidine and RNA secondary structure increase the retrovirus mutation rate. J Virol. 1992 May;66(5):3093–3100. doi: 10.1128/jvi.66.5.3093-3100.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pathak V. K., Temin H. M. Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: deletions and deletions with insertions. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6024–6028. doi: 10.1073/pnas.87.16.6024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pathak V. K., Temin H. M. Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: substitutions, frameshifts, and hypermutations. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6019–6023. doi: 10.1073/pnas.87.16.6019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Patick A. K., Rose R., Greytok J., Bechtold C. M., Hermsmeier M. A., Chen P. T., Barrish J. C., Zahler R., Colonno R. J., Lin P. F. Characterization of a human immunodeficiency virus type 1 variant with reduced sensitivity to an aminodiol protease inhibitor. J Virol. 1995 Apr;69(4):2148–2152. doi: 10.1128/jvi.69.4.2148-2152.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Preston B. D., Poiesz B. J., Loeb L. A. Fidelity of HIV-1 reverse transcriptase. Science. 1988 Nov 25;242(4882):1168–1171. doi: 10.1126/science.2460924. [DOI] [PubMed] [Google Scholar]
  41. Roberts J. D., Bebenek K., Kunkel T. A. The accuracy of reverse transcriptase from HIV-1. Science. 1988 Nov 25;242(4882):1171–1173. doi: 10.1126/science.2460925. [DOI] [PubMed] [Google Scholar]
  42. Shirasaka T., Kavlick M. F., Ueno T., Gao W. Y., Kojima E., Alcaide M. L., Chokekijchai S., Roy B. M., Arnold E., Yarchoan R. Emergence of human immunodeficiency virus type 1 variants with resistance to multiple dideoxynucleosides in patients receiving therapy with dideoxynucleosides. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2398–2402. doi: 10.1073/pnas.92.6.2398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Temin H. M. Retrovirus variation and reverse transcription: abnormal strand transfers result in retrovirus genetic variation. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6900–6903. doi: 10.1073/pnas.90.15.6900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tisdale M., Kemp S. D., Parry N. R., Larder B. A. Rapid in vitro selection of human immunodeficiency virus type 1 resistant to 3'-thiacytidine inhibitors due to a mutation in the YMDD region of reverse transcriptase. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5653–5656. doi: 10.1073/pnas.90.12.5653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Varela-Echavarría A., Garvey N., Preston B. D., Dougherty J. P. Comparison of Moloney murine leukemia virus mutation rate with the fidelity of its reverse transcriptase in vitro. J Biol Chem. 1992 Dec 5;267(34):24681–24688. [PubMed] [Google Scholar]
  47. Wainberg M. A., Drosopoulos W. C., Salomon H., Hsu M., Borkow G., Parniak M., Gu Z., Song Q., Manne J., Islam S. Enhanced fidelity of 3TC-selected mutant HIV-1 reverse transcriptase. Science. 1996 Mar 1;271(5253):1282–1285. doi: 10.1126/science.271.5253.1282. [DOI] [PubMed] [Google Scholar]
  48. Watanabe S., Temin H. M. Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors. Mol Cell Biol. 1983 Dec;3(12):2241–2249. doi: 10.1128/mcb.3.12.2241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wei X., Ghosh S. K., Taylor M. E., Johnson V. A., Emini E. A., Deutsch P., Lifson J. D., Bonhoeffer S., Nowak M. A., Hahn B. H. Viral dynamics in human immunodeficiency virus type 1 infection. Nature. 1995 Jan 12;373(6510):117–122. doi: 10.1038/373117a0. [DOI] [PubMed] [Google Scholar]
  50. el-Farrash M. A., Kuroda M. J., Kitazaki T., Masuda T., Kato K., Hatanaka M., Harada S. Generation and characterization of a human immunodeficiency virus type 1 (HIV-1) mutant resistant to an HIV-1 protease inhibitor. J Virol. 1994 Jan;68(1):233–239. doi: 10.1128/jvi.68.1.233-239.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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