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. 1989 Dec;33(12):2109–2114. doi: 10.1128/aac.33.12.2109

Azidothymidine triphosphate is an inhibitor of both human immunodeficiency virus type 1 reverse transcriptase and DNA polymerase gamma.

H König 1, E Behr 1, J Löwer 1, R Kurth 1
PMCID: PMC172830  PMID: 2482702

Abstract

The reverse transcriptase from human immunodeficiency virus type 1 was purified from the virus to near homogeneity. The enzyme was shown to possess both RNA-dependent and DNA-dependent DNA-synthesizing activity. Activated DNA as a heteropolymeric substrate was used as efficiently as was the homopolymeric substrate poly(rA)-oligo(dT). The Michaelis-Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Azidothymidine triphosphate, a well-known inhibitor of the enzyme, inhibited the enzyme competitively with respect to dTTP and noncompetitively with respect to the other nucleotides. Azidothymidine triphosphate acted as an efficient inhibitor of cellular DNA polymerase gamma, whereas other enzymes of eucaryotic DNA metabolism, namely, DNA polymerase alpha-primase and DNA polymerase beta, were not inhibited. This finding may explain why some acquired immunodeficiency syndrome patients suffer side effects during azidothymidine therapy.

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Selected References

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  1. APOSHIAN H. V., KORNBERG A. Enzymatic synthesis of deoxyribonucleic acid. IX. The polymerase formed after T2 bacteriophage infection of Escherichia coli: a new enzyme. J Biol Chem. 1962 Feb;237:519–525. [PubMed] [Google Scholar]
  2. Barré-Sinoussi F., Chermann J. C., Rey F., Nugeyre M. T., Chamaret S., Gruest J., Dauguet C., Axler-Blin C., Vézinet-Brun F., Rouzioux C. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science. 1983 May 20;220(4599):868–871. doi: 10.1126/science.6189183. [DOI] [PubMed] [Google Scholar]
  3. Coffin J., Haase A., Levy J. A., Montagnier L., Oroszlan S., Teich N., Temin H., Toyoshima K., Varmus H., Vogt P. What to call the AIDS virus? Nature. 1986 May 1;321(6065):10–10. doi: 10.1038/321010a0. [DOI] [PubMed] [Google Scholar]
  4. Dresler S. L., Kimbro K. S. 2',3'-Dideoxythymidine 5'-triphosphate inhibition of DNA replication and ultraviolet-induced DNA repair synthesis in human cells: evidence for involvement of DNA polymerase delta. Biochemistry. 1987 May 19;26(10):2664–2668. doi: 10.1021/bi00384a002. [DOI] [PubMed] [Google Scholar]
  5. Farmerie W. G., Loeb D. D., Casavant N. C., Hutchison C. A., 3rd, Edgell M. H., Swanstrom R. Expression and processing of the AIDS virus reverse transcriptase in Escherichia coli. Science. 1987 Apr 17;236(4799):305–308. doi: 10.1126/science.2436298. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Hansen J., Schulze T., Mellert W., Moelling K. Identification and characterization of HIV-specific RNase H by monoclonal antibody. EMBO J. 1988 Jan;7(1):239–243. doi: 10.1002/j.1460-2075.1988.tb02805.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hansen J., Schulze T., Moelling K. RNase H activity associated with bacterially expressed reverse transcriptase of human T-cell lymphotropic virus III/lymphadenopathy-associated virus. J Biol Chem. 1987 Sep 15;262(26):12393–12396. [PubMed] [Google Scholar]
  9. Hirano H., Nishimura T., Iwamura T. High-flow-rate hydroxylapatites. Anal Biochem. 1985 Oct;150(1):228–234. doi: 10.1016/0003-2697(85)90463-4. [DOI] [PubMed] [Google Scholar]
  10. Hizi A., McGill C., Hughes S. H. Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1218–1222. doi: 10.1073/pnas.85.4.1218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hu S. Z., Wang T. S., Korn D. DNA primase from KB cells. Evidence for a novel model of primase catalysis by a highly purified primase/polymerase-alpha complex. J Biol Chem. 1984 Feb 25;259(4):2602–2609. [PubMed] [Google Scholar]
  12. König H., Riedel H. D., Knippers R. Reactions in vitro of the DNA polymerase-primase from Xenopus laevis eggs. A role for ATP in chain elongation. Eur J Biochem. 1983 Oct 3;135(3):435–442. doi: 10.1111/j.1432-1033.1983.tb07670.x. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Larder B., Purifoy D., Powell K., Darby G. AIDS virus reverse transcriptase defined by high level expression in Escherichia coli. EMBO J. 1987 Oct;6(10):3133–3137. doi: 10.1002/j.1460-2075.1987.tb02623.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Le Grice S. F., Beuck V., Mous J. Expression of biologically active human T-cell lymphotropic virus type III reverse transcriptase in Bacillus subtilis. Gene. 1987;55(1):95–103. doi: 10.1016/0378-1119(87)90252-6. [DOI] [PubMed] [Google Scholar]
  16. Matthes E., Lehmann C., Scholz D., von Janta-Lipinski M., Gaertner K., Rosenthal H. A., Langen P. Inhibition of HIV-associated reverse transcriptase by sugar-modified derivatives of thymidine 5'-triphosphate in comparison to cellular DNA polymerases alpha and beta. Biochem Biophys Res Commun. 1987 Oct 14;148(1):78–85. doi: 10.1016/0006-291x(87)91078-3. [DOI] [PubMed] [Google Scholar]
  17. Mosbaugh D. W. Purification and characterization of porcine liver DNA polymerase gamma: utilization of dUTP and dTTP during in vitro DNA synthesis. Nucleic Acids Res. 1988 Jun 24;16(12):5645–5659. doi: 10.1093/nar/16.12.5645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mous J., Heimer E. P., Le Grice S. F. Processing protease and reverse transcriptase from human immunodeficiency virus type I polyprotein in Escherichia coli. J Virol. 1988 Apr;62(4):1433–1436. doi: 10.1128/jvi.62.4.1433-1436.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Oguro M., Shioda M., Nagano H., Mano Y., Hanaoka F., Yamada M. The mode of action of aphidicolin on DNA synthesis in isolated nuclei. Biochem Biophys Res Commun. 1980 Jan 15;92(1):13–19. doi: 10.1016/0006-291x(80)91512-0. [DOI] [PubMed] [Google Scholar]
  20. Popovic M., Sarngadharan M. G., Read E., Gallo R. C. Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science. 1984 May 4;224(4648):497–500. doi: 10.1126/science.6200935. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Riedel H. D., König H., Knippers R. ATP and the processivity of Xenopus laevis DNA polymerase alpha. Biochim Biophys Acta. 1984 Nov 22;783(2):158–165. doi: 10.1016/0167-4781(84)90008-3. [DOI] [PubMed] [Google Scholar]
  23. St Clair M. H., Richards C. A., Spector T., Weinhold K. J., Miller W. H., Langlois A. J., Furman P. A. 3'-Azido-3'-deoxythymidine triphosphate as an inhibitor and substrate of purified human immunodeficiency virus reverse transcriptase. Antimicrob Agents Chemother. 1987 Dec;31(12):1972–1977. doi: 10.1128/aac.31.12.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tasheva B., Dessev G. Artifacts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to 2-mercaptoethanol. Anal Biochem. 1983 Feb 15;129(1):98–102. doi: 10.1016/0003-2697(83)90057-x. [DOI] [PubMed] [Google Scholar]
  25. Vrang L., Bazin H., Remaud G., Chattopadhyaya J., Oberg B. Inhibition of the reverse transcriptase from HIV by 3'-azido-3'-deoxythymidine triphosphate and its threo analogue. Antiviral Res. 1987 Mar;7(3):139–149. doi: 10.1016/0166-3542(87)90002-7. [DOI] [PubMed] [Google Scholar]
  26. Vrang L., Oberg B. PPi analogs as inhibitors of human T-lymphotropic virus type III reverse transcriptase. Antimicrob Agents Chemother. 1986 May;29(5):867–872. doi: 10.1128/aac.29.5.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Waqar M. A., Evans M. J., Manly K. F., Hughes R. G., Huberman J. A. Effects of 2',3'-dideoxynucleosides on mammalian cells and viruses. J Cell Physiol. 1984 Nov;121(2):402–408. doi: 10.1002/jcp.1041210218. [DOI] [PubMed] [Google Scholar]
  28. Weber J., Grosse F. Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural DNA. Nucleic Acids Res. 1989 Feb 25;17(4):1379–1393. doi: 10.1093/nar/17.4.1379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wondrak E. M., Löwer J., Kurth R. Inhibition of HIV-1 RNA-dependent DNA polymerase and cellular DNA polymerases alpha, beta and gamma by phosphonoformic acid and other drugs. J Antimicrob Chemother. 1988 Feb;21(2):151–161. doi: 10.1093/jac/21.2.151. [DOI] [PubMed] [Google Scholar]
  30. Yamaguchi M., Matsukage A., Takahashi T. Chick embryo DNA polymerase gamma. Purification and structural analysis of nearly homogeneous enzyme. J Biol Chem. 1980 Jul 25;255(14):7002–7009. [PubMed] [Google Scholar]

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