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. 1989 Jul;86(13):4803–4807. doi: 10.1073/pnas.86.13.4803

Infectious potential of human immunodeficiency virus type 1 reverse transcriptase mutants with altered inhibitor sensitivity.

B A Larder 1, S D Kemp 1, D J Purifoy 1
PMCID: PMC297503  PMID: 2472634

Abstract

There is considerable interest in the potential of human immunodeficiency virus type 1 (HIV-1) to develop drug resistance, especially as 3'-azido-3'-deoxythymidine (Retrovir) is now in widespread clinical use to treat people with AIDS and AIDS-related complex (ARC). To address this possibility, mutations in the HIV reverse transcriptase [deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase (RNA-directed), EC 2.7.7.49] gene have been introduced by site-directed mutagenesis of cloned constructs in Escherichia coli. Analysis of the recombinant mutant reverse transcriptase from a number of these constructs revealed enzymes that maintained enzyme activity but had a reduced ability to recognize inhibitors such as azidothymidine triphosphate. To assess the infectivity of these mutants, several constructs of proviral HIV clones with mutant reverse transcriptase genes have been made and used to transfect T cells. All five mutants tested have lower infectious potential, suggesting considerable levels of reverse transcriptase activity are required for efficient virus replication. Viable virus recovered from two clones showed decreased sensitivity to the antiviral compound phosphonoformate, thus demonstrating the potential for drug-resistant HIV to replicate. However, although the reverse transcriptase from these mutant viruses showed decreased sensitivity to azidothymidine triphosphate, paradoxically these viruses were hypersensitive to azidothymidine when tested in culture.

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Selected References

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

  1. 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]
  2. Chesebro B., Wehrly K. Development of a sensitive quantitative focal assay for human immunodeficiency virus infectivity. J Virol. 1988 Oct;62(10):3779–3788. doi: 10.1128/jvi.62.10.3779-3788.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cole S. P. Rapid chemosensitivity testing of human lung tumor cells using the MTT assay. Cancer Chemother Pharmacol. 1986;17(3):259–263. doi: 10.1007/BF00256695. [DOI] [PubMed] [Google Scholar]
  4. Fischl M. A., Richman D. D., Grieco M. H., Gottlieb M. S., Volberding P. A., Laskin O. L., Leedom J. M., Groopman J. E., Mildvan D., Schooley R. T. The efficacy 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):185–191. doi: 10.1056/NEJM198707233170401. [DOI] [PubMed] [Google Scholar]
  5. Fisher A. G., Collalti E., Ratner L., Gallo R. C., Wong-Staal F. A molecular clone of HTLV-III with biological activity. Nature. 1985 Jul 18;316(6025):262–265. doi: 10.1038/316262a0. [DOI] [PubMed] [Google Scholar]
  6. Fisher A. G., Ensoli B., Looney D., Rose A., Gallo R. C., Saag M. S., Shaw G. M., Hahn B. H., Wong-Staal F. Biologically diverse molecular variants within a single HIV-1 isolate. Nature. 1988 Aug 4;334(6181):444–447. doi: 10.1038/334444a0. [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. 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]
  9. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Larder B. A., Purifoy D. J., Powell K. L., Darby G. Site-specific mutagenesis of AIDS virus reverse transcriptase. 1987 Jun 25-Jul 1Nature. 327(6124):716–717. doi: 10.1038/327716a0. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Levy J. A., Hoffman A. D., Kramer S. M., Landis J. A., Shimabukuro J. M., Oshiro L. S. Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS. Science. 1984 Aug 24;225(4664):840–842. doi: 10.1126/science.6206563. [DOI] [PubMed] [Google Scholar]
  14. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16;65(1-2):55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
  15. Parker A. C., Craig J. I., Collins P., Oliver N., Smith I. Acyclovir-resistant herpes simplex virus infection due to altered DNA polymerase. Lancet. 1987 Dec 19;2(8573):1461–1461. doi: 10.1016/s0140-6736(87)91157-3. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Saag M. S., Hahn B. H., Gibbons J., Li Y., Parks E. S., Parks W. P., Shaw G. M. Extensive variation of human immunodeficiency virus type-1 in vivo. Nature. 1988 Aug 4;334(6181):440–444. doi: 10.1038/334440a0. [DOI] [PubMed] [Google Scholar]
  18. Smith M. S., Brian E. L., Pagano J. S. Resumption of virus production after human immunodeficiency virus infection of T lymphocytes in the presence of azidothymidine. J Virol. 1987 Dec;61(12):3769–3773. doi: 10.1128/jvi.61.12.3769-3773.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Smithies O., Gregg R. G., Boggs S. S., Koralewski M. A., Kucherlapati R. S. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature. 1985 Sep 19;317(6034):230–234. doi: 10.1038/317230a0. [DOI] [PubMed] [Google Scholar]
  20. Temin H. M., Baltimore D. RNA-directed DNA synthesis and RNA tumor viruses. Adv Virus Res. 1972;17:129–186. doi: 10.1016/s0065-3527(08)60749-6. [DOI] [PubMed] [Google Scholar]
  21. Wong T. K., Neumann E. Electric field mediated gene transfer. Biochem Biophys Res Commun. 1982 Jul 30;107(2):584–587. doi: 10.1016/0006-291x(82)91531-5. [DOI] [PubMed] [Google Scholar]
  22. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]

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