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. 1992 Nov 25;20(22):5999–6006. doi: 10.1093/nar/20.22.5999

In vitro effect of antisense oligonucleotides on human immunodeficiency virus type 1 reverse transcription.

B Bordier 1, C Hélène 1, P J Barr 1, S Litvak 1, L Sarih-Cottin 1
PMCID: PMC334466  PMID: 1281317

Abstract

The molecular events involved in antisense-mediated inhibition of retroviral transcription were studied by analyzing the in vitro effect of antisense oligodeoxynucleotides on reverse transcription by Human Immunodeficiency Virus type 1 (HIV-1) reverse transcriptase (RT). Oligonucleotides have been designed to be complementary to three targets located in the 5' region of the HIV-1 RNA genome: the transactivating response element (TAR), the U5 region and a sequence contiguous to the primer binding site (PrePBS). Antisense oligodeoxynucleotides were used with their 3'-OH end either free or blocked by a dideoxynucleotide in order to avoid cDNA synthesis. Experiments with two recombinant forms of HIV RT, carrying or not RNase H activity, showed that antisense oligonucleotides can arrest reverse transcription by an RNase H-independent mechanism. The AntiTAR oligonucleotide did not affect reverse transcription. In contrast, the AntiU5 and AntiPrePBS oligonucleotides led to an efficient inhibition of both forms of HIV RT. In the case of the AntiU5, the inhibition obtained in the absence of the RNase H activity indicates that this effect can be related to features of the RNA secondary structure. The AntiPrePBS oligonucleotide did bind to its target only in the presence of PBS primer. Use of shifted oligonucleotides showed that the AntiPrePBS inhibitory effect depends on a cooperative annealing with the AntiPBS primer on the template.

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Selected References

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

  1. Agris C. H., Blake K. R., Miller P. S., Reddy M. P., Ts'o P. O. Inhibition of vesicular stomatitis virus protein synthesis and infection by sequence-specific oligodeoxyribonucleoside methylphosphonates. Biochemistry. 1986 Oct 7;25(20):6268–6275. doi: 10.1021/bi00368a065. [DOI] [PubMed] [Google Scholar]
  2. Aldovini A., Young R. A. Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J Virol. 1990 May;64(5):1920–1926. doi: 10.1128/jvi.64.5.1920-1926.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barber A. M., Hizi A., Maizel J. V., Jr, Hughes S. H. HIV-1 reverse transcriptase: structure predictions for the polymerase domain. AIDS Res Hum Retroviruses. 1990 Sep;6(9):1061–1072. doi: 10.1089/aid.1990.6.1061. [DOI] [PubMed] [Google Scholar]
  4. Bathurst I. C., Moen L. K., Lujan M. A., Gibson H. L., Feucht P. H., Pichuantes S., Craik C. S., Santi D. V., Barr P. J. Characterization of the human immunodeficiency virus type-1 reverse transcriptase enzyme produced in yeast. Biochem Biophys Res Commun. 1990 Sep 14;171(2):589–595. doi: 10.1016/0006-291x(90)91187-w. [DOI] [PubMed] [Google Scholar]
  5. Berkhout B., Silverman R. H., Jeang K. T. Tat trans-activates the human immunodeficiency virus through a nascent RNA target. Cell. 1989 Oct 20;59(2):273–282. doi: 10.1016/0092-8674(89)90289-4. [DOI] [PubMed] [Google Scholar]
  6. Birg F., Praseuth D., Zerial A., Thuong N. T., Asseline U., Le Doan T., Hélène C. Inhibition of simian virus 40 DNA replication in CV-1 cells by an oligodeoxynucleotide covalently linked to an intercalating agent. Nucleic Acids Res. 1990 May 25;18(10):2901–2908. doi: 10.1093/nar/18.10.2901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boiziau C., Thuong N. T., Toulmé J. J. Mechanisms of the inhibition of reverse transcription by antisense oligonucleotides. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):768–772. doi: 10.1073/pnas.89.2.768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bordier B., Tarrago-Litvak L., Sallafranque-Andreola M. L., Robert D., Tharaud D., Fournier M., Barr P. J., Litvak S., Sarih-Cottin L. Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA(Lys). Nucleic Acids Res. 1990 Feb 11;18(3):429–436. doi: 10.1093/nar/18.3.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bushman F. D., Craigie R. Activities of human immunodeficiency virus (HIV) integration protein in vitro: specific cleavage and integration of HIV DNA. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1339–1343. doi: 10.1073/pnas.88.4.1339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cobrinik D., Soskey L., Leis J. A retroviral RNA secondary structure required for efficient initiation of reverse transcription. J Virol. 1988 Oct;62(10):3622–3630. doi: 10.1128/jvi.62.10.3622-3630.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gatignol A., Buckler-White A., Berkhout B., Jeang K. T. Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR. Science. 1991 Mar 29;251(5001):1597–1600. doi: 10.1126/science.2011739. [DOI] [PubMed] [Google Scholar]
  12. Gilboa E., Mitra S. W., Goff S., Baltimore D. A detailed model of reverse transcription and tests of crucial aspects. Cell. 1979 Sep;18(1):93–100. doi: 10.1016/0092-8674(79)90357-x. [DOI] [PubMed] [Google Scholar]
  13. Goodchild J., Agrawal S., Civeira M. P., Sarin P. S., Sun D., Zamecnik P. C. Inhibition of human immunodeficiency virus replication by antisense oligodeoxynucleotides. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5507–5511. doi: 10.1073/pnas.85.15.5507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guo Q., Lu M., Churchill M. E., Tullius T. D., Kallenbach N. R. Asymmetric structure of a three-arm DNA junction. Biochemistry. 1990 Dec 11;29(49):10927–10934. doi: 10.1021/bi00501a010. [DOI] [PubMed] [Google Scholar]
  15. Hélène C. Rational design of sequence-specific oncogene inhibitors based on antisense and antigene oligonucleotides. Eur J Cancer. 1991;27(11):1466–1471. doi: 10.1016/0277-5379(91)90033-a. [DOI] [PubMed] [Google Scholar]
  16. Hélène C., Toulmé J. J. Specific regulation of gene expression by antisense, sense and antigene nucleic acids. Biochim Biophys Acta. 1990 Jun 21;1049(2):99–125. doi: 10.1016/0167-4781(90)90031-v. [DOI] [PubMed] [Google Scholar]
  17. Kulka M., Smith C. C., Aurelian L., Fishelevich R., Meade K., Miller P., Ts'o P. O. Site specificity of the inhibitory effects of oligo(nucleoside methylphosphonate)s complementary to the acceptor splice junction of herpes simplex virus type 1 immediate early mRNA 4. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6868–6872. doi: 10.1073/pnas.86.18.6868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Laspia M. F., Rice A. P., Mathews M. B. HIV-1 Tat protein increases transcriptional initiation and stabilizes elongation. Cell. 1989 Oct 20;59(2):283–292. doi: 10.1016/0092-8674(89)90290-0. [DOI] [PubMed] [Google Scholar]
  19. Le S. Y., Chen J. H., Braun M. J., Gonda M. A., Maizel J. V. Stability of RNA stem-loop structure and distribution of non-random structure in the human immunodeficiency virus (HIV-I). Nucleic Acids Res. 1988 Jun 10;16(11):5153–5168. doi: 10.1093/nar/16.11.5153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lemaitre M., Bayard B., Lebleu B. Specific antiviral activity of a poly(L-lysine)-conjugated oligodeoxyribonucleotide sequence complementary to vesicular stomatitis virus N protein mRNA initiation site. Proc Natl Acad Sci U S A. 1987 Feb;84(3):648–652. doi: 10.1073/pnas.84.3.648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lever A., Gottlinger H., Haseltine W., Sodroski J. Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions. J Virol. 1989 Sep;63(9):4085–4087. doi: 10.1128/jvi.63.9.4085-4087.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Loreau N., Boiziau C., Verspieren P., Shire D., Toulmé J. J. Blockage of AMV reverse transcriptase by antisense oligodeoxynucleotides. FEBS Lett. 1990 Nov 12;274(1-2):53–56. doi: 10.1016/0014-5793(90)81327-k. [DOI] [PubMed] [Google Scholar]
  23. Majumdar C., Stein C. A., Cohen J. S., Broder S., Wilson S. H. Stepwise mechanism of HIV reverse transcriptase: primer function of phosphorothioate oligodeoxynucleotide. Biochemistry. 1989 Feb 7;28(3):1340–1346. doi: 10.1021/bi00429a060. [DOI] [PubMed] [Google Scholar]
  24. Matsukura M., Shinozuka K., Zon G., Mitsuya H., Reitz M., Cohen J. S., Broder S. Phosphorothioate analogs of oligodeoxynucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7706–7710. doi: 10.1073/pnas.84.21.7706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Muesing M. A., Smith D. H., Capon D. J. Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein. Cell. 1987 Feb 27;48(4):691–701. doi: 10.1016/0092-8674(87)90247-9. [DOI] [PubMed] [Google Scholar]
  26. Sallafranque-Andreola M. L., Robert D., Barr P. J., Fournier M., Litvak S., Sarih-Cottin L., Tarrago-Litvak L. Human immunodeficiency virus reverse transcriptase expressed in transformed yeast cells. Biochemical properties and interactions with bovine tRNALys. Eur J Biochem. 1989 Sep 15;184(2):367–374. doi: 10.1111/j.1432-1033.1989.tb15028.x. [DOI] [PubMed] [Google Scholar]
  27. Varmus H. Retroviruses. Science. 1988 Jun 10;240(4858):1427–1435. doi: 10.1126/science.3287617. [DOI] [PubMed] [Google Scholar]
  28. Zamecnik P. C., Goodchild J., Taguchi Y., Sarin P. S. Inhibition of replication and expression of human T-cell lymphotropic virus type III in cultured cells by exogenous synthetic oligonucleotides complementary to viral RNA. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4143–4146. doi: 10.1073/pnas.83.12.4143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zamecnik P. C., Stephenson M. L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci U S A. 1978 Jan;75(1):280–284. doi: 10.1073/pnas.75.1.280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zerial A., Thuong N. T., Hélène C. Selective inhibition of the cytopathic effect of type A influenza viruses by oligodeoxynucleotides covalently linked to an intercalating agent. Nucleic Acids Res. 1987 Dec 10;15(23):9909–9919. doi: 10.1093/nar/15.23.9909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. van der Krol A. R., Mol J. N., Stuitje A. R. Modulation of eukaryotic gene expression by complementary RNA or DNA sequences. Biotechniques. 1988 Nov-Dec;6(10):958–976. [PubMed] [Google Scholar]

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