Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1995 Mar;69(3):1794–1801. doi: 10.1128/jvi.69.3.1794-1801.1995

Sequence-specific inhibition of gene expression by a novel antisense oligodeoxynucleotide phosphorothioate directed against a nonregulatory region of the human immunodeficiency virus type 1 genome.

M I Anazodo 1, M A Wainberg 1, A D Friesen 1, J A Wright 1
PMCID: PMC188787  PMID: 7853519

Abstract

Previous studies have demonstrated that oligodeoxynucleotide phosphorothioates complementary to human immunodeficiency virus type 1 (HIV-1) RNA are more nuclease resistant and are effective inhibitors of HIV-1 replication than their unmodified counterpart. In this study, antisense oligodeoxynucleotide sequences were evaluated for therapeutic potential in the treatment of HIV infections. The use of HIV-infected lymphocytes to test the efficacy of a drug is very complex, and therefore it is difficult to draw conclusions about the mechanism. We used a COS-like Monkey kidney cell line (CMT3) stably transfected with plasmids pCMVgagpol-rre-r (containing gag and pol genes) and pCMVrev (containing the rev gene of HIV-1), derived from cDNA clone BH10, as a model. A biologically active provirus that transcribes and translates their nucleotide sequences into viral proteins p24, p39/41, p55, and p160 was generated. Sequence-specific and dose-dependent inhibition of HIV-1 viral protein synthesis and significant inhibition at the mRNA level were demonstrated by antisense construct GPI2A, directed against a nonregulatory region of the HIV-1 genome. Also, our studies demonstrated enhancement of the antisense effect through encapsulation in a cationic lipid preparation. The observed attenuation of HIV-1 mRNA levels suggests that, at least in part, the mechanism of action of GPI2A was at the transcript level. Further studies have also shown antiviral activity of this construct as determined by the reverse transcriptase assay using acutely and chronically infected cells of lymphoid origin (H9 cells). Toxicological studies involving cell growth characteristics, colony-forming ability, effects on cellular proteins, specific activities of labeled proteins, and DNA synthesis in cell culture showed no cytotoxic effects of GPI2A.

Full Text

The Full Text of this article is available as a PDF (495.9 KB).

Selected References

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

  1. Agrawal S. Antisense oligonucleotides as antiviral agents. Trends Biotechnol. 1992 May;10(5):152–158. doi: 10.1016/0167-7799(92)90203-8. [DOI] [PubMed] [Google Scholar]
  2. Agrawal S., Goodchild J., Civeira M. P., Thornton A. H., Sarin P. S., Zamecnik P. C. Oligodeoxynucleoside phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency virus. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7079–7083. doi: 10.1073/pnas.85.19.7079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Agrawal S., Ikeuchi T., Sun D., Sarin P. S., Konopka A., Maizel J., Zamecnik P. C. Inhibition of human immunodeficiency virus in early infected and chronically infected cells by antisense oligodeoxynucleotides and their phosphorothioate analogues. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7790–7794. doi: 10.1073/pnas.86.20.7790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Agrawal S., Mayrand S. H., Zamecnik P. C., Pederson T. Site-specific excision from RNA by RNase H and mixed-phosphate-backbone oligodeoxynucleotides. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1401–1405. doi: 10.1073/pnas.87.4.1401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Agrawal S., Temsamani J., Tang J. Y. Pharmacokinetics, biodistribution, and stability of oligodeoxynucleotide phosphorothioates in mice. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7595–7599. doi: 10.1073/pnas.88.17.7595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Akhtar S., Kole R., Juliano R. L. Stability of antisense DNA oligodeoxynucleotide analogs in cellular extracts and sera. Life Sci. 1991;49(24):1793–1801. doi: 10.1016/0024-3205(91)90480-y. [DOI] [PubMed] [Google Scholar]
  7. Amara F. M., Chen F. Y., Wright J. A. A novel transforming growth factor-beta 1 responsive cytoplasmic trans-acting factor binds selectively to the 3'-untranslated region of mammalian ribonucleotide reductase R2 mRNA: role in message stability. Nucleic Acids Res. 1993 Oct 11;21(20):4803–4809. doi: 10.1093/nar/21.20.4803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Arya S. K., Guo C., Josephs S. F., Wong-Staal F. Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). Science. 1985 Jul 5;229(4708):69–73. doi: 10.1126/science.2990040. [DOI] [PubMed] [Google Scholar]
  9. Battles J. K., Hu M. Y., Rasmussen L., Tobin G. J., Gonda M. A. Immunological characterization of the gag gene products of bovine immunodeficiency virus. J Virol. 1992 Dec;66(12):6868–6877. doi: 10.1128/jvi.66.12.6868-6877.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bennett C. F., Chiang M. Y., Chan H., Shoemaker J. E., Mirabelli C. K. Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol Pharmacol. 1992 Jun;41(6):1023–1033. [PubMed] [Google Scholar]
  11. Boulerice F., Bour S., Geleziunas R., Lvovich A., Wainberg M. A. High frequency of isolation of defective human immunodeficiency virus type 1 and heterogeneity of viral gene expression in clones of infected U-937 cells. J Virol. 1990 Apr;64(4):1745–1755. doi: 10.1128/jvi.64.4.1745-1755.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bour S., Boulerice F., Wainberg M. A. Inhibition of gp160 and CD4 maturation in U937 cells after both defective and productive infections by human immunodeficiency virus type 1. J Virol. 1991 Dec;65(12):6387–6396. doi: 10.1128/jvi.65.12.6387-6396.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chan A. K., Litchfield D. W., Wright J. A. Phosphorylation of ribonucleotide reductase R2 protein: in vivo and in vitro evidence of a role for p34cdc2 and CDK2 protein kinases. Biochemistry. 1993 Nov 30;32(47):12835–12840. doi: 10.1021/bi00210a036. [DOI] [PubMed] [Google Scholar]
  14. Cohen J. S. Designing antisense oligonucleotides as pharmaceutical agents. Trends Pharmacol Sci. 1989 Nov;10(11):435–437. doi: 10.1016/S0165-6147(89)80004-5. [DOI] [PubMed] [Google Scholar]
  15. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Garvey K. J., Oberste M. S., Elser J. E., Braun M. J., Gonda M. A. Nucleotide sequence and genome organization of biologically active proviruses of the bovine immunodeficiency-like virus. Virology. 1990 Apr;175(2):391–409. doi: 10.1016/0042-6822(90)90424-p. [DOI] [PubMed] [Google Scholar]
  17. Gerard R. D., Gluzman Y. New host cell system for regulated simian virus 40 DNA replication. Mol Cell Biol. 1985 Nov;5(11):3231–3240. doi: 10.1128/mcb.5.11.3231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gonda M. A., Braun M. J., Carter S. G., Kost T. A., Bess J. W., Jr, Arthur L. O., Van der Maaten M. J. Characterization and molecular cloning of a bovine lentivirus related to human immunodeficiency virus. 1987 Nov 26-Dec 2Nature. 330(6146):388–391. doi: 10.1038/330388a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Gough N. M. Rapid and quantitative preparation of cytoplasmic RNA from small numbers of cells. Anal Biochem. 1988 Aug 15;173(1):93–95. doi: 10.1016/0003-2697(88)90164-9. [DOI] [PubMed] [Google Scholar]
  21. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  22. Hammarskjöld M. L., Heimer J., Hammarskjöld B., Sangwan I., Albert L., Rekosh D. Regulation of human immunodeficiency virus env expression by the rev gene product. J Virol. 1989 May;63(5):1959–1966. doi: 10.1128/jvi.63.5.1959-1966.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hawkins J. W. Antisense technology: commercial implications. Antisense Res Dev. 1991 Fall;1(3):283–284. [PubMed] [Google Scholar]
  24. Hurta R. A., Wright J. A. Alterations in the activity and regulation of mammalian ribonucleotide reductase by chlorambucil, a DNA damaging agent. J Biol Chem. 1992 Apr 5;267(10):7066–7071. [PubMed] [Google Scholar]
  25. Jeang K. T., Rawlins D. R., Rosenfeld P. J., Shero J. H., Kelly T. J., Hayward G. S. Multiple tandemly repeated binding sites for cellular nuclear factor 1 that surround the major immediate-early promoters of simian and human cytomegalovirus. J Virol. 1987 May;61(5):1559–1570. doi: 10.1128/jvi.61.5.1559-1570.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Juliano R. L., Akhtar S. Liposomes as a drug delivery system for antisense oligonucleotides. Antisense Res Dev. 1992 Summer;2(2):165–176. doi: 10.1089/ard.1992.2.165. [DOI] [PubMed] [Google Scholar]
  27. Lee M. H., Sano K., Morales F. E., Imagawa D. T. Sensitive reverse transcriptase assay to detect and quantitate human immunodeficiency virus. J Clin Microbiol. 1987 Sep;25(9):1717–1721. doi: 10.1128/jcm.25.9.1717-1721.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Leonetti J. P., Machy P., Degols G., Lebleu B., Leserman L. Antibody-targeted liposomes containing oligodeoxyribonucleotides complementary to viral RNA selectively inhibit viral replication. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2448–2451. doi: 10.1073/pnas.87.7.2448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lewis N., Williams J., Rekosh D., Hammarskjöld M. L. Identification of a cis-acting element in human immunodeficiency virus type 2 (HIV-2) that is responsive to the HIV-1 rev and human T-cell leukemia virus types I and II rex proteins. J Virol. 1990 Apr;64(4):1690–1697. doi: 10.1128/jvi.64.4.1690-1697.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lisziewicz J., Sun D., Klotman M., Agrawal S., Zamecnik P., Gallo R. Specific inhibition of human immunodeficiency virus type 1 replication by antisense oligonucleotides: an in vitro model for treatment. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11209–11213. doi: 10.1073/pnas.89.23.11209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lisziewicz J., Sun D., Metelev V., Zamecnik P., Gallo R. C., Agrawal S. Long-term treatment of human immunodeficiency virus-infected cells with antisense oligonucleotide phosphorothioates. Proc Natl Acad Sci U S A. 1993 May 1;90(9):3860–3864. doi: 10.1073/pnas.90.9.3860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Matsukura M., Zon G., Shinozuka K., Robert-Guroff M., Shimada T., Stein C. A., Mitsuya H., Wong-Staal F., Cohen J. S., Broder S. Regulation of viral expression of human immunodeficiency virus in vitro by an antisense phosphorothioate oligodeoxynucleotide against rev (art/trs) in chronically infected cells. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4244–4248. doi: 10.1073/pnas.86.11.4244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Noé C., Hernandez-Borrell J., Kinsky S. C., Matsuura E., Leserman L. Inhibition of cell proliferation with antibody-targeted liposomes containing methotrexate-gamma-dimyristoylphosphatidylethanolamine. Biochim Biophys Acta. 1988 Dec 22;946(2):253–260. doi: 10.1016/0005-2736(88)90400-2. [DOI] [PubMed] [Google Scholar]
  34. Petteway S. R., Jr, Lambert D. M., Metcalf B. W. The chronically infected cell as a target for the treatment of HIV infection and AIDS. Trends Pharmacol Sci. 1991 Jan;12(1):28–34. doi: 10.1016/0165-6147(91)90485-b. [DOI] [PubMed] [Google Scholar]
  35. Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
  36. Rekosh D., Nygren A., Flodby P., Hammarskjöld M. L., Wigzell H. Coexpression of human immunodeficiency virus envelope proteins and tat from a single simian virus 40 late replacement vector. Proc Natl Acad Sci U S A. 1988 Jan;85(2):334–338. doi: 10.1073/pnas.85.2.334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Smith A. J., Cho M. I., Hammarskjöld M. L., Rekosh D. Human immunodeficiency virus type 1 Pr55gag and Pr160gag-pol expressed from a simian virus 40 late replacement vector are efficiently processed and assembled into viruslike particles. J Virol. 1990 Jun;64(6):2743–2750. doi: 10.1128/jvi.64.6.2743-2750.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Spearman M., Taylor W. R., Greenberg A. H., Wright J. A. Antisense oligodeoxyribonucleotide inhibition of TGF-beta 1 gene expression and alterations in the growth and malignant properties of mouse fibrosarcoma cells. Gene. 1994 Nov 4;149(1):25–29. doi: 10.1016/0378-1119(94)90408-1. [DOI] [PubMed] [Google Scholar]
  39. Stein C. A., Cheng Y. C. Antisense oligonucleotides as therapeutic agents--is the bullet really magical? Science. 1993 Aug 20;261(5124):1004–1012. doi: 10.1126/science.8351515. [DOI] [PubMed] [Google Scholar]
  40. Stevenson M., Bukrinsky M., Haggerty S. HIV-1 replication and potential targets for intervention. AIDS Res Hum Retroviruses. 1992 Feb;8(2):107–117. doi: 10.1089/aid.1992.8.107. [DOI] [PubMed] [Google Scholar]
  41. Wickstrom E. Oligodeoxynucleotide stability in subcellular extracts and culture media. J Biochem Biophys Methods. 1986 Sep;13(2):97–102. doi: 10.1016/0165-022x(86)90021-7. [DOI] [PubMed] [Google Scholar]
  42. Wright J. A., Chan A. K., Choy B. K., Hurta R. A., McClarty G. A., Tagger A. Y. Regulation and drug resistance mechanisms of mammalian ribonucleotide reductase, and the significance to DNA synthesis. Biochem Cell Biol. 1990 Dec;68(12):1364–1371. doi: 10.1139/o90-199. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Zon G. Oligonucleotide analogues as potential chemotherapeutic agents. Pharm Res. 1988 Sep;5(9):539–549. doi: 10.1023/a:1015985728434. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES