Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Nov 15;89(22):10802–10806. doi: 10.1073/pnas.89.22.10802

Inhibition of human immunodeficiency virus type 1 expression by a hairpin ribozyme.

J O Ojwang 1, A Hampel 1, D J Looney 1, F Wong-Staal 1, J Rappaport 1
PMCID: PMC50430  PMID: 1438280

Abstract

Ribozymes are RNAs that possess the dual properties of RNA sequence-specific recognition, analogous to conventional antisense molecules, and RNA substrate destruction via site-specific cleavage. The cleavage reaction is catalytic in that more than one substrate molecule is processed per ribozyme molecule. We have designed a hairpin ribozyme that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in the leader sequence (at nucleotides +111/112 relative to the transcription initiation site). The ribozyme was tested in vitro and gave efficient and specific cleavage of RNA containing the leader sequence. To test the antiviral efficacy of this ribozyme, we have cotransfected into HeLa cells HIV-1 proviral DNA and a plasmid expressing the ribozyme from the human beta-actin promoter. HIV-1 expression was inhibited as measured by p24 antigen levels and reduced Tat activity. The antiviral effect of the ribozyme appears to be specific and results from directed RNA cleavage; activity requires both a target sequence and a functional RNA catalytic center. These results suggest that this HIV-1-directed hairpin ribozyme may be useful as a therapeutic agent.

Full text

PDF
10806

Images in this article

10804Image
on p.10804
10804Image
on p.10804
10804Image
on p.10804
10805Image
on p.10805
10805Image
on p.10805
10805Image
on p.10805

Selected References

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

  1. 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]
  2. 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]
  3. Baltimore D. Gene therapy. Intracellular immunization. Nature. 1988 Sep 29;335(6189):395–396. doi: 10.1038/335395a0. [DOI] [PubMed] [Google Scholar]
  4. Buzayan J. M., Gerlach W. L., Bruening G. Satellite tobacco ringspot virus RNA: A subset of the RNA sequence is sufficient for autolytic processing. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8859–8862. doi: 10.1073/pnas.83.23.8859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cameron F. H., Jennings P. A. Specific gene suppression by engineered ribozymes in monkey cells. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9139–9143. doi: 10.1073/pnas.86.23.9139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chrisey L., Rossi J., Sarver N. Meeting Report. Ribozymes: progress and prospects of catalytic RNA as therapeutic agents. Antisense Res Dev. 1991 Spring;1(1):57–63. doi: 10.1089/ard.1991.1.57. [DOI] [PubMed] [Google Scholar]
  7. Dropulić B., Lin N. H., Martin M. A., Jeang K. T. Functional characterization of a U5 ribozyme: intracellular suppression of human immunodeficiency virus type 1 expression. J Virol. 1992 Mar;66(3):1432–1441. doi: 10.1128/jvi.66.3.1432-1441.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Gunning P., Leavitt J., Muscat G., Ng S. Y., Kedes L. A human beta-actin expression vector system directs high-level accumulation of antisense transcripts. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4831–4835. doi: 10.1073/pnas.84.14.4831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hampel A., Tritz R., Hicks M., Cruz P. 'Hairpin' catalytic RNA model: evidence for helices and sequence requirement for substrate RNA. Nucleic Acids Res. 1990 Jan 25;18(2):299–304. doi: 10.1093/nar/18.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hampel A., Tritz R. RNA catalytic properties of the minimum (-)sTRSV sequence. Biochemistry. 1989 Jun 13;28(12):4929–4933. doi: 10.1021/bi00438a002. [DOI] [PubMed] [Google Scholar]
  13. Haseloff J., Gerlach W. L. Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature. 1988 Aug 18;334(6183):585–591. doi: 10.1038/334585a0. [DOI] [PubMed] [Google Scholar]
  14. Hutchins C. J., Rathjen P. D., Forster A. C., Symons R. H. Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res. 1986 May 12;14(9):3627–3640. doi: 10.1093/nar/14.9.3627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Izant J. G., Weintraub H. Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis. Cell. 1984 Apr;36(4):1007–1015. doi: 10.1016/0092-8674(84)90050-3. [DOI] [PubMed] [Google Scholar]
  16. Leavitt J., Gunning P., Porreca P., Ng S. Y., Lin C. S., Kedes L. Molecular cloning and characterization of mutant and wild-type human beta-actin genes. Mol Cell Biol. 1984 Oct;4(10):1961–1969. doi: 10.1128/mcb.4.10.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Melton D. A. Injected anti-sense RNAs specifically block messenger RNA translation in vivo. Proc Natl Acad Sci U S A. 1985 Jan;82(1):144–148. doi: 10.1073/pnas.82.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mitsuya H., Yarchoan R., Broder S. Molecular targets for AIDS therapy. Science. 1990 Sep 28;249(4976):1533–1544. doi: 10.1126/science.1699273. [DOI] [PubMed] [Google Scholar]
  22. Prody G. A., Bakos J. T., Buzayan J. M., Schneider I. R., Bruening G. Autolytic processing of dimeric plant virus satellite RNA. Science. 1986 Mar 28;231(4745):1577–1580. doi: 10.1126/science.231.4745.1577. [DOI] [PubMed] [Google Scholar]
  23. Ratner L., Fisher A., Jagodzinski L. L., Mitsuya H., Liou R. S., Gallo R. C., Wong-Staal F. Complete nucleotide sequences of functional clones of the AIDS virus. AIDS Res Hum Retroviruses. 1987 Spring;3(1):57–69. doi: 10.1089/aid.1987.3.57. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sarver N., Cantin E. M., Chang P. S., Zaia J. A., Ladne P. A., Stephens D. A., Rossi J. J. Ribozymes as potential anti-HIV-1 therapeutic agents. Science. 1990 Mar 9;247(4947):1222–1225. doi: 10.1126/science.2107573. [DOI] [PubMed] [Google Scholar]
  27. Sczakiel G., Pawlita M. Inhibition of human immunodeficiency virus type 1 replication in human T cells stably expressing antisense RNA. J Virol. 1991 Jan;65(1):468–472. doi: 10.1128/jvi.65.1.468-472.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sczakiel G., Pawlita M., Kleinheinz A. Specific inhibition of human immunodeficiency virus type 1 replication by RNA transcribed in sense and antisense orientation from the 5'-leader/gag region. Biochem Biophys Res Commun. 1990 Jun 15;169(2):643–651. doi: 10.1016/0006-291x(90)90379-2. [DOI] [PubMed] [Google Scholar]
  29. Trono D., Feinberg M. B., Baltimore D. HIV-1 Gag mutants can dominantly interfere with the replication of the wild-type virus. Cell. 1989 Oct 6;59(1):113–120. doi: 10.1016/0092-8674(89)90874-x. [DOI] [PubMed] [Google Scholar]
  30. Weerasinghe M., Liem S. E., Asad S., Read S. E., Joshi S. Resistance to human immunodeficiency virus type 1 (HIV-1) infection in human CD4+ lymphocyte-derived cell lines conferred by using retroviral vectors expressing an HIV-1 RNA-specific ribozyme. J Virol. 1991 Oct;65(10):5531–5534. doi: 10.1128/jvi.65.10.5531-5534.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES