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. 1994 Oct 11;91(21):9715–9719. doi: 10.1073/pnas.91.21.9715

Ribozyme-mediated suppression of Moloney murine leukemia virus and human immunodeficiency virus type I replication in permissive cell lines.

L Q Sun 1, D Warrilow 1, L Wang 1, C Witherington 1, J Macpherson 1, G Symonds 1
PMCID: PMC44887  PMID: 7937878

Abstract

Several hammerhead ribozymes targeted to different sites within the retroviral packaging (psi) sequences of the Moloney murine leukemia virus (Mo-MLV) and the human immunodeficiency virus type 1 (HIV-1) were designed and shown to cleave target RNA in vitro at the chosen sites. The engineered ribozymes, as well as antisense sequence complementary to the Mo-MLV psi packaging region, were cloned into the 3' untranslated region of the neomycin-resistance gene (neo). This was coupled to the simian virus 40 early promoter within the pSV2neo vector. For the ribozymes against the Mo-MLV psi site, the constructs were transfected into Mo-MLV-infected and virus-producing mouse NIH 3T3 cells. With the exception of one of the single ribozymes (the one least effective in cutting target RNA in vitro), all of the constructs effectively (70-80%) suppressed retrovirus production. These results demonstrate a direct correlation between in vitro cleavage and in vivo ribozyme-mediated virus suppression. In addition, a ribozyme targeted to the HIV-1 psi packaging site was engineered into the same vector and transfected into the human T-cell line SupT1. The transfectants were cloned and then challenged with HIV-1. When compared to vector-transfected control cells, a significant reduction in HIV-1 production was observed as measured by p24 and syncytia formation assays. This study demonstrates a feasible approach to the suppression of retrovirus replication by targeting the psi packaging site with hammerhead ribozymes.

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Selected References

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

  1. Adam M. A., Miller A. D. Identification of a signal in a murine retrovirus that is sufficient for packaging of nonretroviral RNA into virions. J Virol. 1988 Oct;62(10):3802–3806. doi: 10.1128/jvi.62.10.3802-3806.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alford R. L., Honda S., Lawrence C. B., Belmont J. W. RNA secondary structure analysis of the packaging signal for Moloney murine leukemia virus. Virology. 1991 Aug;183(2):611–619. doi: 10.1016/0042-6822(91)90990-s. [DOI] [PubMed] [Google Scholar]
  3. Chen C. J., Banerjea A. C., Harmison G. G., Haglund K., Schubert M. Multitarget-ribozyme directed to cleave at up to nine highly conserved HIV-1 env RNA regions inhibits HIV-1 replication--potential effectiveness against most presently sequenced HIV-1 isolates. Nucleic Acids Res. 1992 Sep 11;20(17):4581–4589. doi: 10.1093/nar/20.17.4581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Gautsch J. W., Meier H. A short-term quantitative XC assay for murine leukemia virus. Virology. 1976 Jul 15;72(2):509–513. doi: 10.1016/0042-6822(76)90179-3. [DOI] [PubMed] [Google Scholar]
  6. Han L., Yun J. S., Wagner T. E. Inhibition of Moloney murine leukemia virus-induced leukemia in transgenic mice expressing antisense RNA complementary to the retroviral packaging sequences. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4313–4317. doi: 10.1073/pnas.88.10.4313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Harrison G. P., Lever A. M. The human immunodeficiency virus type 1 packaging signal and major splice donor region have a conserved stable secondary structure. J Virol. 1992 Jul;66(7):4144–4153. doi: 10.1128/jvi.66.7.4144-4153.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Homann M., Tzortzakaki S., Rittner K., Sczakiel G., Tabler M. Incorporation of the catalytic domain of a hammerhead ribozyme into antisense RNA enhances its inhibitory effect on the replication of human immunodeficiency virus type 1. Nucleic Acids Res. 1993 Jun 25;21(12):2809–2814. doi: 10.1093/nar/21.12.2809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Lo K. M., Biasolo M. A., Dehni G., Palú G., Haseltine W. A. Inhibition of replication of HIV-1 by retroviral vectors expressing tat-antisense and anti-tat ribozyme RNA. Virology. 1992 Sep;190(1):176–183. doi: 10.1016/0042-6822(92)91203-7. [DOI] [PubMed] [Google Scholar]
  12. Mann R., Baltimore D. Varying the position of a retrovirus packaging sequence results in the encapsidation of both unspliced and spliced RNAs. J Virol. 1985 May;54(2):401–407. doi: 10.1128/jvi.54.2.401-407.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ojwang J. O., Hampel A., Looney D. J., Wong-Staal F., Rappaport J. Inhibition of human immunodeficiency virus type 1 expression by a hairpin ribozyme. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10802–10806. doi: 10.1073/pnas.89.22.10802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Reed K. C., Mann D. A. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 1985 Oct 25;13(20):7207–7221. doi: 10.1093/nar/13.20.7207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rossi J. J., Cantin E. M., Sarver N., Chang P. F. The potential use of catalytic RNAs in therapy of HIV infection and other diseases. Pharmacol Ther. 1991;50(2):245–254. doi: 10.1016/0163-7258(91)90016-f. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Sioud M., Drlica K. Prevention of human immunodeficiency virus type 1 integrase expression in Escherichia coli by a ribozyme. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7303–7307. doi: 10.1073/pnas.88.16.7303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Warrilow D., Takayama Y., Symonds G. A rapid protocol for the introduction of large, multiple oligonucleotide-mediated insertions. Biotechniques. 1992 Jul;13(1):42–45. [PubMed] [Google Scholar]
  19. 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]
  20. Yu M., Ojwang J., Yamada O., Hampel A., Rapapport J., Looney D., Wong-Staal F. A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6340–6344. doi: 10.1073/pnas.90.13.6340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]

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