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. 1990 Aug;87(15):5817–5821. doi: 10.1073/pnas.87.15.5817

RNA transcripts of the human immunodeficiency virus transactivation response element can inhibit action of the viral transactivator.

G J Graham 1, J J Maio 1
PMCID: PMC54419  PMID: 1696012

Abstract

Tandem repeats of the transactivation response element (TAR) of the human immunodeficiency virus 1 (HIV-1) were generated using a specially constructed "tandemizing" plasmid, pGem-Tan. This plasmid exploits the rotational nonequivalence of Ava I restriction sites to generate multiple copies of an inserted sequence. Twelve tandem repeats of the TAR were then placed in sense and antisense orientations behind a strong human beta-actin gene promoter. The TAR constructs were transfected with an appropriate HIV-1-driven reporter and tat gene expression plasmids into NT2/D1 cells, a pluripotential human embryonic teratocarcinoma cell line. Twelve tandem TAR repeats in the sense orientation suppressed 85-90% of the transactivating function of the virus-encoded tat protein, whereas the antisense construct or constructs containing single copies of TAR in either sense or antisense orientations were relatively ineffective. The suppression was specific for reporter gene constructs containing an intact HIV-1 long terminal repeat: Reporter genes driven by other promoters or by an HIV-1 long terminal repeat lacking the TAR were not suppressed. Suppression of activation by tat required transcription into RNA: Similar constructs containing the TAR repeats but lacking a eukaryotic promoter failed to suppress tat activation. In the absence of tat, the TAR DNA stimulated 2- to 5-fold the expression of gene constructs driven not only by the HIV-1 long terminal repeat but also by the human beta-actin gene and the simian virus 40 promoters.

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Selected References

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

  1. Andrews P. W. Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro. Dev Biol. 1984 Jun;103(2):285–293. doi: 10.1016/0012-1606(84)90316-6. [DOI] [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. Cullen B. R., Greene W. C. Regulatory pathways governing HIV-1 replication. Cell. 1989 Aug 11;58(3):423–426. doi: 10.1016/0092-8674(89)90420-0. [DOI] [PubMed] [Google Scholar]
  5. Edery I., Petryshyn R., Sonenberg N. Activation of double-stranded RNA-dependent kinase (dsl) by the TAR region of HIV-1 mRNA: a novel translational control mechanism. Cell. 1989 Jan 27;56(2):303–312. doi: 10.1016/0092-8674(89)90904-5. [DOI] [PubMed] [Google Scholar]
  6. Gaynor R., Soultanakis E., Kuwabara M., Garcia J., Sigman D. S. Specific binding of a HeLa cell nuclear protein to RNA sequences in the human immunodeficiency virus transactivating region. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4858–4862. doi: 10.1073/pnas.86.13.4858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gendler S., Taylor-Papadimitriou J., Duhig T., Rothbard J., Burchell J. A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats. J Biol Chem. 1988 Sep 15;263(26):12820–12823. [PubMed] [Google Scholar]
  8. Gentz R., Chen C. H., Rosen C. A. Bioassay for trans-activation using purified human immunodeficiency virus tat-encoded protein: trans-activation requires mRNA synthesis. Proc Natl Acad Sci U S A. 1989 Feb;86(3):821–824. doi: 10.1073/pnas.86.3.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Hambor J. E., Hauer C. A., Shu H. K., Groger R. K., Kaplan D. R., Tykocinski M. L. Use of an Epstein-Barr virus episomal replicon for anti-sense RNA-mediated gene inhibition in a human cytotoxic T-cell clone. Proc Natl Acad Sci U S A. 1988 Jun;85(11):4010–4014. doi: 10.1073/pnas.85.11.4010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hartley J. L., Gregori T. J. Cloning multiple copies of a DNA segment. Gene. 1981 May;13(4):347–353. doi: 10.1016/0378-1119(81)90014-7. [DOI] [PubMed] [Google Scholar]
  12. Jones K. A., Luciw P. A., Duchange N. Structural arrangements of transcription control domains within the 5'-untranslated leader regions of the HIV-1 and HIV-2 promoters. Genes Dev. 1988 Sep;2(9):1101–1114. doi: 10.1101/gad.2.9.1101. [DOI] [PubMed] [Google Scholar]
  13. Maio J. J., Brown F. L. Regulation of expression driven by human immunodeficiency virus type 1 and human T-cell leukemia virus type I long terminal repeats in pluripotential human embryonic cells. J Virol. 1988 Apr;62(4):1398–1407. doi: 10.1128/jvi.62.4.1398-1407.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Maio J. J. DNA strand reassociation and polyribonucleotide binding in the African green monkey, Cercopithecus aethiops. J Mol Biol. 1971 Mar 28;56(3):579–595. doi: 10.1016/0022-2836(71)90403-7. [DOI] [PubMed] [Google Scholar]
  15. Perez-Stable C., Shen C. K. Competitive and cooperative functioning of the anterior and posterior promoter elements of an Alu family repeat. Mol Cell Biol. 1986 Jun;6(6):2041–2052. doi: 10.1128/mcb.6.6.2041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Scholer H., Haslinger A., Heguy A., Holtgreve H., Karin M. In vivo competition between a metallothionein regulatory element and the SV40 enhancer. Science. 1986 Apr 4;232(4746):76–80. doi: 10.1126/science.3006253. [DOI] [PubMed] [Google Scholar]
  17. Sodroski J., Rosen C., Wong-Staal F., Salahuddin S. Z., Popovic M., Arya S., Gallo R. C., Haseltine W. A. Trans-acting transcriptional regulation of human T-cell leukemia virus type III long terminal repeat. Science. 1985 Jan 11;227(4683):171–173. doi: 10.1126/science.2981427. [DOI] [PubMed] [Google Scholar]
  18. Takeshita S., Tezuka K., Takahashi M., Honkawa H., Matsuo A., Matsuishi T., Hashimoto-Gotoh T. Tandem gene amplification in vitro for rapid and efficient expression in animal cells. Gene. 1988 Nov 15;71(1):9–18. doi: 10.1016/0378-1119(88)90072-8. [DOI] [PubMed] [Google Scholar]
  19. 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]

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