Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Negative feedback inhibition of HIV-1 by TAT-inducible expression of siRNA

Nature Biotechnology volume 22, pages 1573–1578 (2004)Cite this article

Abstract

Here we demonstrate that an inducible anti-HIV short hairpin RNA (shRNA) expressed from a Pol II promoter inhibits HIV-1 gene expression in mammalian cells. Our strategy is based on a promoter system in which the HIV-1 LTR is fused to the Drosophila hsp70 minimal heat shock promoter. This system is inducible by HIV-1 TAT, which functions in a negative feedback loop to activate transcription of an shRNA directed against HIV-1 rev. Upon induction the shRNA is processed to an siRNA that guides inhibition of HIV replication in cultured T-lymphocytes and hematopoietic stem cell–derived monocytes. The fusion promoter system may be safer than drug-inducible systems for shRNA-mediated gene therapy against HIV as the shRNAs are only expressed following HIV infection.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Expression of shRNA from a Pol II promoter (a) shRNA sequence and expression cassettes.
Figure 2: HIV-1 TAT inducible siRNA system (a) Schematic representation of LTRhsp-shRNA.
Figure 3: Inhibition of HIV-1 replication.

Similar content being viewed by others

References

  1. Grishok, A. et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23–34 (2001).

    Article  CAS  Google Scholar 

  2. Hutvagner, G. et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293, 834–838 (2001).

    Article  CAS  Google Scholar 

  3. Wianny, F. & Zernica-Goetz, M. Specific interference with gene function by double stranded RNA in early mouse development. Nat. Cell Biol. 2, 70–75 (2000).

    Article  CAS  Google Scholar 

  4. Kennerdell, J. & Carthew, R. Use of dsRNA-mediated genetic interference to demonstrate that frizzed and frizzled 2 act in the wingless pathway. Cell 95, 1017–1026 (1998).

    Article  CAS  Google Scholar 

  5. Fire, A. et al. Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans . Nature 391, 806–811 (1998).

    Article  CAS  Google Scholar 

  6. Lipardi, C., Wei, Q. & Patterson, B. RNAi as randomly degraded PCR. siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107, 297–307 (2001).

    Article  CAS  Google Scholar 

  7. Sijen, T. et al. On the role of RNA amplification in dsRNA –triggered gene silencing. Cell 107, 465–476 (2001).

    Article  CAS  Google Scholar 

  8. Caplan, N.J., Parrish, S., Imani, F., Fire, A. & Morgan, R.A. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA 98, 9742–9747 (2001).

    Article  Google Scholar 

  9. Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  Google Scholar 

  10. Brummelkamp, T.R., Bernards, R. & Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553 (2002).

    Article  CAS  Google Scholar 

  11. Lee, N.S. et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat. Biotechnol. 20, 500–505 (2002).

    Article  CAS  Google Scholar 

  12. Miyagishi, M. & Taira, K. U6 promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat. Biotechnol. 20, 497–500 (2002).

    Article  CAS  Google Scholar 

  13. Xia, H., Paulson, H. & Davidson, BL. siRNA-mediated gene silencing in vitro and in vivo . Nat. Biotechnol. 20, 1006–1010 (2002).

    Article  CAS  Google Scholar 

  14. An, D. et al. Efficient lentiviral vectors for short hairpin RNA delivery into human cells. Hum. Gene Ther. 14, 1207–1212 (2003).

    Article  CAS  Google Scholar 

  15. Yi, R., Qin, Y., Macara, I. & Cullen, B. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev. 17, 3011–3016 (2003).

    Article  CAS  Google Scholar 

  16. Lee, H. et al. DNA sequence requirements for generating paused polymerase at the start of hsp70. Genes Dev. 6, 284–295 (1992).

    Article  CAS  Google Scholar 

  17. Dropulic, B., Hermankova, M. & Pitha, P. A conditionally replicating HIV-1 vector interferes with wild-type HIV-1 replication and spread. Proc. Natl. Acad. Sci. USA 93, 11103–11108 (1996).

    Article  CAS  Google Scholar 

  18. Paik, S. et al. Defective HIV-1 provirus encoding a multi-target ribozyme inhibits accumulation of spliced and unspliced HIV-1 mRNAs, reduces infectivity of viral progeny, and protects the cells from pathogenesis. Hum. Gene Ther. 8, 1115–1123 (1997).

    Article  CAS  Google Scholar 

  19. Karn, J. Tackling Tat. J. Mol. Biol. 293, 235–254 (1999).

    Article  CAS  Google Scholar 

  20. Kobor, M. & Greenblatt, J. Regulation of transcription elongation by phosphorylation. Biochim. Biophys. Acta 1577, 261–275 (2002).

    Article  CAS  Google Scholar 

  21. Brand, A. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415 (1993).

    CAS  PubMed  Google Scholar 

  22. No, D., Yao, T. & Evans, R. Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci. USA 93, 3346–3351 (1996).

    Article  CAS  Google Scholar 

  23. Krumm, A., Meulia, T. & Groudine, M. Common mechanisms for the control of eukaryotic transcriptional elongation. Bioessays 15, 659–665 (1993).

    Article  CAS  Google Scholar 

  24. Kretz-Remy, C. & Arrigo, A. The kinetics of HIV-1 long terminal repeat transcriptional activation resemble those of hsp70 promoter in heat-shock treated HeLa cells. FEBS Lett. 351, 191–196 (1994).

    Article  CAS  Google Scholar 

  25. Palangat, M., Meier, T., Keene, R. & Landick, R. Transcriptional pausing at +62 of the HIV-1 nascent RNA modulates formation of the TAR RNA structure. Mol. Cell 1, 1033–1042 (1998).

    Article  CAS  Google Scholar 

  26. Mason, P. & Lis, J. Cooperative and competitive protein interactions at the hsp70 promoter. J. Biol. Chem. 272, 33227–33233 (1997).

    Article  CAS  Google Scholar 

  27. Hsu, M. et al. Human fatty acid synthase gene: evidence for the presence of two promoters and their functional interaction. J. Biol. Chem. 271, 13584–13592 (1996).

    Article  CAS  Google Scholar 

  28. Han, P., Brown, R. & Barsoum, J. Transactivation of heterologous promoters by HIV-1 Tat. Nucleic Acids Res. 19, 7225–7229 (1991).

    Article  CAS  Google Scholar 

  29. Southgate, C. & Green, M. The HIV-1 tat protein activates transcription from an upstream DNA binding site: implications for tat function. Genes Dev. 5, 2496–2507 (1991).

    Article  CAS  Google Scholar 

  30. Wiznerowicz, M. & Trono, D. Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference. J. Virol. 77, 8957–8961 (2003).

    Article  CAS  Google Scholar 

  31. Matsukura, S., Jones, P. & Takai, D. Establishment of conditional vectors for hairpin siRNA knockdowns. Nucleic Acids Res. 31, e77 (2003).

    Article  Google Scholar 

  32. Van De Wetering, M. et al. Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep. 4, 609–615 (2003).

    Article  CAS  Google Scholar 

  33. Moss, E. & Taylor, J. Small-interfering RNAs in the radar of the interferon system. Nat. Cell Biol. 5, 771–772 (2003).

    Article  CAS  Google Scholar 

  34. Sledz, C. et al. Activation of the interferon system by short-interfering RNAs. Nat. Cell Biol. 5, 834–839 (2003).

    Article  CAS  Google Scholar 

  35. Bridge, A. et al. Induction of an interferon response by RNAi vectors in mammalian cells. Nat. Genet. 34, 263–264 (2003).

    Article  CAS  Google Scholar 

  36. Gasmi, M. et al. Requirements for efficient production and transduction of human immunodeficiency virus type 1-based vectors. J. Virol. 73, 1828–1834 (1999).

    Article  CAS  Google Scholar 

  37. Graham, F. & van der Eb, A. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456–467 (1973).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research was supported by National Institutes of Health grants AI 29329, AI42552 and HL074704.

Author information

Authors and Affiliations

  1. Division of Molecular Biology, Beckman Research Institute of The City of Hope, Duarte, 91010, California, USA

    Hoshang J Unwalla, Ming-Jie Li, James D Kim, Hai Tang Li, Ali Ehsani, Jessica Alluin & John J Rossi

Authors
  1. Hoshang J Unwalla
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ming-Jie Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. James D Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hai Tang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ali Ehsani
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jessica Alluin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. John J Rossi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John J Rossi.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Transcription occurs from both, HIV-1 LTR and mhsp70 TATA boxes. (PDF 79 kb)

Supplementary Fig. 2

Partial processing of shRNA loop. (PDF 88 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Unwalla, H., Li, MJ., Kim, J. et al. Negative feedback inhibition of HIV-1 by TAT-inducible expression of siRNA. Nat Biotechnol 22, 1573–1578 (2004). https://doi.org/10.1038/nbt1040

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1040

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing