Modulation of HIV-1 replication by RNA interference

Abstract

RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells1,2. In mammalian cells, RNAi can be triggered by 21-nucleotide duplexes of small interfering RNA (siRNA)3. Here we describe inhibition of early and late steps of HIV-1 replication in human cell lines and primary lymphocytes by siRNAs targeted to various regions of the HIV-1 genome. We demonstrate that synthetic siRNA duplexes or plasmid-derived siRNAs inhibit HIV-1 infection by specifically degrading genomic HIV-1 RNA, thereby preventing formation of viral complementary-DNA intermediates. These results demonstrate the utility of RNAi for modulating the HIV replication cycle and provide evidence that genomic HIV-1 RNA, as it exists within a nucleoprotein reverse-transcription complex, is amenable to siRNA-mediated degradation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Small interfering RNAs inhibit late events in HIV replication by promoting degradation of HIV-1 RNA.
Figure 2: Small interfering RNAs block early events in HIV replication by promoting degradation of genomic HIV RNA.
Figure 3: Inhibition of HIV replication by siRNAs derived from plasmid DNA templates.

References

  1. 1

    Sharp, P. A. RNA interference—2001. Genes Dev. 15, 485–490 (2001)

    CAS  Article  Google Scholar 

  2. 2

    Hutvagner, G. & Zamore, P. D. RNAi: nature abhors a double-strand. Curr. Opin. Genet. Dev. 12, 225–232 (2002)

    CAS  Article  Google Scholar 

  3. 3

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

    ADS  CAS  Article  Google Scholar 

  4. 4

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

    ADS  CAS  Article  Google Scholar 

  5. 5

    Zamore, P. D., Tuschl, T., Sharp, P. A. & Bartel, D. P. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25–33 (2000)

    CAS  Article  Google Scholar 

  6. 6

    Elbashir, S. M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001)

    CAS  Article  Google Scholar 

  7. 7

    Hammond, S. M., Bernstein, E., Beach, D. & Hannon, G. J. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293–296 (2000)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Bernstein, E., Caudy, A. A., Hammond, S. M. & Hannon, G. J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366 (2001)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Nykanen, A., Haley, B. & Zamore, P. D. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309–321 (2001)

    CAS  Article  Google Scholar 

  10. 10

    Caplen, N. J., Fleenor, J., Fire, A. & Morgan, R. A. dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference. Gene 252, 95–105 (2000)

    CAS  Article  Google Scholar 

  11. 11

    Ui-Tei, K., Zenno, S., Miyata, Y. & Saigo, K. Sensitive assay of RNA interference in Drosophila and Chinese hamster cultured cells using firefly luciferase gene as target. FEBS Lett. 479, 79–82 (2000)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Welker, R., Harris, M., Cardel, B. & Krausslich, H. G. Virion incorporation of human immunodeficiency virus type 1 Nef is mediated by a bipartite membrane-targeting signal: analysis of its role in enhancement of viral infectivity. J. Virol. 72, 8833–8840 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Kimpton, J. & Emerman, M. Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated β-galactosidase gene. J. Virol. 66, 2232–2239 (1992)

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Bitko, V. & Barik, S. Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses. BMC Microbiol. 1, 34–45 (2001)

    CAS  Article  Google Scholar 

  15. 15

    Li, Y. et al. Molecular characterization of human immunodeficiency virus type 1 cloned directly from uncultured human brain tissue: Identification of replication-competent and -defective viral genomes. J. Virol. 65, 3973–3985 (1991)

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Moore, J. & Stevenson, M. New targets for inhibitors of HIV-1 replication. Nature Rev. Mol. Cell Biol. 1, 40–49 (2000)

    CAS  Article  Google Scholar 

  17. 17

    Bitko, V. & Barik, S. An endoplasmic reticulum-specific stress-activated caspase (caspase-12) is implicated in the apoptosis of A549 epithelial cells by respiratory syncytial virus. J. Cell Biochem. 80, 441–454 (2001)

    CAS  Article  Google Scholar 

  18. 18

    Wang, M. B. & Waterhouse, P. M. High-efficiency silencing of a β-glucuronidase gene in rice is correlated with repetitive transgene structure but is independent of DNA methylation. Plant Mol. Biol. 43, 67–82 (2000)

    CAS  Article  Google Scholar 

  19. 19

    Varshawesley, S. et al. Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J. 27, 581–590 (2001)

    Article  Google Scholar 

  20. 20

    Dornburg, R. & Pomerantz, R. J. HIV-1 gene therapy: promise for the future. Adv. Pharmacol. 49, 229–261 (2000)

    CAS  Article  Google Scholar 

  21. 21

    Ketting, R. F., Haverkamp, T. H., van Luenen, H. G. & Plasterk, R. H. Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD. Cell 99, 133–141 (1999)

    CAS  Article  Google Scholar 

  22. 22

    Tabara, H., Hill, R. J., Mello, C. C., Priess, J. R. & Kohara, Y. pos-1 encodes a cytoplasmic zinc-finger protein essential for germline specification in C. elegans. Development 126, 1–11 (1999)

    CAS  Google Scholar 

  23. 23

    Caplen, 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)

    ADS  CAS  Article  Google Scholar 

  24. 24

    Billy, E., Brondani, V., Zhang, H., Muller, U. & Filipowicz, W. Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc. Natl Acad. Sci. USA 98, 14428–14433 (2001)

    ADS  CAS  Article  Google Scholar 

  25. 25

    Paddison, P. J., Caudy, A. A. & Hannon, G. J. Stable suppression of gene expression by RNAi in mammalian cells. Proc. Natl Acad. Sci. USA 99, 1443–1448 (2002)

    ADS  CAS  Article  Google Scholar 

  26. 26

    Yang, S., Tutton, S., Pierce, E. & Yoon, K. Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells. Mol. Cell. Biol. 21, 7807–7816 (2001)

    CAS  Article  Google Scholar 

  27. 27

    Sharkey, M. et al. Persistence of episomal HIV-1 infection intermediates in patients on highly active antiretroviral therapy. Nature Med. 6, 76–81 (2000)

    CAS  Article  Google Scholar 

  28. 28

    Brichacek, B. & Stevenson, M. Quantitative competitive RNA PCR for quantitation of virion associated HIV-1 RNA. Methods 12, 294–299 (1997)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank A. Mann for research support, C. Mello and P. Zamore for discussions, B. Mellor for preparation of the figures, and T. Pinkos for manuscript preparation. We also acknowledge assay support provided by the University of Massachusetts Center for AIDS Research. HIVYU-2 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), from B. Hahn and G. Shaw. This study was supported by grants from the NIH and the Jenner Foundation to M.S.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Mario Stevenson.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Jacque, J., Triques, K. & Stevenson, M. Modulation of HIV-1 replication by RNA interference. Nature 418, 435–438 (2002). https://doi.org/10.1038/nature00896

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.