Advertisement

Molecular Biotechnology

, Volume 39, Issue 3, pp 231–238 | Cite as

Knockdown of Cellular RNA Helicase DDX3 by Short Hairpin RNAs Suppresses HIV-1 Viral Replication Without Inducing Apoptosis

  • Musarat Ishaq
  • Jiajie Hu
  • Xiaoyun Wu
  • Qiong Fu
  • Yalin Yang
  • Qingzhen Liu
  • Deyin Guo
Research

Abstract

The targeting of a cellular co-factor, rather than the HIV-1-specific RNAs, by small interfering RNAs holds promise as the rapid mutational ability of the HIV-1 genome may obviate the potential clinical use of RNAi against this virus. The DEAD-box RNA helicase DDX3 is an essential Rev co-factor in the CRM1-Rev-RRE complex that promotes the export of unspliced and single-spliced HIV-1 RNAs from the nucleus to cytoplasm. In this report, human DDX3 was targeted by specific short hairpin RNAs, and the down-regulation of cell's endogenous DDX3 suppressed the nuclear export of unspliced HIV-1 RNAs but did not affect the cell viability. We further showed that the knockdown of cellular DDX3 could effectively inhibit the replication of HIV-1. Therefore, the current results suggest that the RNA helicase DDX3 may become a potential target by RNAi for future genetic therapy of HIV/AIDS.

Keywords

HIV-1 RNA helicase DDX3 RNAi Short hairpin RNA RNA nuclear export 

Notes

Acknowledgments

We thank Dr. Kuan-The Jeang for providing DDX3-HA plasmid, Dr. John Rossi for shRNA Rev, and Dr Ke Zhuang for Ghost (CD4/CCR5) cells. This study was supported by China “863” programme (grant 2006AA02Z123), China “973” programme (grant 2006CB504305), and the MOE “111” project #B06018.

References

  1. 1.
    McManus, M. T., & Sharp, P. A. (2002). Gene silencing in mammals by small interfering RNAs. Nature Reviews Genetics, 3, 737–747.CrossRefGoogle Scholar
  2. 2.
    Chiu, Y. L., & Rana, T. M. (2002). RNAi in human cells: basic structural and functional features of small interfering RNA. Molecular Cell, 10, 549–561.CrossRefGoogle Scholar
  3. 3.
    Lee, N. S., Dohjima, T., Bauer, G., Li, H., Li, M. J., Ehsani, A., Salvaterra, P., & Rossi, J. (2002). Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnology, 20, 500–505.Google Scholar
  4. 4.
    Novina, C. D., Murray, M. F., Dykxhoorn, D. M., Beresford, P. J., Riess, J., Lee, S. K., Collman, R. G., Lieberman, J., Shankar, P., & Sharp, P. A. (2002). siRNA-directed inhibition of HIV-1 infection. Nature Medicine, 8, 681–686.Google Scholar
  5. 5.
    Anderson, J., Banerjea, A., & Akkina, R. (2003). Bispecific short hairpin siRNA constructs targeted to CD4, CXCR4, and CCR5 confer HIV-1 resistance. Oligonucleotides, 13, 303–312.CrossRefGoogle Scholar
  6. 6.
    Fang, J., Kubotaa, S., Yanga, B., Zhoua, N., Zhanga, H., Godbout, R., & Pomerantz, R. J. (2004). A DEAD box protein facilitates HIV-1 replication as a cellular co-factor of Rev. Virology, 330, 471–480.CrossRefGoogle Scholar
  7. 7.
    Li, Z., Xiong, Y., Peng, Y., Pan, J., Chen, Y., Wu, X., Hussain, S., Tien, P., & Guo, D. (2005). Specific inhibition of HIV-1 replication by short hairpin RNAs targeting human cyclin T1 without inducing apoptosis. FEBS Letters, 579, 3100–3106.CrossRefGoogle Scholar
  8. 8.
    Rocak, S., & Linder, P. (2004). DEAD-box proteins: the driving forces behind RNA metabolism. Nature Reviews Molecular Cell Biology, 5, 232–241.CrossRefGoogle Scholar
  9. 9.
    Nekhai, S., & Jeang, K. T. (2006). Transcriptional and post-transcriptional regulation of HIV-1 gene expression: role of cellular factors for Tat and Rev. Future Microbiology, 1, 417–426.CrossRefGoogle Scholar
  10. 10.
    Jeang, K. T., & Yedavalli, V. (2005). Role of RNA helicases in HIV-1 replication. Nucleic Acids Research, 34, 4198–4205.CrossRefGoogle Scholar
  11. 11.
    Yedavalli, V. S., Neuveut, C., Chi, Y. H., Kleiman, L., & Jeang, K. T. (2004). Requirement of DDX3 DEAD box RNA helicase for HIV-1 Rev-RRE export function. Cell, 119, 381–392.CrossRefGoogle Scholar
  12. 12.
    Roy, B. B., Hu, J., Guo, X., Russell, R. S., Guo, F., Kleiman, L., & Liang, C. (2006). Association of RNA helicase A with human immunodeficiency virus type 1 particles. Journal of Biological Chemistry, 281, 12625–12635.CrossRefGoogle Scholar
  13. 13.
    Cocude, C., Truong, M. J., Billaut-Mulot, O., Delsart, V., Darcissac, E., Capron, A., Mouton, Y., & Bahr, G. M. (2003). A novel cellular RNA helicase, RH116, differentially regulates cell growth, programmed cell death and human immunodeficiency virus type 1 replication. Journal of General Virology, 84, 3215–3225.CrossRefGoogle Scholar
  14. 14.
    Malim, M. H., Hauber, J., Le, S. Y., Maizel, J. V., & Cullen, B. R. (1989). The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature, 338, 254–257.CrossRefGoogle Scholar
  15. 15.
    Sodroski, J., Goh, W. C., Rosen, C., Dayton, A., Terwilliger, E., & Haseltine, W. (1986). A second post-transcriptional trans-activator gene required for HTLV-III replication. Nature, 321, 412–417.CrossRefGoogle Scholar
  16. 16.
    Felber, B. K., Hadzopoulou-Cladaras, M., Cladaras, C., Copeland, T., & Pavlakis, G. N. (1989). Rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proceedings of National Academy of Sciences USA, 86, 1495–1499.CrossRefGoogle Scholar
  17. 17.
    Holland, S. M., Ahmad, N., Maitra, R. K., Wingfield, P., & Venkatesan, S. (1990). Human immunodeficiency virus rev protein recognizes a target sequence in rev-responsive element RNA within the context of RNA secondary structure. Journal of Virology, 64, 5966–5975.Google Scholar
  18. 18.
    Hope, T. J., McDonald, D., Huang, X. J., Low, J., & Parslow, T. G. (1990). Mutational analysis of the human immunodeficiency virus type 1 Rev transactivator: essential residues near the amino terminus. Journal of Virology, 64, 5360–5366.Google Scholar
  19. 19.
    Henderson, B. R., & Percipalle, P. (1997). Interactions between HIV Rev and nuclear import and export factors: the Rev nuclear localization signal mediates specific binding to human importin beta. Journal of Molecular Biology, 274, 693–707.CrossRefGoogle Scholar
  20. 20.
    Fischer, U., Pllard, V. W., Luhrmann, R., Teufel, M., Michael, M. W., Dreyfuss, G., & Malim, M. H. (1999). Rev mediated nuclear export of RNA is dominated over nuclear retention and is coupled to the Ran-GTPase cycle. Nucleic Acids Research, 27, 4128–4134.CrossRefGoogle Scholar
  21. 21.
    Michaelson, J. S., & Leder, P. (2003). RNAi reveals antiapoptotic and transcriptionally repressive activities of DAXX. Journal of Cell Science, 116, 345–352.CrossRefGoogle Scholar
  22. 22.
    He, J., Choe, S., Walker, R., Di Marzio, P., Morgan, D. O., & Landau, N. R. (1995). Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. Journal of Virology, 69, 6705–6711.Google Scholar
  23. 23.
    Connor, R. I., Chen, B. K., Choe, S., & Landau, N. R. (1995). Vpr is required for efficient replication of human immunodeficiency virus type 1 in mononuclear phagocytes. Virology, 206, 935–944.CrossRefGoogle Scholar
  24. 24.
    Suhasini, M., Badri, K. R., Holland, T. C., & Reddy, T. R. (2005). Sam68 is absolutely required for Rev function and HIV-1 production. Nucleic Acids Research, 33, 873–879.CrossRefGoogle Scholar
  25. 25.
    Kellam, P., Holzerlandt, R., Gramoustianou, E., Jenner, R., & Kwan, A. (2003). Viral bioinformatics: computational views of host and pathogen. Novartis Foundation Symposium, 254, 234–52.Google Scholar
  26. 26.
    DeFilippis, V., Raggo, C., Moses, A., & Fruh, K. (2003). Functional genomics in virology and antiviral drug discovery. Trends in Biotechnology, 21, 452–457.CrossRefGoogle Scholar
  27. 27.
    Krishnan, V., & Zeichner, L. S. (2004). Alteration in the expression of DEAD-box and other RNA binding proteins during HIV-1 replication. Reterovirology, 1, 42.CrossRefGoogle Scholar

Copyright information

© Humana Press 2008

Authors and Affiliations

  • Musarat Ishaq
    • 1
  • Jiajie Hu
    • 1
  • Xiaoyun Wu
    • 1
  • Qiong Fu
    • 1
  • Yalin Yang
    • 1
  • Qingzhen Liu
    • 1
  • Deyin Guo
    • 1
  1. 1.State Key Laboratory of Virology and Modern Virology Research Center, College of Life SciencesWuhan UniversityWuhanChina

Personalised recommendations