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.

Killing HIV-infected cells by transduction with an HIV protease-activated caspase-3 protein

Abstract

At present, treatment of HIV infection uses small inhibitory molecules that target HIV protease; however, the emergence of resistant HIV strains is increasingly problematic. To circumvent this, we report here a new 'Trojan horse' strategy to kill HIV-infected cells by exploiting HIV protease. We engineered a transducing, modified, apoptosis-promoting caspase-3 protein, TAT–Casp3, that substitutes HIV proteolytic cleavage sites for endogenous ones and efficiently transduces about 100% of cells, but remains inactive in uninfected cells. In HIV-infected cells, TAT–Casp3 becomes processed into an active form by HIV protease, resulting in apoptosis of the infected cell. This strategy could also be applied to other pathogens encoding specific proteases, such as hepatitis C virus, cytomegalovirus and malaria.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Generation and transduction with TAT fusion proteins.
Figure 2: In vivo substrate processing in co-transduced cells.
Figure 3: Activation of TAT–Casp3 and apoptotic induction in co-transduced cells.
Figure 4: HIV protease activates TAT–Casp3WT protein.
Figure 5: Specific killing of HIV-infected cells.

References

  1. Lillehoj, E.P. et al. Purification and structural characterization of the putative gag-pol protease of human immunodeficiency virus. J. Virol. 62, 3053–3058 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Kohl, N.E. et al. Active human immunodeficiency virus protease is required for viral infectivity. Proc. Natl. Acad. Sci. USA 85, 4686–4690 (1988).

    CAS  Article  Google Scholar 

  3. Göttlinger, H.G., Sodroski, J.G. & Haseltine, W.A. Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 86, 5781– 5785 (1989).

    Article  Google Scholar 

  4. Kaplan, A.H. et al. Selection of multiple human immunodeficiency virus type 1 variants that encode viral proteases with decreased sensitivity to an inhibitor of the viral protease. Proc. Natl. Acad. Sci. USA 91 , 5597–5601 (1994).

    CAS  Article  Google Scholar 

  5. Gatlin, J., Arrigo, S.J. & Schmidt, MG. Regulation of intracellular human immunodeficiency virus type-1 protease activity. Virology 244, 87–96 (1998).

    CAS  Article  Google Scholar 

  6. Coffin, J.M., Hughes, S.H. & Varmus, H.E. Retroviruses (Cold Spring Harbor Press, Cold Spring Harbor, New York, 1997).

  7. Condra, J.H. et al. In vivo emergence of HIV-1 variants resistant to multiple protease inhibitors. Nature 374, 569– 571 (1995).

    CAS  Article  Google Scholar 

  8. Gulnik, S.V. et al. Kinetic characterization and cross-resistance patterns of HIV-1 protease mutants selected under drug pressure. Biochemistry 34, 9282–9287 ( 1995).

    CAS  Article  Google Scholar 

  9. Tisdale, M. et al. Cross-resistance analysis of human immunodeficiency virus type 1 variants individually selected for resistance to five different protease inhibitors. Antimicrob. Agents Chemother. 39, 1704–1710 (1995).

    CAS  Article  Google Scholar 

  10. Salvesen, G.S. & Dixit, V.M. caspases: intracellular signaling by proteolysis. Cell 91, 443– 446 (1997).

    CAS  Article  Google Scholar 

  11. Henkart, P.A. ICE family proteases: mediators of all apoptotic cell death? Immunity 4, 195–201 ( 1996).

    CAS  Article  Google Scholar 

  12. Cohen, G.M. caspases: the executioners of apoptosis. J. Biochem. 326, 1–16 (1997).

    CAS  Article  Google Scholar 

  13. Woo, M. et al. Essential contribution of caspase 3/Casp3 to apoptosis and its associated nuclear changes. Genes Dev. 12, 806–819 (1998).

    CAS  Article  Google Scholar 

  14. Enari, M. et al. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43– 50 (1998).

    CAS  Article  Google Scholar 

  15. Liu, X. et al. DFF40 induces DNA fragmentation and chromatin condensation during apoptosis. Proc. Natl. Acad. Sci. USA 15, 8461–8466 (1998).

    Article  Google Scholar 

  16. Ratner, L. et al. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313, 277–284 (1985).

    CAS  Article  Google Scholar 

  17. Rice, C.M. in Fields Virology (eds. Fields, B.N., Knipe, D.M. & Howley, P.M.) 931–960 (Lippincott-Raven, Philadelphia, 1996).

    Google Scholar 

  18. Welch, A.R., Woods, A.S., McNally, L.M., Cotter, R.J. & Gibson, W. A. Herpesvirus maturational protease, assemblin: identification of its gene, putative active site domain, and cleavage site. Proc. Natl. Acad. Sci. USA 88, 10792 –10796 (1991).

    CAS  Article  Google Scholar 

  19. Francis, S.E., Sullivan, D.J. Jr. & Goldberg, D.E. Hemoglobin metabolism in the malaria parasite Plasmodium falciparum. Annu. Rev. Microbiol. 51, 97–123 (1997).

    CAS  Article  Google Scholar 

  20. Barrie, K.A. et al. Natural variation in HIV-1 protease, gag p7 and p6, and protease cleavage sites within gag/pol polyproteins: amino acid substitutions in the absence of protease inhibitors in mothers and children infected by human immunodeficiency virus type 1. Virology 219, 407– 416 (1996).

    CAS  Article  Google Scholar 

  21. Ezhevsky, S.A. et al. Hypo-phosphorylation of the retinoblastoma protein by cyclin D:Cdk4/6 complexes results in active pRb. Proc. Natl. Acad. Sci. USA 94, 10699–10704 ( 1997).

    CAS  Article  Google Scholar 

  22. Lissy, N.A. et al. TCR-antigen induced cell death (AID) occurs from a late G 1 phase cell cycle check point. Immunity 8, 57–65 (1998).

    CAS  Article  Google Scholar 

  23. Nagahara, H. et al. Highly efficient transduction of full length TAT fusion proteins directly into mammalian cells: p27Kip1 mediates cell migration. Nature Med. 4, 1449–1452 (1998).

    CAS  Article  Google Scholar 

  24. Vocero-Akbani, A. et al. Transduction of full length TAT fusion proteins directly into mammalian cells:analysis of TCR-activation induced cell death (AID) in Methods in Enzymology (ed. Reed, J.C.) (Academic, San Diego, in the press).

  25. Coates, P.J. Molecular methods for the identification of apoptosis in tissues. J. Histotechnol. 17, 261–267 (1994).

    Article  Google Scholar 

  26. Xiang, J., Chao, D.T. & Korsmeyer, S.J. Bax-induced cell death may not require interleukin 1B-converting enzyme-like proteases. Proc. Natl. Acad. Sci. USA 93, 14559–14563 ( 1996).

    CAS  Article  Google Scholar 

  27. Westervelt, P., Genedelman, H.E. & Ratner, L. Identification of a determinant within the HIV-1 surface envelope glycoprotein critical for productive infection of cultured primary monocytes. Proc. Natl. Acad. Sci USA 88, 3097–3101 (1991).

    CAS  Article  Google Scholar 

  28. Lou, X., Budihardjo, I., Zou, H., Slaughter, C. & Wang, X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94, 481– 490 (1998).

    Article  Google Scholar 

  29. Li, H., Zhu, C.-j. & Yuan, J. Cleavage of BID by caspase 8 mediates the mitochonrial damage in Fas pathway of apoptosis. Cell 95, 491–501 (1998).

    Article  Google Scholar 

  30. Wong, J.K. et al. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 278, 1291– 1295 (1997).

    CAS  Article  Google Scholar 

  31. Finzi, D. et al. Identification of a reservior for HIV-1 patients on highly active antiretroviral therapy. Science 278, 1295 –1300 (1997).

    CAS  Article  Google Scholar 

  32. Wu, X. et al. Functional RT and IN incorporated into HIV-1 particles independently of the Gag/Pol precursor protein. EMBO J. 16, 5113–5122 (1997).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank E.S. Alnemri for the human Casp3 cDNA; K. Wang for help with DEVD-AFC reactions; Abbott Labs for Ritonavir; S. Horning for doing the TUNEL assays; and D. Goldberg, C. Rice, S. Virgin and all the members of the Dowdy and Ratner labs for critical input. This work was supported by the N.I.H. (L.R.) and the Howard Hughes Medical Institute (S.F.D.). S.F.D. is an Assistant Investigator of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Steven F. Dowdy.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Vocero-Akbani, A., Heyden, N., Lissy, N. et al. Killing HIV-infected cells by transduction with an HIV protease-activated caspase-3 protein. Nat Med 5, 29–33 (1999). https://doi.org/10.1038/4710

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/4710

Further reading

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