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.

  • Research Article
  • Published:

Integration complexes derived from HIV vectors for rapid assays in vitro

Nature Biotechnology volume 17, pages 578–582 (1999)Cite this article

Abstract

Of three enzymes encoded by HIV–reverse transcriptase, protease, and integrase—only the first two have been exploited clinically as inhibitor targets. Efforts to develop inhibitors of purified integrase protein have yielded many compounds, but none with clinical utility. A different source of integration activity for studies in vitro is provided by replication intermediates isolated from HIV-infected cells. These preintegration complexes (PICs) can direct integration of the endogenously synthesized viral cDNA into an added target DNA in vitro. Despite their authentic activities, assays of PICs have not been widely used due to technical obstacles, particularly the requirement for handling large amounts of infectious HIV. Here, we describe greatly improved methods for producing PICs using HIV-based vectors that are capable of establishing an integrated provirus but not a spreading infection. We also report the development of a PIC integration assay using DNA-coated microtiter plates, which speeds assays of PIC integration in vitro. We used this method to screen a library of chemicals related to known integrase inhibitors and found a new compound, quinalizarin sulfate, that displayed enhanced activity against PICs.

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: DNA cutting and joining reactions mediating cDNA integration.
Figure 2: Diagram of the DNA constructions used for the production of HIV-based vector particles.
Figure 3: Integration activity of vector-based PICs analyzed by Southern blot.
Figure 4: Diagram of a microtiter plate-based assay for detection of integration by PICs.
Figure 5: Quantification of integrated HIV genomes by real-time fluorescence-monitored PCR and characterization of integration inhibitors.

Similar content being viewed by others

References

  1. Coffin, J.M., Hughes, S.H. & Varmus, H.E. Retroviruses (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1997).

    Google Scholar 

  2. Bushman, F.D., Landau, N.R. & Emini, E.A. New developments in the biology and treatment of HIV. Proc. Natl. Acad. Sci. USA 95, 11041– 11042 (1998).

    Article  CAS  Google Scholar 

  3. Pommier, Y., Pilon, A.A., Bajaj, K., Mazumder, A. & Neamati, N. HIV-1 integrase as a target for antiviral drugs. Antivir. Chem. Chemother. 8, 463–483 (1997).

    Article  CAS  Google Scholar 

  4. Hansen, M.S.T., Carteau, S., Hoffmann, C., Li, L. & Bushman, F. in Genetic engineering. Principles and methods (ed. Setlow, J.K.) 41–62 (Plenum, New York and London, 1998).

    Google Scholar 

  5. Patel, P.H. & Preston, B.D. Marked infidelity of human immunodeficiency virus type 1 reverse transcriptase at RNA and DNA template ends. Proc. Natl. Acad. Sci. USA 91, 549– 553 (1994).

    Article  CAS  Google Scholar 

  6. Miller, M.D., Farnet, C.M. & Bushman, F.D. Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition. J. Virol. 71, 5382–5390 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Brown, P.O., Bowerman, B., Varmus, H.E. & Bishop, J.M. Correct integration of retroviral DNA in vitro. Cell 49, 347–356 (1987).

    Article  CAS  Google Scholar 

  8. Farnet, C.M. & Haseltine, W.A. Integration of human immunodeficiency virus type 1 DNA in vitro. Proc. Natl. Acad. Sci. USA 87, 4164–4168 (1990).

    Article  CAS  Google Scholar 

  9. Ellison, V.H., Abrams, H., Roe, T., Lifson, J. & Brown, P.O. Human immunodeficiency virus integration in a cell-free system. J. Virol. 64, 2711– 2715 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Hansen, M. & Bushman, F.D. HIV-2 preintegration complexes: activities in vitro and response to inhibitors. J. Virol. 71, 3351–3356 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Lee, Y.M.H. & Coffin, J.M. Relationship of avian retrovirus DNA synthesis to integration in vitro. Mol. Cell Biol. 11, 1419–1430 (1991).

    Article  CAS  Google Scholar 

  12. Bukrinsky, M.I. et al. Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection. Proc. Natl. Acad. Sci. USA 90, 6125–6129 (1993).

    Article  CAS  Google Scholar 

  13. Gallay, P., Swingler, S., Song, J., Bushman, F. & Trono, D. HIV nuclear import is governed by the phosphotyrosine-mediated binding of matrix to the core domain of integrase. Cell 17, 569–576 (1995).

    Article  Google Scholar 

  14. Farnet, C.M. & Haseltine, W.A. Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex. J. Virol. 65, 1910–1915 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Farnet, C. & Bushman, F.D. HIV-1 cDNA integration: requirement of HMG I(Y) protein for function of preintegration complexes in vitro. Cell 88, 1–20 ( 1997).

    Article  Google Scholar 

  16. Wei, S.-Q., Mizuuchi, K. & Craigie, R. Footprints of the viral DNA ends in Moloney murine leukemia virus preintegration complexes reflect a specific association with integrase. Proc. Natl. Acad. Sci. USA 95, 10535– 10540 (1998).

    Article  CAS  Google Scholar 

  17. Lee, Y.M.H. & Coffin, J.M. Efficient autointegration of avian retrovirus DNA in vitro. J. Virol. 64, 5958 –5965 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Bushman, F.D., Fujiwara, T. & Craigie, R. Retroviral DNA integration directed by HIV integration protein in vitro. Science 249, 1555– 1558 (1990).

    Article  CAS  Google Scholar 

  19. Bushman, F.D. & Craigie, R. Activities of human immunodeficiency virus (HIV) integration protein in vitro: specific cleavage and integration of HIV DNA. Proc. Natl. Acad. Sci. USA 88, 1339–1343 (1991).

    Article  CAS  Google Scholar 

  20. Hazuda, D.J., Felock, P.J., Hastings, J.C., Pramanik, B. & Wolfe, A.L. Differential divalent cation requirements uncouple the assembly and catalytic reactions of human immunodeficiency virus type 1 integrase. J. Virol. 71, 7005– 7011 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Hindmarsh, P. et al. HMG protein family members stimulate human immunodeficiency virus type 1 avain sarcoma virus concerted DNA integration in vitro. J. Virol. 73, 2994–3003 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Farnet, C., Lipford, R., Wang, B. & Bushman, F.D. Differential inhibition of HIV-1 preintegration complexes and purified integrase protein by small molecules. Proc. Natl. Acad. Sci. USA 93, 9742–9747 (1996).

    Article  CAS  Google Scholar 

  23. Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263 –267 (1996).

    Article  CAS  Google Scholar 

  24. Kafri, T., van Praag, H., Ouyang, L., Gage, F.H. & Verma, I.M. A packaging cell line for lentiviral vectors. J. Virol. 73, 573– 584 (1999).

    Google Scholar 

  25. Gossen, M. et al. Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766–1769 ( 1995).

    Article  CAS  Google Scholar 

  26. Gibson, U., Heid, C.A. & Williams, P.M. A novel method for real time quantitative RT PCR. Genome Res. 6, 995–1001 (1996).

    Article  CAS  Google Scholar 

  27. Heid, C.A., Stevens, J., Livak, K.J. & Williams, P.M. Real time quantitative PCR. Genome Res. 6, 986– 994 (1996).

    Article  CAS  Google Scholar 

  28. Cushman, M. & Sherman, P. Inhibition of HIV-1 integration protein by aurintricarboxylic acid monomers, monomer analogs, and polymer fractions. Biochem. Biophys. Res. Commun. 185, 85–90 (1992).

    Article  CAS  Google Scholar 

  29. Robinson, W.E., Reinecke, M.G., Abdel-Malek, S., Jia, Q. & Chow, S.A. Inhibitors of HIV-1 replication that inhibit HIV integrase. Proc. Natl. Acad. Sci. USA 93 , 6326–6331 (1996).

    Article  CAS  Google Scholar 

  30. Farnet, C.M. et al. Human immunodeficiency virus type 1 integration: new aromatic hydroxylated inhibitors and studies of the inhibition mechanism. Antimicrob. Agents Chemother. 42, 2245–2253 (1998).

    Article  CAS  Google Scholar 

  31. Bartz, S.R. & Vodicka, M.A. Production of high-titer human immunodeficiency virus yype 1 pseudotyped with vesicular stomatitis virus glycoprotein. Methods 12, 337– 342 (1997).

    Article  CAS  Google Scholar 

  32. King, P.J. & Robinson, W.E.J. Resistance to the anti-human immunodeficiency virus type 1 compound L-chicoric acid results from a single mutation at amino acid residue 140 of integrase. J. Virol. 72, 8420–8424 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Miller, M.D., Wang, B. & Bushman, F.D. HIV-1 preintegration complexes containing discontinuous plus strands are competent to integrate in vitro. J. Virol. 69, 3938–3944 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Ghosh, S.S. & Musso, G.F. Covalent attachment of oligonucleotides to solid supports. Nucleic Acids Res. 15, 5353–5372 (1987).

    Article  CAS  Google Scholar 

  35. Lund, V., Schmid, R., Rickwood, D. & Hornes, E. Assessment of methods for covalent binding of nucleic acids to magnetic beads, dynabeads, and the characteristics of the bound nucleic acids in hybridization reactions. Nucleic Acids Res. 16, 10861–10879 (1988).

    Article  CAS  Google Scholar 

  36. Rasmussen, S.R., Larsen, M.R. & Rasmussen, S.E. Covalent immobilization of DNA onto polystyrene microwells: the molecules are only bound at the 5´ end. Anal. Biochem . 198, 138–142 ( 1991).

    Article  CAS  Google Scholar 

  37. Chu, B.C.F., Wahl, G.M. & Orgel, L.E. Derivitization of unprotected polynucleotides. Nucleic Acids Res. 11, 6513–6529 (1983).

    Article  CAS  Google Scholar 

  38. Hazuda, D.J., Hastings, J.C., Wolfe, A.L. & Emini, E.A. A novel assay for the DNA strand-transfer reaction of HIV-1 integrase. Nucleic Acids Res. 22, 1121–1122 (1994).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank members of the Bushman lab, Barbara Chu, Leslie Orgel, Matthew Helf, Francois Ferre, and Inder Verma for help and discussions and Alison Bocksruker for assistance with the manuscript. F.D.B. is a Scholar of the Leukemia Society of America. This work was supported by grants GM56553 and AI34786 to F.D.B.

Author information

Authors and Affiliations

  1. Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, 92037, CA

    Mark S.T. Hansen & Frederic D. Bushman

  2. Althea Technologies, San Diego, 92121, CA

    George J. Smith III

  3. Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, 92037, CA

    Tal Kafri

  4. Department of Chemistry, University of California at San Diego, La Jolla, 92024, CA

    Valentina Molteni & Jay S. Siegel

Authors
  1. Mark S.T. Hansen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. George J. Smith III
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tal Kafri
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Valentina Molteni
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jay S. Siegel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Frederic D. Bushman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Frederic D. Bushman.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hansen, M., Smith, G., Kafri, T. et al. Integration complexes derived from HIV vectors for rapid assays in vitro . Nat Biotechnol 17, 578–582 (1999). https://doi.org/10.1038/9886

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/9886

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