A quantitative assay for HIV DNA integration in vivo (original) (raw)

Nature Medicine volume 7, pages 631–634 (2001)Cite this article

Abstract

Early steps of infection by HIV-1 involve entry of the viral core into cells, reverse transcription to form the linear viral DNA, and integration of that DNA into a chromosome of the host. The unintegrated DNA can also follow non-productive pathways, in which it is circularized by recombination between DNA long-terminal repeats (LTRs), circularized by ligation of the DNA ends or degraded. Here we report quantitative methods that monitor formation of reverse transcription products, two-LTR circles and integrated proviruses. The integration assay employs a novel quantitative form of Alu-PCR that should be generally applicable to studies of integrating viruses and gene transfer vectors.

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

Relevant articles

Open Access articles citing this article.

Access options

Subscribe to this journal

Receive 12 print issues and online access

$209.00 per year

only $17.42 per issue

Buy this article

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

Additional access options:

References

  1. 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
  2. Lewin, S.R. et al. Use of real-time PCR and molecular beacons to detect virus replication in Human Immunodeficiency Virus type 1-infected individuals on prolonged effective antiretroviral therapy. J. Virol. 73, 6099–6103 (1999).
    CAS PubMed PubMed Central Google Scholar
  3. Sharkey, M.E. et al. Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy. Nature Med. 6, 76–81 (2000).
    Article CAS Google Scholar
  4. Benkirane, M., Corbeau, P., Housset, V. & Devaux, C. An antibody that binds the imunoglobulin CDR3-like region of the CD4 molecule inhibits provirus transcription in HIV-infected T cells. EMBO J. 12, 4909–4921 (1993).
    Article CAS Google Scholar
  5. Courcoul, M. et al. Peripheral blood mononuclear cells produce normal amounts of defective Vif- human immunodeficiency virus type 1 particles which are restricted for the preretrotranscription steps. J. Virol. 69, 2068–2074 (1995).
    CAS PubMed PubMed Central Google Scholar
  6. Sonza, S. et al. Human immunodeficiency virus type 1 replication is blocked prior to revere transcription and integration in freshly isolated peripheral blood monocytes. J. Virol. 70, 3863–3869 (1996).
    CAS PubMed PubMed Central Google Scholar
  7. Chun, T.-W. et al. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc. Natl. Acad. Sci. USA 94, 13193–13197 (1997).
    Article CAS Google Scholar
  8. Carteau, S., Hoffmann, C. & Bushman, F.D. Chromosome structure and HIV-1 cDNA integration: Centromeric alphoid repeats are a disfavored target. J. Virol. 72, 4005–4014 (1998).
    CAS PubMed PubMed Central Google Scholar
  9. Furtado, M.R. et al. Persistence of HIV-1 transcription in peripheral blood mononuclear cells in patients receiving potent antiretroviral therapy. New Engl. J. Med. 340, 1614–1622 (1999).
    Article CAS Google Scholar
  10. 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
  11. Kafri, T., van Praag, H., Ouyang, L., Gage, F.H. & Verma, I.M. A packaging cell line for lentiviral vectors. J. Virol. 73, 576–584 (1999).
    CAS PubMed PubMed Central Google Scholar
  12. Zennou, V. et al. HIV-1 genome nuclear import is mediated by a central DNA flap. Cell 101, 173–185 (2000).
    Article CAS Google Scholar
  13. Follenzi, A., Ailes, L.E., Bakovic, S., Gueuna, M. & Naldini, L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nature Genet. 25, 217–222 (2000).
    Article CAS Google Scholar
  14. Hazuda, D.J. et al. Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells. Science 287, 646–650 (2000).
    Article CAS Google Scholar

Download references

Acknowledgements

This work was supported by NIH grants GM56553 and AI34786 to F.D.B., the James B. Pendleton Charitable Trust, the Berger Foundation, and Cornelia Mackey.

Author information

Authors and Affiliations

  1. Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA
    Scott L. Butler, Mark S.T. Hansen & Frederic D. Bushman

Authors

  1. Scott L. Butler
    You can also search for this author inPubMed Google Scholar
  2. Mark S.T. Hansen
    You can also search for this author inPubMed Google Scholar
  3. Frederic D. Bushman
    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence toFrederic D. Bushman.

Rights and permissions

About this article

Cite this article

Butler, S., Hansen, M. & Bushman, F. A quantitative assay for HIV DNA integration in vivo.Nat Med 7, 631–634 (2001). https://doi.org/10.1038/87979

Download citation

This article is cited by