Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase

Published 6/16/2008
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Biology

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Summary

Current HIV-1 strategies act to suppress the viral life cycle but do not effectively eradicate infection. Here, we demonstrate that an engineered recombinase can efficiently excise integrated HIV-1 proviral DNA from the genome of infected cells.

Cite this Article

Copy Citation

Hauber, J. Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase. J. Vis. Exp. (16), e793, doi:10.3791/793 (2008).

Abstract

HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies primarily target virus enzymes or virus-cell fusion, suppressing the viral life cycle without eradicating the infection. Since the integrated provirus is not targeted by these approaches, new resistant strains of HIV-1 may emerge. Here, we report that the engineered recombinase Tre (see Molecular evolution of the Tre recombinase , Buchholz, F., Max Planck Institute for Cell Biology and Genetics, Dresden) efficiently excises integrated HIV-1 proviral DNA from the genome of infected cells. We produced loxLTR containing viral pseudotypes and infected HeLa cells to examine whether Tre recombinase can excise the provirus from the genome of HIV-1 infected human cells. A virus particle-releasing cell line was cloned and transfected with a plasmid expressing Tre or with a parental control vector. Recombinase activity and virus production were monitored. All assays demonstrated the efficient deletion of the provirus from infected cells without visible cytotoxic effects. These results serve as proof of principle that it is possible to evolve a recombinase to specifically target an HIV-1 LTR and that this recombinase is capable of excising the HIV-1 provirus from the genome of HIV-1-infected human cells.

Before an engineered recombinase could enter the therapeutic arena, however, significant obstacles need to be overcome. Among the most critical issues, that we face, are an efficient and safe delivery to targeted cells and the absence of side effects.

Protocol

Please view related videos entitled Principles of Site-Specific Recombinase (SSR) Technology and Molecular Evolution of the Tre Recombinase for more information about site-specific recombination and engineering the site-specificity of recombinase enzymes.

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Disclosures

The authors have nothing to disclose.

Comments

10 Comments

  1. Please this must be in clinical trials asap..  

    Reply
    Posted by: Anonymous
    June 24, 2008 - 7:26 PM
  2. Moving faster with recombinase. To tell the truth i don' t trust completely in the results of the research, any kind of research, humans trial included. Kp-1461 has been halted almost at the end of phase ² humans trial.. and now seems that kp-1461 can not work in vitro too.. The koronispharma managers have to pay for this.. A LOT OF MONEY.

    Reply
    Posted by: Anonymous
    June 25, 2008 - 8:56 AM
  3. how can I download a video protocol in my flash disc?  

    Reply
    Posted by: Anonymous
    September 20, 2008 - 4:36 PM
  4. Dear Anonymous, please feel free to contact us at support@jove.com and we could discuss your problem.

    Reply
    Posted by: Anonymous
    October 6, 2008 - 6:34 PM
  5. How can I get in touch with doctor Joachim Hauber? I am HIV positive, there isn´t a single day ever since I was diagnosed this disease that I don´t feel like my life just ended. I would do anything to get cured.

    Reply
    Posted by: Anonymous
    October 20, 2008 - 1:37 PM
  6. Reply
    Posted by: Anonymous
    November 3, 2008 - 8:41 AM
  7. Interesting presentation on remarkable research. Worthy of further discussion

    Reply
    Posted by: Anonymous
    February 23, 2009 - 1:48 PM
  8. Have you sequenced the host cell genome in the wild type control vs. the HIV-1 infected vs Tre recombinase cells to
    definitively show where the HIV-1 genome is inserting, and that it is removed?

    Reply
    Posted by: Douglas C.
    October 21, 2009 - 9:41 AM
  9. why can't i get an update on this work?? i have looked everywhere. it has gotten so little publicity..i dont get it

    Reply
    Posted by: Anonymous
    June 25, 2010 - 1:04 AM
  10. hi
    i need full text this article
    plz
    tanx

    Reply
    Posted by: Anonymous
    October 10, 2010 - 4:23 PM

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