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Nucleic Acids Research Advance Access published online on January 3, 2008
Nucleic Acids Research, doi:10.1093/nar/gkm1127
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

Chromatinized templates reveal the requirement for the LEDGF/p75 PWWP domain during HIV-1 integration in vitro

Yaïr Botbol1, Nidhanapati K. Raghavendra2, Shaila Rahman2, Alan Engelman2,* and Marc Lavigne1,*

1Department of Virology, Unit of Structural Virology, Pasteur Institute, 25 rue du Dr Roux, 75724 Paris cedex 15, France and 2Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Division of AIDS, Harvard Medical School, Boston, Massachusetts 02115, USA

*To whom correspondence should be addressed. Tel: +33 1 40 61 36 57; Fax: +33 1 45 68 89 93; Email: mlavigne{at}pasteur.fr Correspondence may also be addressed to Alan Engelman. Tel: +1 617 632 4361; Fax: +1 627 632 3113; Email: alan_engelman{at}dfci.harvard.edu

Received July 20, 2007. Revised December 3, 2007. Accepted December 4, 2007.

Integration is an essential step in the retroviral lifecycle, and the lentiviral integrase binding protein lens epithelium-derived growth factor (LEDGF)/p75 plays a crucial role during human immunodeficiency virus type 1 (HIV-1) cDNA integration. In vitro, LEDGF/p75 stimulates HIV-1 integrase activity into naked target DNAs. Here, we demonstrate that this chromatin-associated protein also stimulates HIV-1 integration into reconstituted polynucleosome templates. Activation of integration depended on the LEDGF/p75-integrase interaction with either type of template. A differential requirement for the dominant DNA and chromatin-binding elements of LEDGF/p75 was however observed when using naked DNA versus polynucleosomes. With naked DNA, the complete removal of these N-terminal elements was required to abate cofactor function. With polynucleosomes, activation mainly depended on the PWWP domain, and to a lesser extent on nearby AT-hook DNA-binding motifs. GST pull-down assays furthermore revealed a role for the PWWP domain in binding to nucleosomes. These results are completely consistent with recent ex vivo studies that characterized the PWWP and integrase-binding domains of LEDGF/p75 as crucial for restoring HIV-1 infection to LEDGF-depleted cells. Our studies therefore establish novel in vitro conditions, highlighting chromatinized DNA as target acceptor templates, for physiologically relevant studies of LEDGF/p75 in lentiviral cDNA integration.


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