Nucleic Acids Research Advance Access published online on January 3, 2008
Nucleic Acids Research, doi:10.1093/nar/gkm1127
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Molecular Biology |
Chromatinized templates reveal the requirement for the LEDGF/p75 PWWP domain during HIV-1 integration in vitro
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.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M.-C. Shun, Y. Botbol, X. Li, F. Di Nunzio, J. E. Daigle, N. Yan, J. Lieberman, M. Lavigne, and A. Engelman Identification and Characterization of PWWP Domain Residues Critical for LEDGF/p75 Chromatin Binding and Human Immunodeficiency Virus Type 1 Infectivity J. Virol., December 1, 2008; 82(23): 11555 - 11567. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. A. Brown-Bryan, L. S. Leoh, V. Ganapathy, F. J. Pacheco, M. Mediavilla-Varela, M. Filippova, T. A. Linkhart, R. Gijsbers, Z. Debyser, and C. A. Casiano Alternative Splicing and Caspase-Mediated Cleavage Generate Antagonistic Variants of the Stress Oncoprotein LEDGF/p75 Mol. Cancer Res., August 1, 2008; 6(8): 1293 - 1307. [Abstract] [Full Text] [PDF]
|
|