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

Yair M. Botbol, Nidhanapati K. Raghavendra, Shaila Rahman, Alan Engelman, Marc Lavigne

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1237-1246
Number of pages10
JournalNucleic Acids Research
Volume36
Issue number4
DOIs
StatePublished - Mar 2008
Externally publishedYes

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Virus Integration
HIV-1
Integrases
Nucleosomes
DNA
Chromatin
AT-Hook Motifs
Complementary DNA
Nucleotide Motifs
Virus Diseases
lens epithelium-derived growth factor
In Vitro Techniques
Carrier Proteins

ASJC Scopus subject areas

  • Genetics

Cite this

Chromatinized templates reveal the requirement for the LEDGF/p75 PWWP domain during HIV-1 integration in vitro. / Botbol, Yair M.; Raghavendra, Nidhanapati K.; Rahman, Shaila; Engelman, Alan; Lavigne, Marc.

In: Nucleic Acids Research, Vol. 36, No. 4, 03.2008, p. 1237-1246.

Research output: Contribution to journalArticle

Botbol, Yair M. ; Raghavendra, Nidhanapati K. ; Rahman, Shaila ; Engelman, Alan ; Lavigne, Marc. / Chromatinized templates reveal the requirement for the LEDGF/p75 PWWP domain during HIV-1 integration in vitro. In: Nucleic Acids Research. 2008 ; Vol. 36, No. 4. pp. 1237-1246.
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