TY - JOUR
T1 - Chromatinized templates reveal the requirement for the LEDGF/p75 PWWP domain during HIV-1 integration in vitro
AU - Botbol, Yaïr
AU - Raghavendra, Nidhanapati K.
AU - Rahman, Shaila
AU - Engelman, Alan
AU - Lavigne, Marc
N1 - Funding Information:
This work was supported by grant AI39394 from the US National Institutes of Health (to A.E.) and by grants 2005/004 and 2006/124 from the Agence Nationale de Recherche sur le SIDA (to M.L.). M.L. and Y.B. thank Prof. Moshe Yaniv, Dr Félix Rey and Dr Jean Franc¸ ois Mouscadet for support, advice and valuable discussion. Y.B. thanks Dr Eric Batsché for help with real-time PCR. Funding to pay the Open Access publication charges for this article was provided by the Pasteur Institute of Paris, France.
PY - 2008/3
Y1 - 2008/3
N2 - 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.
AB - 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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U2 - 10.1093/nar/gkm1127
DO - 10.1093/nar/gkm1127
M3 - Article
C2 - 18174227
AN - SCOPUS:40249106009
SN - 0305-1048
VL - 36
SP - 1237
EP - 1246
JO - Nucleic acids research
JF - Nucleic acids research
IS - 4
ER -