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

Botbol Y, Raghavendra NK, Rahman S, Engelman A, Lavigne M - Nucleic Acids Res. (2008)

Bottom Line: Here, we demonstrate that this chromatin-associated protein also stimulates HIV-1 integration into reconstituted polynucleosome templates.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Unit of Structural Virology, Pasteur Institute, 25 rue du Dr Roux, 75724 Paris cedex 15, France.

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.

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Characterization of the DNA and nucleosome-binding domains of LEDGF/p75. (A) Scheme of the 530 residue human LEDGFp75 protein, highlighting the PWWP domain, charged regions (CRs) 1–3, the NLS, AT-hooks and IBD (27,28). Residues retained in the GST fusion proteins are indicated by bold lines. Relative levels of DNA binding at 50 mM NaCl are summarized at the right. (B) Wild-type and mutant LEDGF/p75 binding to 5S-G5E4 DNA (lanes 1–9) and PN templates (lanes 10–18). Purified GST was substituted for LEDGF/p75 in the reactions (50 mM NaCl) in lanes 1 and 10. Lanes 2 and 11 contained wild-type LEDGF1–530; lanes 3 and 12, LEDGF1–325; lanes 4 and 13, LEDGF326–530; lanes 5 and 14, LEDGF1–471; lanes 6 and 15, LEDGF93–471; lanes 7 and 16, LEDGF226–471; lanes 8 and 17, LEDGF326–471; lanes 9 and 18, LEDGF347–471. The lower panel on the right was developed using anti-histone H3 antibodies. (C) TPT DNA (lanes 1–9) and MN (lanes 10–18) binding. The reactions in lanes 1–18 were identical to those in panel B except for the identity of the indicated DNA and nucleosome templates. (D) Same as in panel C, except that binding of LEDGF mutants to TPT DNA (lanes 1–5, 11–15 and 21–25) or MNs (lanes 6–10, 16–20 and 26–30) was performed at 50 mM NaCl (lanes 1–10), 100 mM NaCl (lanes 11–20) or 150 mM NaCl (lanes 21–30). GST alone was analyzed in lanes 1, 6, 11, 16, 21 and 26. Lanes 2, 7, 12, 17, 22 and 27 contained LEDGF1–471; lanes 3, 8, 13, 18, 23 and 28 harbored LEDGF93–471; lanes 4, 9, 14, 19, 24 and 29 used LEDGF226–471; lanes 5, 10, 15, 20, 25 and 30 used LEDGF326–471. (E) Same as in panel D, except that binding assays were conducted using 5S-G5E4 DNA (lanes 1–5, 11–15 and 21–25) or PNs (lanes 6–10, 16–20 and 26–30) in the presence of 150, 200 or 250 mM NaCl, as indicated. (F) Capture of free 5S-G5E4 DNA (lanes 1–5) or PN templates (lanes 6–10) by GST-LEDGF1–100 at different salt concentrations. The right panel shows a plot of quantified radiolabeled DNA recovery (naked or assembled in PN) as a function of salt concentration; error bars represent the variation obtained from four independent experiments.
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Figure 4: Characterization of the DNA and nucleosome-binding domains of LEDGF/p75. (A) Scheme of the 530 residue human LEDGFp75 protein, highlighting the PWWP domain, charged regions (CRs) 1–3, the NLS, AT-hooks and IBD (27,28). Residues retained in the GST fusion proteins are indicated by bold lines. Relative levels of DNA binding at 50 mM NaCl are summarized at the right. (B) Wild-type and mutant LEDGF/p75 binding to 5S-G5E4 DNA (lanes 1–9) and PN templates (lanes 10–18). Purified GST was substituted for LEDGF/p75 in the reactions (50 mM NaCl) in lanes 1 and 10. Lanes 2 and 11 contained wild-type LEDGF1–530; lanes 3 and 12, LEDGF1–325; lanes 4 and 13, LEDGF326–530; lanes 5 and 14, LEDGF1–471; lanes 6 and 15, LEDGF93–471; lanes 7 and 16, LEDGF226–471; lanes 8 and 17, LEDGF326–471; lanes 9 and 18, LEDGF347–471. The lower panel on the right was developed using anti-histone H3 antibodies. (C) TPT DNA (lanes 1–9) and MN (lanes 10–18) binding. The reactions in lanes 1–18 were identical to those in panel B except for the identity of the indicated DNA and nucleosome templates. (D) Same as in panel C, except that binding of LEDGF mutants to TPT DNA (lanes 1–5, 11–15 and 21–25) or MNs (lanes 6–10, 16–20 and 26–30) was performed at 50 mM NaCl (lanes 1–10), 100 mM NaCl (lanes 11–20) or 150 mM NaCl (lanes 21–30). GST alone was analyzed in lanes 1, 6, 11, 16, 21 and 26. Lanes 2, 7, 12, 17, 22 and 27 contained LEDGF1–471; lanes 3, 8, 13, 18, 23 and 28 harbored LEDGF93–471; lanes 4, 9, 14, 19, 24 and 29 used LEDGF226–471; lanes 5, 10, 15, 20, 25 and 30 used LEDGF326–471. (E) Same as in panel D, except that binding assays were conducted using 5S-G5E4 DNA (lanes 1–5, 11–15 and 21–25) or PNs (lanes 6–10, 16–20 and 26–30) in the presence of 150, 200 or 250 mM NaCl, as indicated. (F) Capture of free 5S-G5E4 DNA (lanes 1–5) or PN templates (lanes 6–10) by GST-LEDGF1–100 at different salt concentrations. The right panel shows a plot of quantified radiolabeled DNA recovery (naked or assembled in PN) as a function of salt concentration; error bars represent the variation obtained from four independent experiments.

Mentions: The PWWP domain in large part mediates LEDGF/p75 binding to chromatin in live cells (27,28), but the mechanism underlying this interaction is unknown. Unclear is whether the PWWP domain principally interacts with DNA or a protein component(s) of chromatin: one study failed to detect significant binding between the isolated domain and DNA in vitro (27), whereas a separate study indicated that a 58 amino acid residue stretch within the domain contributed to DNA binding (29). The results of the previous experiments revealed a significant role for the PWWP domain in LEDGF/p75-dependent activation of integration into PNs, but not naked DNA. To ascertain if the PWWP domain mediated binding to nucleosomes, an interaction assay was designed to simultaneously monitor LEDGF/p75 binding to free DNA versus nucleosomes. For this, full-length or truncated LEDGF/p75 GST fusion proteins (Figure 4A) pre-bound to glutathione-sepharose beads were incubated with radiolabeled DNA or assembled nucleosomes. Bound DNA was detected following deproteinization, gel electrophoresis and phosporImager analysis. Bound nucleoprotein complexes were analyzed for core histone H3 content by western blotting or for DNA content after deproteinization and agarose gel electrophoresis.Figure 4.


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

Botbol Y, Raghavendra NK, Rahman S, Engelman A, Lavigne M - Nucleic Acids Res. (2008)

Characterization of the DNA and nucleosome-binding domains of LEDGF/p75. (A) Scheme of the 530 residue human LEDGFp75 protein, highlighting the PWWP domain, charged regions (CRs) 1–3, the NLS, AT-hooks and IBD (27,28). Residues retained in the GST fusion proteins are indicated by bold lines. Relative levels of DNA binding at 50 mM NaCl are summarized at the right. (B) Wild-type and mutant LEDGF/p75 binding to 5S-G5E4 DNA (lanes 1–9) and PN templates (lanes 10–18). Purified GST was substituted for LEDGF/p75 in the reactions (50 mM NaCl) in lanes 1 and 10. Lanes 2 and 11 contained wild-type LEDGF1–530; lanes 3 and 12, LEDGF1–325; lanes 4 and 13, LEDGF326–530; lanes 5 and 14, LEDGF1–471; lanes 6 and 15, LEDGF93–471; lanes 7 and 16, LEDGF226–471; lanes 8 and 17, LEDGF326–471; lanes 9 and 18, LEDGF347–471. The lower panel on the right was developed using anti-histone H3 antibodies. (C) TPT DNA (lanes 1–9) and MN (lanes 10–18) binding. The reactions in lanes 1–18 were identical to those in panel B except for the identity of the indicated DNA and nucleosome templates. (D) Same as in panel C, except that binding of LEDGF mutants to TPT DNA (lanes 1–5, 11–15 and 21–25) or MNs (lanes 6–10, 16–20 and 26–30) was performed at 50 mM NaCl (lanes 1–10), 100 mM NaCl (lanes 11–20) or 150 mM NaCl (lanes 21–30). GST alone was analyzed in lanes 1, 6, 11, 16, 21 and 26. Lanes 2, 7, 12, 17, 22 and 27 contained LEDGF1–471; lanes 3, 8, 13, 18, 23 and 28 harbored LEDGF93–471; lanes 4, 9, 14, 19, 24 and 29 used LEDGF226–471; lanes 5, 10, 15, 20, 25 and 30 used LEDGF326–471. (E) Same as in panel D, except that binding assays were conducted using 5S-G5E4 DNA (lanes 1–5, 11–15 and 21–25) or PNs (lanes 6–10, 16–20 and 26–30) in the presence of 150, 200 or 250 mM NaCl, as indicated. (F) Capture of free 5S-G5E4 DNA (lanes 1–5) or PN templates (lanes 6–10) by GST-LEDGF1–100 at different salt concentrations. The right panel shows a plot of quantified radiolabeled DNA recovery (naked or assembled in PN) as a function of salt concentration; error bars represent the variation obtained from four independent experiments.
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: Characterization of the DNA and nucleosome-binding domains of LEDGF/p75. (A) Scheme of the 530 residue human LEDGFp75 protein, highlighting the PWWP domain, charged regions (CRs) 1–3, the NLS, AT-hooks and IBD (27,28). Residues retained in the GST fusion proteins are indicated by bold lines. Relative levels of DNA binding at 50 mM NaCl are summarized at the right. (B) Wild-type and mutant LEDGF/p75 binding to 5S-G5E4 DNA (lanes 1–9) and PN templates (lanes 10–18). Purified GST was substituted for LEDGF/p75 in the reactions (50 mM NaCl) in lanes 1 and 10. Lanes 2 and 11 contained wild-type LEDGF1–530; lanes 3 and 12, LEDGF1–325; lanes 4 and 13, LEDGF326–530; lanes 5 and 14, LEDGF1–471; lanes 6 and 15, LEDGF93–471; lanes 7 and 16, LEDGF226–471; lanes 8 and 17, LEDGF326–471; lanes 9 and 18, LEDGF347–471. The lower panel on the right was developed using anti-histone H3 antibodies. (C) TPT DNA (lanes 1–9) and MN (lanes 10–18) binding. The reactions in lanes 1–18 were identical to those in panel B except for the identity of the indicated DNA and nucleosome templates. (D) Same as in panel C, except that binding of LEDGF mutants to TPT DNA (lanes 1–5, 11–15 and 21–25) or MNs (lanes 6–10, 16–20 and 26–30) was performed at 50 mM NaCl (lanes 1–10), 100 mM NaCl (lanes 11–20) or 150 mM NaCl (lanes 21–30). GST alone was analyzed in lanes 1, 6, 11, 16, 21 and 26. Lanes 2, 7, 12, 17, 22 and 27 contained LEDGF1–471; lanes 3, 8, 13, 18, 23 and 28 harbored LEDGF93–471; lanes 4, 9, 14, 19, 24 and 29 used LEDGF226–471; lanes 5, 10, 15, 20, 25 and 30 used LEDGF326–471. (E) Same as in panel D, except that binding assays were conducted using 5S-G5E4 DNA (lanes 1–5, 11–15 and 21–25) or PNs (lanes 6–10, 16–20 and 26–30) in the presence of 150, 200 or 250 mM NaCl, as indicated. (F) Capture of free 5S-G5E4 DNA (lanes 1–5) or PN templates (lanes 6–10) by GST-LEDGF1–100 at different salt concentrations. The right panel shows a plot of quantified radiolabeled DNA recovery (naked or assembled in PN) as a function of salt concentration; error bars represent the variation obtained from four independent experiments.
Mentions: The PWWP domain in large part mediates LEDGF/p75 binding to chromatin in live cells (27,28), but the mechanism underlying this interaction is unknown. Unclear is whether the PWWP domain principally interacts with DNA or a protein component(s) of chromatin: one study failed to detect significant binding between the isolated domain and DNA in vitro (27), whereas a separate study indicated that a 58 amino acid residue stretch within the domain contributed to DNA binding (29). The results of the previous experiments revealed a significant role for the PWWP domain in LEDGF/p75-dependent activation of integration into PNs, but not naked DNA. To ascertain if the PWWP domain mediated binding to nucleosomes, an interaction assay was designed to simultaneously monitor LEDGF/p75 binding to free DNA versus nucleosomes. For this, full-length or truncated LEDGF/p75 GST fusion proteins (Figure 4A) pre-bound to glutathione-sepharose beads were incubated with radiolabeled DNA or assembled nucleosomes. Bound DNA was detected following deproteinization, gel electrophoresis and phosporImager analysis. Bound nucleoprotein complexes were analyzed for core histone H3 content by western blotting or for DNA content after deproteinization and agarose gel electrophoresis.Figure 4.

Bottom Line: Here, we demonstrate that this chromatin-associated protein also stimulates HIV-1 integration into reconstituted polynucleosome templates.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Unit of Structural Virology, Pasteur Institute, 25 rue du Dr Roux, 75724 Paris cedex 15, France.

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.

Show MeSH
Related in: MedlinePlus