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Functional coupling between HIV-1 integrase and the SWI/SNF chromatin remodeling complex for efficient in vitro integration into stable nucleosomes.

Lesbats P, Botbol Y, Chevereau G, Vaillant C, Calmels C, Arneodo A, Andreola ML, Lavigne M, Parissi V - PLoS Pathog. (2011)

Bottom Line: We show here that stably associated nucleosomes strongly inhibit in vitro two viral-end integration by decreasing the accessibility of DNA to integrase.These effects are not observed after remodeling by other human remodeling factors such as SNF2H or BRG1 lacking the integrase binding protein INI1.Our data indicate that some chromatin structures can be refractory for integration and that coupling between nucleosome remodeling and HIV-1 integration is required to overcome this natural barrier.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire MCMP, UMR 5234 CNRS-Université Victor Segalen Bordeaux 2, Bordeaux, France.

ABSTRACT
Establishment of stable HIV-1 infection requires the efficient integration of the retroviral genome into the host DNA. The molecular mechanism underlying the control of this process by the chromatin structure has not yet been elucidated. We show here that stably associated nucleosomes strongly inhibit in vitro two viral-end integration by decreasing the accessibility of DNA to integrase. Remodeling of the chromatinized template by the SWI/SNF complex, whose INI1 major component interacts with IN, restores and redirects the full-site integration into the stable nucleosome region. These effects are not observed after remodeling by other human remodeling factors such as SNF2H or BRG1 lacking the integrase binding protein INI1. This suggests that the restoration process depends on the direct interaction between IN and the whole SWI/SNF complex, supporting a functional coupling between the remodeling and integration complexes. Furthermore, in silico comparison between more than 40,000 non-redundant cellular integration sites selected from literature and nucleosome occupancy predictions also supports that HIV-1 integration is promoted in the genomic region of weaker intrinsic nucleosome density in the infected cell. Our data indicate that some chromatin structures can be refractory for integration and that coupling between nucleosome remodeling and HIV-1 integration is required to overcome this natural barrier.

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Related in: MedlinePlus

Effect of chromatin remodeling activity of the SWI/SNF complex on the in vitro integration in nucleosomal templates.Naked or chromatinized pBSK-zeo-5S-G5EA vector was used as acceptor substrate in a concerted integration assay performed after SWI/SNF treatment in presence of ATP (0 to 120 minutes) with 12 pmoles IN, 10 ng donor and 100 ng acceptor. Efficient remodeling of the vector was previously checked by REA assay (see Figure S6A). The reaction products were loaded on 1% agarose gel and an example of the result obtained with the 1/1.67 PN template is reported in (A). The position the different products obtained after half-site (HSI), full-site (FSI) and donor/donor integration (d/d) are shown in addition to donor (d). The circular FSI products obtained with vectors assembled with increasing amounts of histones expressed in DNA/histones mass ratio (µg/µg) (1/33, 1/167, 1/2) with or without SWI/SNF pre-treatment were specifically quantified by cloning in bacteria and reported as the number of ampicillin-, kanamycin- and tetracycline-resistant selected clones (B). 20 correct integration loci were localized in the vector sequence and the number of integration events obtained with the naked or 1/1.67 PN template remodeled by SWI/SNF was quantified and is shown as integration number per 100 bp of DNA for region I containing the stably assembled nucleosomes and region II carrying more labile nucleosomes (C). All the values are the mean ± standard deviation (error bars) of three independent sets of experiments.
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ppat-1001280-g003: Effect of chromatin remodeling activity of the SWI/SNF complex on the in vitro integration in nucleosomal templates.Naked or chromatinized pBSK-zeo-5S-G5EA vector was used as acceptor substrate in a concerted integration assay performed after SWI/SNF treatment in presence of ATP (0 to 120 minutes) with 12 pmoles IN, 10 ng donor and 100 ng acceptor. Efficient remodeling of the vector was previously checked by REA assay (see Figure S6A). The reaction products were loaded on 1% agarose gel and an example of the result obtained with the 1/1.67 PN template is reported in (A). The position the different products obtained after half-site (HSI), full-site (FSI) and donor/donor integration (d/d) are shown in addition to donor (d). The circular FSI products obtained with vectors assembled with increasing amounts of histones expressed in DNA/histones mass ratio (µg/µg) (1/33, 1/167, 1/2) with or without SWI/SNF pre-treatment were specifically quantified by cloning in bacteria and reported as the number of ampicillin-, kanamycin- and tetracycline-resistant selected clones (B). 20 correct integration loci were localized in the vector sequence and the number of integration events obtained with the naked or 1/1.67 PN template remodeled by SWI/SNF was quantified and is shown as integration number per 100 bp of DNA for region I containing the stably assembled nucleosomes and region II carrying more labile nucleosomes (C). All the values are the mean ± standard deviation (error bars) of three independent sets of experiments.

Mentions: To determine the effect of this remodeling activity on concerted integration, both reactions were coupled. Briefly, acceptor plasmids were treated with the purified SWI/SNF complex and then IN was added to the reaction solution under conditions allowing the integration reaction. As shown in Figure 3A, remodeling of the PN templates by SWI/SNF led to recovery of full-site integration on these templates. Same results were obtained with the other PN (S8B). Quantification of the circular FSI products in bacteria confirmed the integration recovery after SWI/SNF treatment (Figure 3B). Additional gel filtration purification of the SWI/SNF complex followed by the assay of the eluted fractions in IN-mediated concerted integration into PN showed that the integration restoration activity co-purified with the active remodeling complex. This led us to rule out the possible contaminant source for the restoration effect (data not shown).


Functional coupling between HIV-1 integrase and the SWI/SNF chromatin remodeling complex for efficient in vitro integration into stable nucleosomes.

Lesbats P, Botbol Y, Chevereau G, Vaillant C, Calmels C, Arneodo A, Andreola ML, Lavigne M, Parissi V - PLoS Pathog. (2011)

Effect of chromatin remodeling activity of the SWI/SNF complex on the in vitro integration in nucleosomal templates.Naked or chromatinized pBSK-zeo-5S-G5EA vector was used as acceptor substrate in a concerted integration assay performed after SWI/SNF treatment in presence of ATP (0 to 120 minutes) with 12 pmoles IN, 10 ng donor and 100 ng acceptor. Efficient remodeling of the vector was previously checked by REA assay (see Figure S6A). The reaction products were loaded on 1% agarose gel and an example of the result obtained with the 1/1.67 PN template is reported in (A). The position the different products obtained after half-site (HSI), full-site (FSI) and donor/donor integration (d/d) are shown in addition to donor (d). The circular FSI products obtained with vectors assembled with increasing amounts of histones expressed in DNA/histones mass ratio (µg/µg) (1/33, 1/167, 1/2) with or without SWI/SNF pre-treatment were specifically quantified by cloning in bacteria and reported as the number of ampicillin-, kanamycin- and tetracycline-resistant selected clones (B). 20 correct integration loci were localized in the vector sequence and the number of integration events obtained with the naked or 1/1.67 PN template remodeled by SWI/SNF was quantified and is shown as integration number per 100 bp of DNA for region I containing the stably assembled nucleosomes and region II carrying more labile nucleosomes (C). All the values are the mean ± standard deviation (error bars) of three independent sets of experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037357&req=5

ppat-1001280-g003: Effect of chromatin remodeling activity of the SWI/SNF complex on the in vitro integration in nucleosomal templates.Naked or chromatinized pBSK-zeo-5S-G5EA vector was used as acceptor substrate in a concerted integration assay performed after SWI/SNF treatment in presence of ATP (0 to 120 minutes) with 12 pmoles IN, 10 ng donor and 100 ng acceptor. Efficient remodeling of the vector was previously checked by REA assay (see Figure S6A). The reaction products were loaded on 1% agarose gel and an example of the result obtained with the 1/1.67 PN template is reported in (A). The position the different products obtained after half-site (HSI), full-site (FSI) and donor/donor integration (d/d) are shown in addition to donor (d). The circular FSI products obtained with vectors assembled with increasing amounts of histones expressed in DNA/histones mass ratio (µg/µg) (1/33, 1/167, 1/2) with or without SWI/SNF pre-treatment were specifically quantified by cloning in bacteria and reported as the number of ampicillin-, kanamycin- and tetracycline-resistant selected clones (B). 20 correct integration loci were localized in the vector sequence and the number of integration events obtained with the naked or 1/1.67 PN template remodeled by SWI/SNF was quantified and is shown as integration number per 100 bp of DNA for region I containing the stably assembled nucleosomes and region II carrying more labile nucleosomes (C). All the values are the mean ± standard deviation (error bars) of three independent sets of experiments.
Mentions: To determine the effect of this remodeling activity on concerted integration, both reactions were coupled. Briefly, acceptor plasmids were treated with the purified SWI/SNF complex and then IN was added to the reaction solution under conditions allowing the integration reaction. As shown in Figure 3A, remodeling of the PN templates by SWI/SNF led to recovery of full-site integration on these templates. Same results were obtained with the other PN (S8B). Quantification of the circular FSI products in bacteria confirmed the integration recovery after SWI/SNF treatment (Figure 3B). Additional gel filtration purification of the SWI/SNF complex followed by the assay of the eluted fractions in IN-mediated concerted integration into PN showed that the integration restoration activity co-purified with the active remodeling complex. This led us to rule out the possible contaminant source for the restoration effect (data not shown).

Bottom Line: We show here that stably associated nucleosomes strongly inhibit in vitro two viral-end integration by decreasing the accessibility of DNA to integrase.These effects are not observed after remodeling by other human remodeling factors such as SNF2H or BRG1 lacking the integrase binding protein INI1.Our data indicate that some chromatin structures can be refractory for integration and that coupling between nucleosome remodeling and HIV-1 integration is required to overcome this natural barrier.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire MCMP, UMR 5234 CNRS-Université Victor Segalen Bordeaux 2, Bordeaux, France.

ABSTRACT
Establishment of stable HIV-1 infection requires the efficient integration of the retroviral genome into the host DNA. The molecular mechanism underlying the control of this process by the chromatin structure has not yet been elucidated. We show here that stably associated nucleosomes strongly inhibit in vitro two viral-end integration by decreasing the accessibility of DNA to integrase. Remodeling of the chromatinized template by the SWI/SNF complex, whose INI1 major component interacts with IN, restores and redirects the full-site integration into the stable nucleosome region. These effects are not observed after remodeling by other human remodeling factors such as SNF2H or BRG1 lacking the integrase binding protein INI1. This suggests that the restoration process depends on the direct interaction between IN and the whole SWI/SNF complex, supporting a functional coupling between the remodeling and integration complexes. Furthermore, in silico comparison between more than 40,000 non-redundant cellular integration sites selected from literature and nucleosome occupancy predictions also supports that HIV-1 integration is promoted in the genomic region of weaker intrinsic nucleosome density in the infected cell. Our data indicate that some chromatin structures can be refractory for integration and that coupling between nucleosome remodeling and HIV-1 integration is required to overcome this natural barrier.

Show MeSH
Related in: MedlinePlus