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In vitro initial attachment of HIV-1 integrase to viral ends: control of the DNA specific interaction by the oligomerization state.

Lesbats P, Métifiot M, Calmels C, Baranova S, Nevinsky G, Andreola ML, Parissi V - Nucleic Acids Res. (2008)

Bottom Line: In addition, we show that IN monomers bound to nonspecific DNA can also fold into functionally different oligomeric complexes displaying nonspecific double-strand DNA break activity in contrast to the well known single strand cut catalyzed by associated IN.Our results imply that the efficient formation of the active integration complex highly requires the early correct positioning of monomeric integrase or the direct binding of preformed dimers on the viral ends.Taken together the data indicates that IN oligomerization controls both the enzyme specificity and activity.

View Article: PubMed Central - PubMed

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

ABSTRACT
HIV-1 integrase (IN) oligomerization and DNA recognition are crucial steps for the subsequent events of the integration reaction. Recent advances described the involvement of stable intermediary complexes including dimers and tetramers in the in vitro integration processes, but the initial attachment events and IN positioning on viral ends are not clearly understood. In order to determine the role of the different IN oligomeric complexes in these early steps, we performed in vitro functional analysis comparing IN preparations having different oligomerization properties. We demonstrate that in vitro IN concerted integration activity on a long DNA substrate containing both specific viral and nonspecific DNA sequences is highly dependent on binding of preformed dimers to viral ends. In addition, we show that IN monomers bound to nonspecific DNA can also fold into functionally different oligomeric complexes displaying nonspecific double-strand DNA break activity in contrast to the well known single strand cut catalyzed by associated IN. Our results imply that the efficient formation of the active integration complex highly requires the early correct positioning of monomeric integrase or the direct binding of preformed dimers on the viral ends. Taken together the data indicates that IN oligomerization controls both the enzyme specificity and activity.

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Oligomerization state (A and B) and functional analyses (C and D) of the associated INZn preparation. One hundred and fifty picomole of IN purified in presence of 50 µm ZnSO4 (INZn, 10 µM) were submitted to gel filtration chromatography. (A) The nature of the IN peaks was determined by comparing profiles obtained with proteins of known molecular weights aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa) and chymotrypsinogen A (25 kDa). One picomole of INZn was submitted to DSS crosslink (0.8 µg DSS, 50 mM HEPES pH 7.5, 30 mM NaCl) for 15 min and 60 min at 22°C and then 12% SDS–PAGE gel followed by western blot using polyclonal anti-IN antibodies. (B) Monomer (Mo), dimer (Di) and tetramer (Te) positions were determined by comparison with a molecular weight marker (MQ). Concerted integration assay (C) was performed without IN (lane -IN) or with 1 pmol of INzn (same final protein and NaCl concentrations of respectively 50 nM and 30 mM) using 100 ng of acceptor DNA (3000 bp) and 10 ng of 32P 5′-labelled donor pre-processed DNA (296 bp). The reaction products were either loaded on 1% agarose gel or cloned in MC1060/P3 E. coli strain. The position and the structure of the different products obtained after half-site (HSI), full-site (FSI) and donor/donor integration (d/d) are reported. The number of resistant selected colonies obtained in absence of IN (-IN) or after integration reaction carried by INZn (mean ± SD of three independent experiments) and the structure of the integration loci from 20 clones in each condition are reported in D.
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Figure 1: Oligomerization state (A and B) and functional analyses (C and D) of the associated INZn preparation. One hundred and fifty picomole of IN purified in presence of 50 µm ZnSO4 (INZn, 10 µM) were submitted to gel filtration chromatography. (A) The nature of the IN peaks was determined by comparing profiles obtained with proteins of known molecular weights aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa) and chymotrypsinogen A (25 kDa). One picomole of INZn was submitted to DSS crosslink (0.8 µg DSS, 50 mM HEPES pH 7.5, 30 mM NaCl) for 15 min and 60 min at 22°C and then 12% SDS–PAGE gel followed by western blot using polyclonal anti-IN antibodies. (B) Monomer (Mo), dimer (Di) and tetramer (Te) positions were determined by comparison with a molecular weight marker (MQ). Concerted integration assay (C) was performed without IN (lane -IN) or with 1 pmol of INzn (same final protein and NaCl concentrations of respectively 50 nM and 30 mM) using 100 ng of acceptor DNA (3000 bp) and 10 ng of 32P 5′-labelled donor pre-processed DNA (296 bp). The reaction products were either loaded on 1% agarose gel or cloned in MC1060/P3 E. coli strain. The position and the structure of the different products obtained after half-site (HSI), full-site (FSI) and donor/donor integration (d/d) are reported. The number of resistant selected colonies obtained in absence of IN (-IN) or after integration reaction carried by INZn (mean ± SD of three independent experiments) and the structure of the integration loci from 20 clones in each condition are reported in D.

Mentions: The oligomerization state of each sample was checked by size exclusion (gel filtration) chromatography. As shown in Figure 1A, we obtained in the presence of Zn an IN solution containing monomers, dimers and tetramers. In contrast, all the IN preparations obtained in the absence of Zn and in the presence of CHAPS were dissociated (Supplementary Material 1). The oligomerization equilibrium of the associated preparation was further checked by crosslink with DSS followed by SDS–PAGE and western blot. As shown in Figure 1B, the data obtained using chromatography were confirmed since monomers, dimers and tetramers were detected using this approach with similar proportions than those obtained after gel filtration. Longer crosslink incubation times did not change the oligomerization profile, indicating that DSS was able to crosslink only preformed oligomers without inducing further multimerization. Same results were obtained when using the SAXS methods (data not shown). Consequently, we assumed that the crosslink analysis reflects the proportion of oligomers in the IN solutions, and thus, this method was used to evaluate all the IN preparations throughout this work.Figure 1.


In vitro initial attachment of HIV-1 integrase to viral ends: control of the DNA specific interaction by the oligomerization state.

Lesbats P, Métifiot M, Calmels C, Baranova S, Nevinsky G, Andreola ML, Parissi V - Nucleic Acids Res. (2008)

Oligomerization state (A and B) and functional analyses (C and D) of the associated INZn preparation. One hundred and fifty picomole of IN purified in presence of 50 µm ZnSO4 (INZn, 10 µM) were submitted to gel filtration chromatography. (A) The nature of the IN peaks was determined by comparing profiles obtained with proteins of known molecular weights aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa) and chymotrypsinogen A (25 kDa). One picomole of INZn was submitted to DSS crosslink (0.8 µg DSS, 50 mM HEPES pH 7.5, 30 mM NaCl) for 15 min and 60 min at 22°C and then 12% SDS–PAGE gel followed by western blot using polyclonal anti-IN antibodies. (B) Monomer (Mo), dimer (Di) and tetramer (Te) positions were determined by comparison with a molecular weight marker (MQ). Concerted integration assay (C) was performed without IN (lane -IN) or with 1 pmol of INzn (same final protein and NaCl concentrations of respectively 50 nM and 30 mM) using 100 ng of acceptor DNA (3000 bp) and 10 ng of 32P 5′-labelled donor pre-processed DNA (296 bp). The reaction products were either loaded on 1% agarose gel or cloned in MC1060/P3 E. coli strain. The position and the structure of the different products obtained after half-site (HSI), full-site (FSI) and donor/donor integration (d/d) are reported. The number of resistant selected colonies obtained in absence of IN (-IN) or after integration reaction carried by INZn (mean ± SD of three independent experiments) and the structure of the integration loci from 20 clones in each condition are reported in D.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602759&req=5

Figure 1: Oligomerization state (A and B) and functional analyses (C and D) of the associated INZn preparation. One hundred and fifty picomole of IN purified in presence of 50 µm ZnSO4 (INZn, 10 µM) were submitted to gel filtration chromatography. (A) The nature of the IN peaks was determined by comparing profiles obtained with proteins of known molecular weights aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa) and chymotrypsinogen A (25 kDa). One picomole of INZn was submitted to DSS crosslink (0.8 µg DSS, 50 mM HEPES pH 7.5, 30 mM NaCl) for 15 min and 60 min at 22°C and then 12% SDS–PAGE gel followed by western blot using polyclonal anti-IN antibodies. (B) Monomer (Mo), dimer (Di) and tetramer (Te) positions were determined by comparison with a molecular weight marker (MQ). Concerted integration assay (C) was performed without IN (lane -IN) or with 1 pmol of INzn (same final protein and NaCl concentrations of respectively 50 nM and 30 mM) using 100 ng of acceptor DNA (3000 bp) and 10 ng of 32P 5′-labelled donor pre-processed DNA (296 bp). The reaction products were either loaded on 1% agarose gel or cloned in MC1060/P3 E. coli strain. The position and the structure of the different products obtained after half-site (HSI), full-site (FSI) and donor/donor integration (d/d) are reported. The number of resistant selected colonies obtained in absence of IN (-IN) or after integration reaction carried by INZn (mean ± SD of three independent experiments) and the structure of the integration loci from 20 clones in each condition are reported in D.
Mentions: The oligomerization state of each sample was checked by size exclusion (gel filtration) chromatography. As shown in Figure 1A, we obtained in the presence of Zn an IN solution containing monomers, dimers and tetramers. In contrast, all the IN preparations obtained in the absence of Zn and in the presence of CHAPS were dissociated (Supplementary Material 1). The oligomerization equilibrium of the associated preparation was further checked by crosslink with DSS followed by SDS–PAGE and western blot. As shown in Figure 1B, the data obtained using chromatography were confirmed since monomers, dimers and tetramers were detected using this approach with similar proportions than those obtained after gel filtration. Longer crosslink incubation times did not change the oligomerization profile, indicating that DSS was able to crosslink only preformed oligomers without inducing further multimerization. Same results were obtained when using the SAXS methods (data not shown). Consequently, we assumed that the crosslink analysis reflects the proportion of oligomers in the IN solutions, and thus, this method was used to evaluate all the IN preparations throughout this work.Figure 1.

Bottom Line: In addition, we show that IN monomers bound to nonspecific DNA can also fold into functionally different oligomeric complexes displaying nonspecific double-strand DNA break activity in contrast to the well known single strand cut catalyzed by associated IN.Our results imply that the efficient formation of the active integration complex highly requires the early correct positioning of monomeric integrase or the direct binding of preformed dimers on the viral ends.Taken together the data indicates that IN oligomerization controls both the enzyme specificity and activity.

View Article: PubMed Central - PubMed

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

ABSTRACT
HIV-1 integrase (IN) oligomerization and DNA recognition are crucial steps for the subsequent events of the integration reaction. Recent advances described the involvement of stable intermediary complexes including dimers and tetramers in the in vitro integration processes, but the initial attachment events and IN positioning on viral ends are not clearly understood. In order to determine the role of the different IN oligomeric complexes in these early steps, we performed in vitro functional analysis comparing IN preparations having different oligomerization properties. We demonstrate that in vitro IN concerted integration activity on a long DNA substrate containing both specific viral and nonspecific DNA sequences is highly dependent on binding of preformed dimers to viral ends. In addition, we show that IN monomers bound to nonspecific DNA can also fold into functionally different oligomeric complexes displaying nonspecific double-strand DNA break activity in contrast to the well known single strand cut catalyzed by associated IN. Our results imply that the efficient formation of the active integration complex highly requires the early correct positioning of monomeric integrase or the direct binding of preformed dimers on the viral ends. Taken together the data indicates that IN oligomerization controls both the enzyme specificity and activity.

Show MeSH
Related in: MedlinePlus