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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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Effect of specific and nonspecific ODN on integrase self association. One picomole of INzn 12.5μm or 0.125 µM (lanes 1 and 2) pre-incubated with specific or nonspecific 21 bp ODN (lanes 3 and 4) were submitted to DSS crosslink for 30 min at 22°C and loaded on 12% SDS–PAGE before western-blotting with polyclonal anti-IN antibody. In all reactions the final NaCl concentration was adjusted to 30 mM. Monomer (Mo), dimer (Di) and tetramer (Te) positions were determined by comparison with a molecular weight marker (MQ).
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Figure 6: Effect of specific and nonspecific ODN on integrase self association. One picomole of INzn 12.5μm or 0.125 µM (lanes 1 and 2) pre-incubated with specific or nonspecific 21 bp ODN (lanes 3 and 4) were submitted to DSS crosslink for 30 min at 22°C and loaded on 12% SDS–PAGE before western-blotting with polyclonal anti-IN antibody. In all reactions the final NaCl concentration was adjusted to 30 mM. Monomer (Mo), dimer (Di) and tetramer (Te) positions were determined by comparison with a molecular weight marker (MQ).

Mentions: One picomole of dissociated INZn was incubated overnight with 1 pmole of the 21 nt ODN presenting the LTR ends or random nonviral ODN under our standard conditions (7.5 mM MnCl2, HEPES 20 mM, pH 7.6). The self association of the protein was analyzed using DSS crosslink before performing concerted integration assays. Results in Figure 6 indicate that preincubation of the dissociated enzyme with either short specific viral ODN or random ODN led to its reassociation into oligomers (mainly dimers). These data have been validated by SAXS experiments (data not shown) confirming that monomers can bind either to specific or nonspecific DNA and associate on it. In order to determine whether the oligomers formed on both ODN were functionally similar, we tested the reassociated fractions in concerted integration assays. Results are reported in Figure 7A and show that the incubation of IN monomers with the 21 nt viral ends led to recovery of integration (lane 3 compared to lane 2). In contrast, no integration was detected after incubation with nonviral ODN (lane 4), indicating that, in this case, the multimer enzyme was inactive. Selection of integrants carrying the circular form of FSI products indicated that this reaction was also recovered, at least partially, when the dissociated enzyme was pre-incubated with specific ODN but not with random sequences (Figure 7B). Sequencing of the integration loci showed a similar profile with associated and reassociated IN. The effect of the viral end structure was also analyzed using the blunt substrate for the concerted integration assay. As reported in Figure 7C and D, the same reactivation was observed under these conditions, confirming that the enzyme dissociation affected the early steps of IN attachment to DNA and not the formation of the final synaptic complex.Figure 6.


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)

Effect of specific and nonspecific ODN on integrase self association. One picomole of INzn 12.5μm or 0.125 µM (lanes 1 and 2) pre-incubated with specific or nonspecific 21 bp ODN (lanes 3 and 4) were submitted to DSS crosslink for 30 min at 22°C and loaded on 12% SDS–PAGE before western-blotting with polyclonal anti-IN antibody. In all reactions the final NaCl concentration was adjusted to 30 mM. Monomer (Mo), dimer (Di) and tetramer (Te) positions were determined by comparison with a molecular weight marker (MQ).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Effect of specific and nonspecific ODN on integrase self association. One picomole of INzn 12.5μm or 0.125 µM (lanes 1 and 2) pre-incubated with specific or nonspecific 21 bp ODN (lanes 3 and 4) were submitted to DSS crosslink for 30 min at 22°C and loaded on 12% SDS–PAGE before western-blotting with polyclonal anti-IN antibody. In all reactions the final NaCl concentration was adjusted to 30 mM. Monomer (Mo), dimer (Di) and tetramer (Te) positions were determined by comparison with a molecular weight marker (MQ).
Mentions: One picomole of dissociated INZn was incubated overnight with 1 pmole of the 21 nt ODN presenting the LTR ends or random nonviral ODN under our standard conditions (7.5 mM MnCl2, HEPES 20 mM, pH 7.6). The self association of the protein was analyzed using DSS crosslink before performing concerted integration assays. Results in Figure 6 indicate that preincubation of the dissociated enzyme with either short specific viral ODN or random ODN led to its reassociation into oligomers (mainly dimers). These data have been validated by SAXS experiments (data not shown) confirming that monomers can bind either to specific or nonspecific DNA and associate on it. In order to determine whether the oligomers formed on both ODN were functionally similar, we tested the reassociated fractions in concerted integration assays. Results are reported in Figure 7A and show that the incubation of IN monomers with the 21 nt viral ends led to recovery of integration (lane 3 compared to lane 2). In contrast, no integration was detected after incubation with nonviral ODN (lane 4), indicating that, in this case, the multimer enzyme was inactive. Selection of integrants carrying the circular form of FSI products indicated that this reaction was also recovered, at least partially, when the dissociated enzyme was pre-incubated with specific ODN but not with random sequences (Figure 7B). Sequencing of the integration loci showed a similar profile with associated and reassociated IN. The effect of the viral end structure was also analyzed using the blunt substrate for the concerted integration assay. As reported in Figure 7C and D, the same reactivation was observed under these conditions, confirming that the enzyme dissociation affected the early steps of IN attachment to DNA and not the formation of the final synaptic complex.Figure 6.

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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