Relationship between the Oligomeric Status of HIV-1 Integrase on DNA and Enzymatic Activity

Elvire Guiot,Kevin Carayon,Olivier Delelis,Françoise Simon,Patrick Tauc,Evgenii Zubin,Marina Gottikh,Jean-François Mouscadet,Jean-Claude Brochon,Eric Deprez

doi:10.1074/jbc.m602198200

Abstract

The 3'-processing of the extremities of viral DNA is the first of two reactions catalyzed by HIV-1 integrase (IN). High order IN multimers (tetramers) are required for complete integration, but it remains unclear which oligomer is responsible for the 3'-processing reaction. Moreover, IN tends to aggregate, and it is unknown whether the polymerization or aggregation of this enzyme on DNA is detrimental or beneficial for activity. We have developed a fluorescence assay based on anisotropy for monitoring release of the terminal dinucleotide product in real-time. Because the initial anisotropy value obtained after DNA binding and before catalysis depends on the fractional saturation of DNA sites and the size of IN.DNA complexes, this approach can be used to study the relationship between activity and binding/multimerization parameters in the same assay. By increasing the IN:DNA ratio, we found that the anisotropy increased but the 3'-processing activity displayed a characteristic bell-shaped behavior. The anisotropy values obtained in the first phase were predictive of subsequent activity and accounted for the number of complexes. Interestingly, activity peaked and then decreased in the second phase, whereas anisotropy continued to increase. Time-resolved fluorescence anisotropy studies showed that the most competent form for catalysis corresponds to a dimer bound to one viral DNA end, whereas higher order complexes such as aggregates predominate during the second phase when activity drops off. We conclude that a single IN dimer at each extremity of viral DNA molecules is required for 3'-processing, with a dimer of dimers responsible for the subsequent full integration.

Highlights

The integration of a DNA copy of the HIV-12 genome into the host genome is a crucial step in the life cycle of the retrovirus
For low IN:DNA ratios, the r values obtained after DNA binding and before catalysis were fully predictive of subsequent IN activity, according to the fractional saturation function
Because r depends on both fractional saturation and the molecular size of complexes at saturation of DNA sites, our results show that high order multimers or aggregated states of IN are detrimental to 3Ј-processing activity

Summary

EXPERIMENTAL PROCEDURES

Oligonucleotides and Nomenclature—Unlabeled and fluorescein-labeled single-stranded (ss) ODNs were purchased from Eurogentec (Liege, Belgium) (except for the 2Ј-aminouridinecontaining ODN) and purified by electrophoresis in acrylamide gels. The uridine-containing ODN (corresponding to strand a of the DNA substrate HIV-aUF) contains a fluorescein attached to the 2Ј-amino group of the 3Ј-terminal 2Ј-aminouridine It was synthesized as follows: the solid support was prepared as previously described [21]. (i) In fixed-time experiments, the reaction was stopped by adding SDS (0.25% final), disrupting all the IN1⁄7DNA complexes in the sample In such experiments, the solution contained two fluorescent species: the nonprocessed ODN and the fluorescein-labeled dinucleotide released by the cleavage reaction. (ii) In real-time conditions, an additional fluorescent population corresponding to IN complexed with the unprocessed ds ODN, is present in the sample In this case, Fdinu was calculated as follows, rt ϭ 0 Ϫ r rmax Ϫ rdinu (Eq 3). Where ␩ is the viscosity, V is the volume of the rotating unit, k is the Boltzmann constant, and T is the temperature (K)

RESULTS

DISCUSSION

IN:DNAa