Small-angle X-ray characterization of the nucleoprotein complexes resulting from DNA-induced oligomerization of HIV-1 integrase

Abstract

HIV-1 integrase (IN) catalyses integration of a DNA copy of the viral genome into the host genome. Specific interactions between retroviral IN and long terminal repeats (LTR) are required for this insertion. To characterize quantitatively the influence of the determinants of DNA substrate specificity on the oligomerization status of IN, we used the small-angle X-ray scattering (SAXS) technique. Under certain conditions in the absence of ODNs IN existed only as monomers. IN preincubation with specific ODNs led mainly to formation of dimers, the relative amount of which correlated well with the increase in the enzyme activity in the 3'-processing reaction. Under these conditions, tetramers were scarce. Non-specific ODNs stimulated formation of catalytically inactive dimers and tetramers. Complexes of monomeric, dimeric and tetrameric forms of IN with specific and non-specific ODNs had varying radii of gyration (R(g)), suggesting that the specific sequence-dependent formation of IN tetramers can probably occur by dimerization of two dimers of different structure. From our data we can conclude that the DNA-induced oligomerization of HIV-1 IN is probably of importance to provide substrate specificity and to increase the enzyme activity.

Figure 1.

Guinier plot representation of the SAXS data. The logarithmic dependencies of the intensity of X-ray scattering, ln I(h) on the diffraction angle, h², are presented. Analysis of data was performed using the equation I(h) = I(0) exp(− h²/3). Curve 1: 67.4 µM IN in the absence of ODNs; curve 2: 30.8 µM 21-COM alone; curve 3: 66.9 µM IN after preincubation with 30.8 µM 21-COM.

Figure 2.

The SAXS roentgenograms in Guinier coordinates. Curve 1: 41 µM IN in the absence of ODNs; curve 2: 40 µM IN preincubated with 30.8 µM GCAGT; curve 3: 29.8 µM IN preincubated with 228.6 µM GCAGT.

Figure 3.

Kinetic schemes of IN interaction with ODNs. Model A and B represent two possible kinetic pathways leading to formation of different nucleoprotein complexes. Oligomeric states of integrase are represented as E: monomer; E₂: dimer; E₄: tetramer. S corresponds to ODN substrate.

Figure 4.

Schematic presentation of IN monomers interaction with specific and non-specific ODNs. The scheme shows the interaction of initial IN monomers with ODNs leading to a formation of the enzyme dimers of two types and a formation of catalytically active tetramers of two different dimers.