HIV-1 integrase crosslinked oligomers are active in vitro

The oligomeric state of active human immunodeficiency virus type 1 (HIV-1) integrase (IN) has not been clearly elucidated. We analyzed the activity of the different purified oligomeric forms of recombinant IN obtained after stabilization by platinum crosslinking. The crosslinked tetramer isolated by gel chromatography was able to catalyze the full-site integration of the two viral LTR ends into a target DNA in vitro, whereas the isolated dimeric form of the enzyme was involved in the processing and integration of only one viral end. Accurate concerted integration by IN tetramers was confirmed by cloning and sequencing. Kinetic studies of DNA-integrase complexes led us to propose a model explaining the formation of an active complex. Our data suggest that the tetrameric IN bound to the viral DNA ends is the minimal complex involved in the concerted integration of both LTRs and should be the oligomeric form targeted by future inhibitors

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

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

DNA Aptamers Derived from HIV-1 RNase H Inhibitors are Strong Anti-integrase Agents

International audienceHIV-1 integrase, the retroviral-encoded enzyme involved in the integration of the retrotranscribed viral genome into the host nuclear DNA, is an attractive and still unexploited target. To date, very few inhibitors of this enzyme with a potential therapeutic value have been described. During the search for new HIV-1 targets, we recently described DNA oligodeoxynucleotide aptamers (ODN 93 and ODN 112) that are strong inhibitors of the RNase H activity associated with HIV-1 reverse transcriptase. The striking structural homology between RNase H and integrase led us to study the effect of the RNase H inhibitors on the integrase. Shorter DNA aptamers derived from ODNs 93 and 112 (ODNs 93del and 112del) were able to inhibit HIV-1 integrase in the nanomolar range. They had G-rich sequences able to form G-quartets stabilized by the presence of K(+). The presence of these ions increased the inhibitory efficiency of these agents dramatically. Inhibition of enzymatic activities by ODN 93del and ODN 112del was observed in a cell-free assay system using a recombinant integrase and HIV-1 replication was abolished in infected human cells. Moreover, cell fusion assays showed that these agents do not block viral cell entry at concentrations where viral replication is stopped