Activity of recombinant HIV-1 integrase on mini-HIV DNA

Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA into th e genome of a human cell is an essential step in the viral replication cycl e. Understanding of the integration process has been facilitated by the dev elopment of in vitro assays using specific oligonucleotides and recombinant integrase. However, understanding of the biology of retroviral integration will require in vitro and in vivo model systems using long DNA substrates that mimic the HIV cDNA. We have now studied the activity of recombinant HI V-1 integrase on a linear 4.7 kb double-stranded DNA, containing flanking r egions of similar to 200 bp that represent the intact ends of the HIV-1 lon g terminal repeat (LTR) sequences (mini-HIV). The strand transfer products of the integration reaction can be directly visualized after separation in agarose gels by ethidium bromide staining. The most prominent reaction prod uct resulted from integration of one LTR end into another LTR end (U5 into U5 and U5 into U3). Sequence analysis of the reaction products showed them to be products of legitimate integration preceded by correct processing of the viral LTR ends. Hotspots for integration were detected. Electron micros copy revealed the presence of a range of reaction products resulting from s ingle or multiple integration events. The binding of HIV-1 integrase to min i-HIV DNA was visualized. Oligomers of integrase seem to induce DNA looping whereby the enzyme often appears to be bound to the DNA substrate that ado pts the structure of a three-site synapsis that is reminiscent of the Mu ph age transposase complex.