TARGET DNA CAPTURE BY HIV-1 INTEGRATION COMPLEXES

Background: The early steps of human immunodeficiency virus 1 (HIV-1) replication involve reverse transcription of the viral RNA and integra tion of the resulting cDNA into a host chromosome. The DNA integration step requires the integration machinery ('preintegration complex') to bind to the host DNA before connecting the viral and host DNAs. Here, we present experiments that distinguish among three possible pathways of target-DNA capture: repeated binding and release of target DNA pri or to the chemical strand-transfer step; binding followed by facilitat ed diffusion along target DNA (sliding); and integration at the initia l target-capture site. The mechanism of target-DNA capture has implica tions for the design of gene therapy methods, and influences the inter pretation of results on the selection of integration target sites in v ivo. Results: We present new in vitro conditions that allow us to asse mble HIV-1 integrase - the virus-encoded recombination enzyme - with a viral DNA and then to trap assembled complexes bound to target DNA. W e find that complexes of integrase and viral DNA do not slide along ta rget DNA substantially after binding. We confirm and extend these resu lts by analyzing target capture by a hybrid protein composed of HIV-1 integrase linked to a sequence-specific DNA-binding domain. We find th at the integrase domain binds quickly and lightly under the above cond itions, thereby obstructing function of the fused sequence-specific DN A-binding domain. We also monitor target-DNA capture by HIV-1 preinteg ration complexes purified from freshly infected cells. Partially purif ied complexes commit quickly and stably to the first target DNA added, whereas preintegration complexes in crude cytoplasmic extracts do not . The addition of extracts from uninfected cells to partially purified complexes blocks quick commitment. Conclusions: Under new conditions favorable for the analysis of target-DNA capture in vitro, HIV-1 integ rase complexes bind quickly and stably to target DNA without subsequen t sliding. parallel studies of preintegration complexes support a mode l in which target-site capture in viva is reversible as a result of th e action of cellular factors.