Nucleoprotein complex intermediates in HIV-1 integration.
Integration of retroviral DNA into the host genome is an essential step in the viral replication cycle. The viral DNA, made by reverse transcription in the cytoplasm, forms part of a large nucleoprotein complex called the preintegration complex (PIC). The viral integrase protein is the enzyme within the PIC that is responsible for integrating the viral DNA into the host genome. Integrase is tightly associated with the viral DNA within the PIC as demonstrated by functional assays. Integrase protein catalyzes the key DNA cutting and joining steps of integration in vitro with DNA substrates that mimic the ends of the viral DNA. Under most in vitro assay conditions the stringency of the reaction is relaxed; most products result from "half-site" integration in which only one viral DNA end is integrated into one strand of target DNA rather than concerted integration of pairs of DNA as occurs with PICs and in vivo. Under these relaxed conditions catalysis appears to occur without formation of the highly stable nucleoprotein complexes that is characteristic of the association of integrase with viral DNA in the PIC. Here we describe methods for the assembly of nucleoprotein complex intermediates in HIV-1 DNA integration from purified HIV-1 integrase and substrates that mimic the viral DNA ends.
Li
Min
M
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Room 301, 5 Center Drive MSC 0560, Bethesda, MD 20892, United States.
Craigie
Robert
R
D004279:DNA, Viral / Q000378:metabolism; D005821:Genetic Techniques; D019427:HIV Integrase / Q000378:metabolism; D019428:HIV Integrase Inhibitors / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D009698:Nucleoproteins / Q000378:metabolism*; D013379:Substrate Specificity / Q000502:physiology; D016662:Virus Integration / Q000502:physiology*
D004279:DNA, Viral; D019428:HIV Integrase Inhibitors; D009698:Nucleoproteins; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1