Displacement of viral DNA termini from stable HIV-1 integrase nucleoprotein complexes induced by secondary DNA-binding interactions.
The human immunodeficiency virus type-1 (HIV-1) integrase is known to form a highly stable interaction with the termini of the linear, pre-integrated retroviral genome, where it catalyzes the 3'-OH processing and strand transfer processes required for their coordinated integration into host DNA. Here, we determine that the association of HIV-1 integrase with the viral DNA termini leads to the formation of two classes of nucleoprotein complexes with distinct properties in vitro. Both bound states are intrinsically stable and highly resistant to exonuclease digestion, but nonetheless they exhibit different stabilities in the presence of single-stranded polynucleotides. While a population of preassembled complexes tolerates elevated polynucleotide concentrations, the remainder forms an unstable ternary (integrase-substrate-polynucleotide) intermediate, leading to the rapid expulsion of the otherwise tightly bound substrate. The distribution of complexes between the two states is influenced by the preincubation time and temperature, increases in either of which favor the formation of the challenge-resistant species. Challenge-resistant complexes are formed more efficiently with Mn2+ than with Mg2+ and are sensitive to the length rather than the sequence of the DNA substrate. Due to the delayed appearance of the challenge-resistant form after the initial stable binding of the DNA substrate, our results may be indicative of a structural change in the preassembled complex which thereby modulates its response to exogenous DNA targets.
Pemberton
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IK
Unite de Physicochimie des Macromolecules Biologiques, CNRS UMR 321, Institut Pasteur, Paris 75724, France. iainpem@pasteur.fr
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Buckle
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D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019427:HIV Integrase / Q000737:chemistry / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D009698:Nucleoproteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D016662:Virus Integration
D002413:Cations, Divalent; D004279:DNA, Viral; D046911:Macromolecular Substances; D009698:Nucleoproteins; D019427:HIV Integrase