title: Multiple integrase functions are required to form the native structure of the human immunodeficiency virus type I intasome.
abstract: Mu-mediated polymerase chain reaction footprinting was used to investigate the protein-DNA structure of human immunodeficiency virus type I (HIV-I) preintegration complexes. Preintegration complexes were partially purified from cells after using an established coculture infection technique as well as a novel technique using cell-free supernatant from transfected cells as the source of virus. Footprinting revealed that bound proteins protected the terminal 200-250 base pairs of each viral end from nuclease attack. Bound proteins also caused strong transpositional enhancements near each end of HIV-I. In contrast, regions of viral DNA internal to the ends did not show evidence of strong protein binding. The end regions of preintegrative HIV-I apparently form a unique nucleoprotein structure, which we term the intasome to distinguish it from the greater preintegration complex. Our novel system also allowed us to analyze the structure and function of preintegration complexes isolated from cells infected with integrase mutant viruses. Complexes were derived from viruses defective for either integrase catalysis, integrase binding to the viral DNA substrate, or an unknown function in the carboxyl-terminal domain of the integrase protein. None of these mutant complexes supported detectable integration activity. Despite the presence of the mutant integrase proteins in purified samples, none of these nucleoprotein complexes displayed the native intasome structure detected in wild-type preintegration complexes. We conclude that multiple integrase functions are required to form the native structure of the HIV-I intasome in infected cells.
authors.affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: H
authors.initials: H
authors.lastname: Chen
authors.forename: S Q
authors.initials: SQ
authors.lastname: Wei
authors.forename: A
authors.initials: A
authors.lastname: Engelman
mesh_terms: D003001:Cloning, Molecular; D018983:DNA Footprinting; D004279:DNA, Viral / Q000378:metabolism*; D019427:HIV Integrase / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011485:Protein Binding; D013379:Substrate Specificity; D016662:Virus Integration
chemical_list: D004279:DNA, Viral; D019427:HIV Integrase