title: In vitro DNA tethering of HIV-1 integrase by the transcriptional coactivator LEDGF/p75.
abstract: Although LEDGF/p75 is believed to act as a cellular cofactor of lentiviral integration by tethering integrase (IN) to chromatin, there is no good in vitro model to analyze this functionality. We designed an AlphaScreen assay to study how LEDGF/p75 modulates the interaction of human immunodeficiency virus type 1 IN with DNA. IN bound with similar affinity to DNA mimicking the long terminal repeat or to random DNA. While LEDGF/p75 bound DNA strongly, a mutant of LEDGF/p75 with compromised nuclear localization signal (NLS)/AT hook interacted weakly, and the LEDGF/p75 PWWP domain did not interact, corroborating previous reports on the role of NLS and AT hooks in charge-dependent DNA binding. LEDGF/p75 stimulated IN binding to DNA 10-fold to 30-fold. Stimulation of IN-DNA binding required a direct interaction between IN and the C-terminus of LEDGF/p75. Addition of either the C-terminus of LEDGF/p75 (amino acids 325-530) or LEDGF/p75 mutated in the NLS/AT hooks interfered with IN binding to DNA. Our results are consistent with an in vitro model of LEDGF/p75-mediated tethering of IN to DNA. The inhibition of IN-DNA interaction by the LEDGF/p75 C-terminus may provide a novel strategy for the inhibition of HIV IN activity and may explain the potent inhibition of HIV replication observed after the overexpression of C-terminal fragments in cell culture.
authors.affiliation: Laboratory for Molecular Virology and Gene Therapy, Molecular Medicine, KULeuven and IRC Kulak, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium.
authors.forename: Melissa
authors.initials: M
authors.lastname: McNeely
authors.forename: Jelle
authors.initials: J
authors.lastname: Hendrix
authors.forename: Katrien
authors.initials: K
authors.lastname: Busschots
authors.forename: Eline
authors.initials: E
authors.lastname: Boons
authors.forename: Angélique
authors.initials: A
authors.lastname: Deleersnijder
authors.forename: Melanie
authors.initials: M
authors.lastname: Gerard
authors.forename: Frauke
authors.initials: F
authors.lastname: Christ
authors.forename: Zeger
authors.initials: Z
authors.lastname: Debyser
mesh_terms: D020856:AT Rich Sequence / Q000235:genetics; D001681:Biological Assay; D004247:DNA / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D019427:HIV Integrase / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012995:Solubility; D013329:Structure-Activity Relationship
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D050505:Mutant Proteins; D019913:Nuclear Localization Signals; D009841:Oligonucleotides; C119380:lens epithelium-derived growth factor; D004247:DNA; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1