title: A high-throughput assay for HIV-1 integrase 3'-processing activity using time-resolved fluorescence.
abstract: HIV-1 integrase (HIV-1 IN), a well-validated antiviral drug target, catalyzes multistep reactions to incorporate viral DNA into the genome of the host cell; these include both a 3'-processing (3'P) reaction and a strand transfer reaction. These enzymatic activities can be measured in vitro with short DNA oligonucleotides that mimic a single viral LTR DNA end and purified IN. A highly sensitive and reproducible time-resolved fluorescence (TRF)-based assay for HIV-1 IN 3'P activity is now reported. This assay was optimized with respect to time and concentrations of metal ions, substrate and enzyme. The assay has now been used successfully to measure HIV-1 IN 3'P activity and has been shown to detect the anti-IN activity of several known 3'P inhibition compounds accurately. This assay, which is amenable to high-throughput screening, will be useful for identification of additional HIV-1 IN 3'P inhibitors.
authors.affiliation: McGill University AIDS Centre, Lady Davis for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada.
authors.forename: Ying-Shan
authors.initials: YS
authors.lastname: Han
authors.forename: Peter
authors.initials: P
authors.lastname: Quashie
authors.forename: Thibault
authors.initials: T
authors.lastname: Mesplede
authors.forename: Hongtao
authors.initials: H
authors.lastname: Xu
authors.forename: Kevork
authors.initials: K
authors.lastname: Mekhssian
authors.forename: Craig
authors.initials: C
authors.lastname: Fenwick
authors.forename: Mark A
authors.initials: MA
authors.lastname: Wainberg
mesh_terms: D005453:Fluorescence; D019427:HIV Integrase / Q000378:metabolism*; D019428:HIV Integrase Inhibitors / Q000032:analysis*; D015497:HIV-1 / Q000201:enzymology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D013997:Time Factors
chemical_list: D019428:HIV Integrase Inhibitors; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1