title: Reporter gene expression from LTR-circles as tool to identify HIV-1 integrase inhibitors.
abstract: Early HIV-1 integrase inhibitors, such as compounds containing a β-diketo acid moiety, were identified by extensive high-throughput screening campaigns. Traditionally, in vitro biochemical assays, measuring the catalytic activities of integrase, have been used for this purpose. However, these assays are confounded by the absence of cellular processes or cofactors that play a role in the integration of HIV-1 DNA in the cellular genome. In contrast to regular cell-based virus inhibition assays, which targets all steps of the viral replication cycle, a novel cellular screening assays was developed to enable the specific identification of integrase inhibitors, employing a readout that is linked with the inhibition of integrase activity. Therefore, a HIV-1 lentiviral vector equipped with the enhanced green fluorescent protein (eGFP) reporter gene was used to detect expression from extrachromosomal viral DNA (1- or 2-long terminal repeat circles), formed when integration of vector DNA into the cellular genome is prevented by an integrase inhibitor. In this assay, eGFP expression from the low residual level of transcriptional activity of extrachromosomal DNA was measured via high-throughput flow cytometry. An algorithm for analysis of eGFP expression histograms enabled the specific identification of integrase inhibitors. This assay is amenable for high throughput screening to identify inhibitors of HIV-1 integrase.
authors.affiliation: Janssen Infectious Diseases BVBA, Turnhoutseweg 30, 2340 Beerse, Belgium. mvloock@its.jnj.com
authors.forename: M
authors.initials: M
authors.lastname: Van Loock
authors.forename: A
authors.initials: A
authors.lastname: Hombrouck
authors.forename: T
authors.initials: T
authors.lastname: Jacobs
authors.forename: B
authors.initials: B
authors.lastname: Winters
authors.forename: G
authors.initials: G
authors.lastname: Meersseman
authors.forename: K
authors.initials: K
authors.lastname: Van Acker
authors.forename: R F
authors.initials: RF
authors.lastname: Clayton
authors.forename: B A
authors.initials: BA
authors.lastname: Malcolm
mesh_terms: D004353:Drug Evaluation, Preclinical / Q000379:methods*; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D019427:HIV Integrase / Q000378:metabolism*; D019428:HIV Integrase Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000187:drug effects*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans
chemical_list: D019428:HIV Integrase Inhibitors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1