The Human Immunodeficiency virus type 1 (HIV-1) integrase (IN) allows the integration of the double-stranded viral DNA, terminal product of reverse transcription, into the cellular chromatin. This process, in the context of the retroviral preintegration complex, is carried out in two spatially and temporally distinct chemical reactions, 3’ processing and DNA strand transfer. In the cytoplasm, IN binds the U3 and U5 ends of the viral DNA and processes their last two 3’ end nucleotides. After nuclear entry, IN uses the 3’-termini generated in the 3’ processing to integrate the viral DNA into the chromatin acceptor site. During nuclear entry, IN interacts with the lens epithelium-derived growth factor LEDGF/p75 through the IN catalytic core domain (CCD) interface cavity and the LEDGF/p75 C-terminal IN-binding domain (IBD). LEDGF/p75 promotes viral integration by tethering the preintegration complex to the chromatin. IN dimerization, enzyme reactions and its interaction with LEDGF/p75 are all essential for viral replication and are validated drug targets. Currently, development of compounds that have an allosteric mechanism of inhibition is a promising and innovative approach to inhibit HIV-1 replication. We have established a biochemical assay using Homogeneous Time Resolved Fluorescence (HTRF) methodology that combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR) to evaluate at the same time the inhibition of i) IN-IN dimerization, ii) IN catalytic reactions and iii) LEDGF-IN binding. In addition, we also established single assays to assess IN-IN subunit exchange, IN 3’ processing and/or strand transfer reactions, LEDGF-IN binding. Overall, we established a simple assay to identify small molecules that can act as allosteric IN inhibitors.
A Homogeneous Time Resolved Fluorescence (HTRF) method for the evaluation of HIV-1 integrase dimerization, enzyme reactions and interaction with LEDGF/p75
ESPOSITO, FRANCESCA;TRAMONTANO, ENZO
2014-01-01
Abstract
The Human Immunodeficiency virus type 1 (HIV-1) integrase (IN) allows the integration of the double-stranded viral DNA, terminal product of reverse transcription, into the cellular chromatin. This process, in the context of the retroviral preintegration complex, is carried out in two spatially and temporally distinct chemical reactions, 3’ processing and DNA strand transfer. In the cytoplasm, IN binds the U3 and U5 ends of the viral DNA and processes their last two 3’ end nucleotides. After nuclear entry, IN uses the 3’-termini generated in the 3’ processing to integrate the viral DNA into the chromatin acceptor site. During nuclear entry, IN interacts with the lens epithelium-derived growth factor LEDGF/p75 through the IN catalytic core domain (CCD) interface cavity and the LEDGF/p75 C-terminal IN-binding domain (IBD). LEDGF/p75 promotes viral integration by tethering the preintegration complex to the chromatin. IN dimerization, enzyme reactions and its interaction with LEDGF/p75 are all essential for viral replication and are validated drug targets. Currently, development of compounds that have an allosteric mechanism of inhibition is a promising and innovative approach to inhibit HIV-1 replication. We have established a biochemical assay using Homogeneous Time Resolved Fluorescence (HTRF) methodology that combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR) to evaluate at the same time the inhibition of i) IN-IN dimerization, ii) IN catalytic reactions and iii) LEDGF-IN binding. In addition, we also established single assays to assess IN-IN subunit exchange, IN 3’ processing and/or strand transfer reactions, LEDGF-IN binding. Overall, we established a simple assay to identify small molecules that can act as allosteric IN inhibitors.
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