Development and application of a high-throughput screening assay for HIV-1 integrase enzyme activities.
Integrase (IN) mediates the covalent insertion of the retroviral genome into its host chromosomal DNA. This enzymatic activity can be reconstituted in vitro with short DNA oligonucleotides, which mimic a single viral DNA end, and purified IN. Herein we report a highly efficient and sensitive high-throughput screen, HIV Integrase Target SRI Assay (HITS), for HIV-1 IN activity using 5' biotin-labeled DNA (5' BIO donor) and 3' digoxygenin-labeled DNA (3' DIG target). Following 3' processing of the 5' BIO donor, strand transfer proceeds with integration of the 5' BIO donor into the 3' DIG target. Products were captured on a streptavidin-coated microplate and the amount of DIG retained in the well was measured. The end point values, measured as absorbance, ranged from 0.9 to 1.5 for IN-mediated reactions as compared with background readings of 0.05 to 0.12. The Z factor for the assay ranged from 0.7 to 0.85. The assay was used to screen drugs in a high-throughput format, and furthermore, we adapted the assay to study mechanistic questions regarding the integration process. For example, using variations of the assay format, we showed high preference of E strand of the long terminal repeat (LTR) viral DNA as a target strand compared with its complementary A strand. The E strand is the strand processed by IN. Furthermore, we explored the reported inhibitory effect of reverse transcriptase on integration.
John
Sinu
S
Graduate Program, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, USA.
Fletcher
Thomas M
TM
Jonsson
Colleen B
CB
D001710:Biotin / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D016679:Genome, Viral; D019427:HIV Integrase / Q000737:chemistry*; D019428:HIV Integrase Inhibitors / Q000494:pharmacology*; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry; D020079:Terminal Repeat Sequences; D014158:Transcription, Genetic
D019428:HIV Integrase Inhibitors; D009841:Oligonucleotides; D001710:Biotin; D004247:DNA; D019809:Streptavidin; D004492:Edetic Acid; D019427:HIV Integrase; D054303:HIV Reverse Transcriptase