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First published on August 15, 2005, doi:10.1177/1087057105276318

Journal of Biomolecular Screening 2005;10:606.

A more recent version of this article appeared on September 1, 2005
 © 2005 Society for Biomolecular Sciences

Article

Development and Application of a High-Throughput Screening Assay for HIV-1 Integrase Enzyme Activities

Sinu John1, Thomas M. Fletcher III2, Colleen B. Jonsson3*

1 Graduate Program, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham.
2 High Throughput Screening and Target Development Program, Southern Research Institute, Birmingham, AL.
3 Department of Biochemistry and Molecular Biology, 2000 9th Avenue South, Southern Research Institute, Birmingham, AL.

* To whom correspondence should be addressed. E-mail: jonsson{at}sri.org.


  Abstract

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 (HITSTM), 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.

Key Words: HIV, integrase (IN), assay development, high-throughput screening (HTS)


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