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BioMed Research International
Volume 2015, Article ID 853891, 7 pages
http://dx.doi.org/10.1155/2015/853891
Research Article

Novel 3′-Processing Integrase Activity Assay by Real-Time PCR for Screening and Identification of HIV-1 Integrase Inhibitors

1Research Institute for Health Sciences, Chiang Mai University, Chiang Mai 50200, Thailand
2Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand
3Biomedical Technology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand

Received 18 February 2015; Revised 13 April 2015; Accepted 17 April 2015

Academic Editor: Olivier Delelis

Copyright © 2015 Supachai Sakkhachornphop et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The 3′-end processing (3′P) of each viral long terminal repeat (LTR) during human immunodeficiency virus type-1 (HIV-1) integration is a vital step in the HIV life cycle. Blocking the 3′P using 3′P inhibitor has recently become an attractive strategy for HIV-1 therapeutic intervention. Recently, we have developed a novel real-time PCR based assay for the detection of 3′P activity in vitro. The methodology usually involves biotinylated HIV-1 LTR, HIV-1 integrase (IN), and specific primers and probe. In this novel assay, we designed the HIV-1 LTR substrate based on a sequence with a homology to HIV-1 LTR labeled at its 3′ end with biotin on the sense strand. Two nucleotides at the 3′ end were subsequently removed by IN activity. Only two nucleotides labeled biotin were captured on an avidin-coated tube; therefore, inhibiting the binding of primers and probe results in late signals in the real-time PCR. This novel assay has successfully detected both the 3′P activity of HIV-1 IN and the anti-IN activity by Raltegravir and sodium azide agent. This real-time PCR assay has been shown to be effective and inexpensive for a high-throughput screening of novel IN inhibitors.