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Novel 3 ‘-Processing Integrase Activity Assay by Real-Time PCR for Screening and Identification of HIV-1 Integrase Inhibitors
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Document Title
Novel 3 '-Processing Integrase Activity Assay by Real-Time PCR for Screening and Identification of HIV-1 Integrase Inhibitors
Author
Sakkhachornphop S, Thongkum W, Tayapiwatana C
Name from Authors Collection
Affiliations
Chiang Mai University; Chiang Mai University; Chiang Mai University; National Science & Technology Development Agency - Thailand; National Center Genetic Engineering & Biotechnology (BIOTEC)
Type
Article
Source Title
BIOMED RESEARCH INTERNATIONAL
ISSN
2314-6133
Year
2015
Volume
2015
Issue
3
Open Access
Green Submitted, Green Published, gold
Publisher
HINDAWI LTD
DOI
10.1155/2015/853891
Format
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.
Funding Sponsor
Health Systems Research Institute [P-13-50163]
License
CC BY
Rights
Authors
Publication Source
WOS