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

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.

journal_name

Biomed Res Int

journal_title

BioMed research international

authors

Sakkhachornphop S,Thongkum W,Tayapiwatana C

doi

10.1155/2015/853891

subject

Has Abstract

pub_date

2015-01-01 00:00:00

pages

853891

eissn

2314-6133

issn

2314-6141

journal_volume

2015

pub_type

杂志文章

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