title: 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.
authors.affiliation: Research Institute for Health Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Supachai
authors.initials: S
authors.lastname: Sakkhachornphop
authors.affiliation: Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Weeraya
authors.initials: W
authors.lastname: Thongkum
authors.affiliation: Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand ; Biomedical 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.
authors.forename: Chatchai
authors.initials: C
authors.lastname: Tayapiwatana
mesh_terms: D015658:HIV Infections / Q000188:drug therapy* / Q000201:enzymology / Q000821:virology; D019427:HIV Integrase / Q000737:chemistry / Q000235:genetics*; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology* / Q000472:pathogenicity; D006801:Humans; D000068898:Raltegravir Potassium / Q000737:chemistry / Q000627:therapeutic use; D019810:Sodium Azide / Q000737:chemistry / Q000627:therapeutic use
chemical_list: D000068898:Raltegravir Potassium; D019810:Sodium Azide; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1