Development and application of a high-throughput screening assay for HIV-1 integrase enzyme activities
Development and application of a high-throughput screening assay for HIV-1 integrase enzyme activities
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DOI：
10.1177/1087057105276318
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发表时间：
2005-09-01
影响因子：
--
通讯作者：
Jonsson, CB
中科院分区：
文献类型：
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作者：
John, S;Fletcher, TM;Jonsson, CB
整合酶（IN）介导逆转录病毒基因组共价插入其宿主染色体DNA中。这种酶活性可以在体外用模拟单个病毒DNA末端的短DNA寡核苷酸和纯化的IN重建。本文报道了一种高效、灵敏的HIV整合酶靶SRI检测方法（HITS（TM）），该方法使用5 ′生物素标记的DNA（5 ′ BIO供体）和3 ′地高辛标记的DNA（3 ′ DIG靶）检测HIV-1 IN活性。在5 'BIO供体的3'处理后，链转移随着5 'BIO供体整合到3' DIG靶中而继续进行。将产物捕获在链霉亲和素包被的微孔板上，并测量保留在孔中的DIG的量。IN介导反应的终点值（以吸光度计）范围为0.9 - 1.5，而背景读数为0.05 - 0.12。测定的Z因子范围为0.7 - 0.85。该试验用于筛选药物的高通量格式，此外，我们适应了该试验，以研究有关的整合过程的机制问题。例如，使用测定形式的变化，我们显示出与其互补A链相比，长末端重复（LTR）病毒DNA的E链作为靶链的高度偏好性。E链是由IN处理的链。此外，我们探讨了逆转录酶对整合的抑制作用。
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 (HITS (TM)), 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.