Rank K B, Fan N, Sharma S K
Pharmacia and Upjohn, Kalamazoo, MI 49007, USA.
Antiviral Res. 1997 Sep;36(1):27-33. doi: 10.1016/s0166-3542(97)00033-8.
The human immunodeficiency virus-1 (HIV-1) integrase catalyzes the specific removal of two nucleotides at either 3' end of each long terminal repeat (LTR) sequence of the proviral DNA duplex. The most commonly used in vitro assays for integrase employ 5' end 32P-labeled double-stranded oligonucleotides and the product of integrase-associated endonuclease activity is visualized by denaturing gel electrophoresis followed by autoradiography. We report here a simple assay system based upon the liberation of [35S]GT dinucleotide from the 3' end of a double-stranded U5 LTR sequence derived from HIV-1. The uncleaved labeled substrate and the unlabeled large product are removed by adsorption to polyethyleneimine cellulose followed by centrifugation. The small labeled GT dinucleotide product released in the supernatant is quantitated in terms of counts released as a function of time. Since the method is rapid and quantitative, it should be useful in the kinetic evaluation of inhibitors of the 3' cleavage activity of HIV-1 integrase.
人类免疫缺陷病毒1型（HIV-1）整合酶催化前病毒DNA双链体每个长末端重复序列（LTR）3'末端两个核苷酸的特异性去除。整合酶最常用的体外测定方法采用5'末端32P标记的双链寡核苷酸，整合酶相关内切核酸酶活性的产物通过变性凝胶电泳，然后进行放射自显影来观察。我们在此报告一种基于从HIV-1衍生的双链U5 LTR序列3'末端释放[35S]GT二核苷酸的简单测定系统。未切割的标记底物和未标记的大产物通过吸附到聚乙烯亚胺纤维素上，然后离心去除。上清液中释放的小标记GT二核苷酸产物根据随时间释放的计数进行定量。由于该方法快速且定量，它应有助于对HIV-1整合酶3'切割活性抑制剂进行动力学评估。
