Gu Z, Quan Y, Li Z, Arts E J, Wainberg M A
McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, Montreal, Quebec, Canada.
J Biol Chem. 1995 Dec 29;270(52):31046-51. doi: 10.1074/jbc.270.52.31046.
We have employed a cell-free human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) assay to study the effects of non-nucleoside inhibitors of RT (NNRTI) by directly monitoring specific HIV DNA products using a HIV-1 genome-derived template and an oligodeoxynucleotide primer. As previously shown by ourselves and others, nucleoside analog triphosphates, e.g. 3'-azido-3'-deoxythymidine triphosphate and 2',3'-dideoxyadenosine triphosphate, could directly inhibit HIV RT RNA-dependent DNA polymerase activity by causing chain termination, as visualized in a RT reaction that yields specific DNA products. In contrast, each of two NNRTIs, nevirapine and delavirdine, directly inhibited RT activity without causing chain termination effects. We also analyzed interactions between nucleoside analogs and NNRTIs or among NNRTIs by chain elongation/dNTP incorporation and/or steady-state kinetic assays. Combinations of nevirapine with the triphosphates of either the (-)-strand of 2',3'-dideoxy-3'-thiacytidine or 2',3'-dideoxyadenosine yielded additive/synergistic effects on RT activity. However, only an additive effect was observed when combinations of nevirapine and 3'-azido-3'-deoxythymidine triphosphate were employed. Combinations of nevirapine and delavirdine had an antagonistic effect on the inhibition of HIV-1 RT activity.
我们采用了一种无细胞的1型人类免疫缺陷病毒（HIV-1）逆转录酶（RT）检测方法，通过使用HIV-1基因组衍生模板和寡脱氧核苷酸引物直接监测特定的HIV DNA产物，来研究RT的非核苷抑制剂（NNRTI）的作用。正如我们自己和其他人之前所表明的，核苷类似物三磷酸酯，例如3'-叠氮-3'-脱氧胸苷三磷酸酯和2',3'-双脱氧腺苷三磷酸酯，可通过导致链终止直接抑制HIV RT RNA依赖性DNA聚合酶活性，这在产生特定DNA产物的RT反应中可以看到。相比之下，两种NNRTI（奈韦拉平和地拉韦定）中的每一种都直接抑制RT活性而不产生链终止效应。我们还通过链延伸/dNTP掺入和/或稳态动力学检测分析了核苷类似物与NNRTI之间或NNRTI之间的相互作用。奈韦拉平与2',3'-双脱氧-3'-硫代胞苷或2',3'-双脱氧腺苷的（-）链三磷酸酯的组合对RT活性产生加性/协同效应。然而，当使用奈韦拉平和3'-叠氮-3'-脱氧胸苷三磷酸酯的组合时，仅观察到加性效应。奈韦拉平和地拉韦定的组合对HIV-1 RT活性的抑制具有拮抗作用。
