Back N K, Nijhuis M, Keulen W, Boucher C A, Oude Essink B O, van Kuilenburg A B, van Gennip A H, Berkhout B
Department of Human Retrovirology, University of Amsterdam, Academic Medical Center, Utrecht, Netherlands.
EMBO J. 1996 Aug 1;15(15):4040-9.
Human immunodeficiency virus type 1 (HIV-1) variants with resistance mutations in the reverse transcriptase (RT) gene appear during drug therapy with the nucleoside analogue 2',3'-dideoxy-3'-thiacytidine (3TC). These resistance mutations alter the methionine (Met) residue of the conserved YMDD motif, which is part of the catalytic core of the RT enzyme. Isoleucine (Ile) variants are initially observed, followed by the appearance and eventual outgrowth of viruses encoding valine (Val). Similar replication kinetics were measured for wild-type and 3TC-resistant HIV-1 viruses in tissue culture infections of a T cell line, but we measured reduced polymerase activity for the two mutant RT enzymes compared with the wild-type enzyme (Ile = 43% and Val = 67%). Gel analysis of the reverse transcription products revealed that both 3TC-resistant RT mutants produce significantly shorter cDNA molecules than the wild-type enzyme [Met (wt)>Val>Ile], indicating that 3TC-resistant RT polymerases are less processive enzymes. Interestingly, these enzyme defects were more pronounced under limiting dNTP concentrations and we therefore assayed virus replication in primary cells that contain relatively low dNTP levels. Under these conditions, we measured significantly reduced replication kinetics for the 3TC-resistant HIV-1 variants [Met (wt)>Val>Ile]. If the level of virus replication can be similarly reduced in 3TC-treated patients that develop drug-resistant HIV-1 variants, this may be of considerable clinical benefit.
在使用核苷类似物2',3'-二脱氧-3'-硫代胞苷（3TC）进行药物治疗期间，会出现人类免疫缺陷病毒1型（HIV-1）逆转录酶（RT）基因带有耐药性突变的变体。这些耐药性突变会改变保守的YMDD基序中的甲硫氨酸（Met）残基，该基序是RT酶催化核心的一部分。最初观察到异亮氨酸（Ile）变体，随后出现并最终出现编码缬氨酸（Val）的病毒并占优势。在T细胞系的组织培养感染中，对野生型和3TC耐药的HIV-1病毒测量了相似的复制动力学，但与野生型酶相比，我们测量了两种突变RT酶的聚合酶活性降低（Ile = 43%，Val = 67%）。逆转录产物的凝胶分析表明，两种3TC耐药的RT突变体产生的cDNA分子均明显短于野生型酶[Met（野生型）>Val>Ile]，这表明3TC耐药的RT聚合酶是持续性较差的酶。有趣的是，这些酶缺陷在有限的dNTP浓度下更为明显，因此我们在含有相对较低dNTP水平的原代细胞中检测了病毒复制。在这些条件下，我们测量了3TC耐药的HIV-1变体的复制动力学显著降低[Met（野生型）>Val>Ile]。如果在出现耐药性HIV-1变体的3TC治疗患者中病毒复制水平能同样降低，这可能具有相当大的临床益处。
