University of Pittsburgh School of Medicine, Department of Medicine, Division of Infectious Diseases, Pittsburgh, PA 15261, USA.
Antimicrob Agents Chemother. 2011 Aug;55(8):3758-64. doi: 10.1128/AAC.00414-11. Epub 2011 Jun 6.
We recently reported that HIV-1 resistant to 3'-azido-3'-deoxythymidine (AZT) is not cross-resistant to 3'-azido-2',3'-dideoxypurines. This finding suggested that the nucleoside base is a major determinant of HIV-1 resistance to nucleoside analogs. To further explore this hypothesis, we conducted in vitro selection experiments by serial passage of HIV-1(LAI) in MT-2 cells in increasing concentrations of 3'-azido-2',3'-dideoxyguanosine (3'-azido-ddG), 3'-azido-2',3'-dideoxycytidine (3'-azido-ddC), or 3'-azido-2',3'-dideoxyadenosine (3'-azido-ddA). 3'-Azido-ddG selected for virus that was 5.3-fold resistant to 3'-azido-ddG compared to wild-type HIV-1(LAI) passaged in the absence of drug. Population sequencing of the entire reverse transcriptase (RT) gene identified L74V, F77L, and L214F mutations in the polymerase domain and K476N and V518I mutations in the RNase H domain. However, when introduced into HIV-1 by site-directed mutagenesis, these 5 mutations only conferred ∼2.0-fold resistance. Single-genome sequencing analyses of the selected virus revealed a complex population of mutants that all contained L74V and L214F linked to other mutations, including ones not identified during population sequencing. Recombinant HIV-1 clones containing RT derived from single sequences exhibited 3.2- to 4.0-fold 3'-azido-ddG resistance. In contrast to 3'-azido-ddG, 3'-azido-ddC selected for the V75I mutation in HIV-1 RT that conferred 5.9-fold resistance, compared to the wild-type virus. Interestingly, we were unable to select HIV-1 that was resistant to 3'-azido-ddA, even at concentrations of 3'-azido-ddA that yielded high intracellular levels of 3'-azido-ddA-5'-triphosphate. Taken together, these findings show that the nucleoside base is a major determinant of HIV-1 resistance mechanisms that can be exploited in the design of novel nucleoside RT inhibitors.
我们最近报道称，对 3'-叠氮-3'-去氧胸苷（AZT）有耐药性的 HIV-1 对 3'-叠氮-2',3'-二脱氧嘌呤核苷没有交叉耐药性。这一发现表明核苷碱基是 HIV-1 对核苷类似物耐药性的主要决定因素。为了进一步探索这一假设，我们通过在递增浓度的 3'-叠氮-2',3'-二脱氧鸟苷（3'-叠氮-ddG）、3'-叠氮-2',3'-二脱氧胞苷（3'-叠氮-ddC）或 3'-叠氮-2',3'-二脱氧腺苷（3'-叠氮-ddA）中连续传代 MT-2 细胞中的 HIV-1（LAI），进行了体外选择实验。与无药物传代的野生型 HIV-1（LAI）相比，3'-叠氮-ddG 选择出的病毒对 3'-叠氮-ddG 的耐药性提高了 5.3 倍。聚合酶域中 L74V、F77L 和 L214F 突变，以及 RNase H 域中的 K476N 和 V518I 突变，对整个逆转录酶（RT）基因的群体测序进行了鉴定。然而，当通过定点突变引入 HIV-1 时，这 5 个突变仅赋予了约 2.0 倍的耐药性。对选择出的病毒的单基因组测序分析显示，存在一个复杂的突变体群体，所有突变体都包含 L74V 和 L214F，与其他突变体相连，包括在群体测序中未鉴定到的突变体。含有源自单个序列的 RT 的重组 HIV-1 克隆表现出对 3'-叠氮-ddG 的 3.2-至 4.0 倍耐药性。与 3'-叠氮-ddG 相反，3'-叠氮-ddC 选择 HIV-1 RT 中的 V75I 突变，与野生型病毒相比，该突变赋予了 5.9 倍的耐药性。有趣的是，我们无法选择对 3'-叠氮-ddA 耐药的 HIV-1，即使在 3'-叠氮-ddA 的浓度下，也无法获得高细胞内 3'-叠氮-ddA-5'-三磷酸水平。总之，这些发现表明核苷碱基是 HIV-1 耐药机制的主要决定因素，可以用于设计新型核苷 RT 抑制剂。
