Rawson Jonathan M O, Daly Michele B, Xie Jiashu, Clouser Christine L, Landman Sean R, Reilly Cavan S, Bonnac Laurent, Kim Baek, Patterson Steven E, Mansky Louis M
Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, USA Molecular, Cellular, Developmental Biology and Genetics Graduate Program, University of Minnesota, Minneapolis, Minnesota, USA.
Center for Drug Discovery, Department of Pediatrics, Emory Center for AIDS Research, Emory University, Atlanta, Georgia, USA.
Antimicrob Agents Chemother. 2016 Mar 25;60(4):2318-25. doi: 10.1128/AAC.03084-15. Print 2016 Apr.
5-Azacytidine (5-aza-C) is a ribonucleoside analog that induces the lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1) by causing predominantly G-to-C transversions during reverse transcription. 5-Aza-C could potentially act primarily as a ribonucleotide (5-aza-CTP) or as a deoxyribonucleotide (5-aza-2'-deoxycytidine triphosphate [5-aza-dCTP]) during reverse transcription. In order to determine the primary form of 5-aza-C that is active against HIV-1, Illumina sequencing was performed using proviral DNA from cells treated with 5-aza-C or 5-aza-dC. 5-Aza-C and 5-aza-dC were found to induce highly similar patterns of mutation in HIV-1 in terms of the types of mutations observed, the magnitudes of effects, and the distributions of mutations at individual sequence positions. Further, 5-aza-dCTP was detected by liquid chromatography-tandem mass spectrometry in cells treated with 5-aza-C, demonstrating that 5-aza-C was a substrate for ribonucleotide reductase. Notably, levels of 5-aza-dCTP were similar in cells treated with equivalent effective concentrations of 5-aza-C or 5-aza-dC. Lastly, HIV-1 reverse transcriptase was found to incorporate 5-aza-CTPin vitroat least 10,000-fold less efficiently than 5-aza-dCTP. Taken together, these data support the model that 5-aza-C enhances the mutagenesis of HIV-1 primarily after reduction to 5-aza-dC, which can then be incorporated during reverse transcription and lead to G-to-C hypermutation. These findings may have important implications for the design of new ribonucleoside analogs directed against retroviruses.
5-氮杂胞苷（5-aza-C）是一种核糖核苷类似物，它通过在逆转录过程中主要引起G到C的颠换来诱导人类免疫缺陷病毒1型（HIV-1）的致死性诱变。在逆转录过程中，5-aza-C可能主要作为核糖核苷酸（5-氮杂胞苷三磷酸[5-aza-CTP]）或脱氧核糖核苷酸（5-氮杂-2'-脱氧胞苷三磷酸[5-aza-dCTP]）起作用。为了确定对HIV-1具有活性的5-aza-C的主要形式，使用来自用5-aza-C或5-氮杂-2'-脱氧胞苷（5-aza-dC）处理的细胞的前病毒DNA进行了Illumina测序。就观察到的突变类型、效应大小和单个序列位置的突变分布而言，发现5-aza-C和5-aza-dC在HIV-1中诱导高度相似的突变模式。此外，在用5-aza-C处理的细胞中通过液相色谱-串联质谱法检测到了5-aza-dCTP，这表明5-aza-C是核糖核苷酸还原酶的底物。值得注意的是，在用等效有效浓度的5-aza-C或5-aza-dC处理的细胞中，5-aza-dCTP的水平相似。最后，发现HIV-1逆转录酶在体外掺入5-aza-CTP的效率比5-aza-dCTP至少低10000倍。综上所述，这些数据支持这样一种模型，即5-aza-C主要在还原为5-aza-dC后增强HIV-1的诱变作用，然后5-aza-dC可以在逆转录过程中掺入并导致G到C的超突变。这些发现可能对针对逆转录病毒的新型核糖核苷类似物的设计具有重要意义。
