Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Center for AIDS Research, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA; Veterans Affairs Medical Center, Decatur, GA, USA.
Antiviral Res. 2014 Jan;101:62-7. doi: 10.1016/j.antiviral.2013.10.017. Epub 2013 Nov 7.
We reported that 3'-azido-2',3'-dideoxyguanosine (3'-azido-ddG) selected for the L74V, F77L, and L214F mutations in the polymerase domain and K476N and V518I mutations in the RNase H domain of HIV-1 reverse transcriptase (RT). In this study, we have defined the molecular mechanisms of 3'-azido-ddG resistance by performing in-depth biochemical analyses of HIV-1 RT containing mutations L74V, F77L, V106I, L214F, R277K, and K476N (SGS3). The SGS3 HIV-1 RT was from a single-genome-derived full-length RT sequence obtained from 3'-azido-ddG resistant HIV-1 selected in vitro. We also analyzed two additional constructs that either lacked the L74V mutation (SGS3-L74V) or the K476N mutation (SGS3-K476N). Pre-steady-state kinetic experiments revealed that the L74V mutation allows RT to effectively discriminate between the natural nucleotide (dGTP) and 3'-azido-ddG-triphosphate (3'-azido-ddGTP). 3'-azido-ddGTP discrimination was primarily driven by a decrease in 3'-azido-ddGTP binding affinity (Kd) and not by a decreased rate of incorporation (kpol). The L74V mutation was found to severely impair RT's ability to excise the chain-terminating 3'-azido-ddG-monophosphate (3'-azido-ddGMP) moiety. However, the K476N mutation partially restored the enzyme's ability to excise 3'-azido-ddGMP on an RNA/DNA, but not on a DNA/DNA, template/primer by selectively decreasing the frequency of secondary RNase H cleavage events. Collectively, these data provide strong additional evidence that the nucleoside base structure is major determinant of HIV-1 resistance to the 3'-azido-2',3'-dideoxynucleosides.
我们曾报道，3'-叠氮-2',3'-双脱氧鸟苷（3'-叠氮-ddG）在 HIV-1 逆转录酶（RT）的聚合酶结构域中选择 L74V、F77L 和 L214F 突变，在 RNase H 结构域中选择 K476N 和 V518I 突变，对其具有抗性。在这项研究中，我们通过对 HIV-1 RT 进行深入的生化分析，定义了 3'-叠氮-ddG 耐药的分子机制，该 RT 含有突变 L74V、F77L、V106I、L214F、R277K 和 K476N（SGS3）。SGS3 HIV-1 RT 来自从体外筛选出的 3'-叠氮-ddG 耐药 HIV-1 中获得的全长 RT 序列的单基因组衍生。我们还分析了另外两个构建体，它们要么缺乏 L74V 突变（SGS3-L74V），要么缺乏 K476N 突变（SGS3-K476N）。预稳态动力学实验表明，L74V 突变使 RT 能够有效地区分天然核苷酸（dGTP）和 3'-叠氮-ddG-三磷酸（3'-叠氮-ddGTP）。3'-叠氮-ddGTP 的区分主要是由于 3'-叠氮-ddGTP 结合亲和力（Kd）降低，而不是由于掺入速率（kpol）降低。发现 L74V 突变严重损害了 RT 切除链终止的 3'-叠氮-ddG-单磷酸（3'-叠氮-ddGMP）部分的能力。然而，K476N 突变通过选择性降低次要 RNase H 切割事件的频率，部分恢复了酶在 RNA/DNA 但不在 DNA/DNA 模板/引物上切除 3'-叠氮-ddGMP 的能力。总之，这些数据提供了强有力的额外证据，证明核苷碱基结构是 HIV-1 对 3'-叠氮-2',3'-双脱氧核苷耐药的主要决定因素。
