Rezende L F, Drosopoulos W C, Prasad V R
Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Nucleic Acids Res. 1998 Jun 15;26(12):3066-72. doi: 10.1093/nar/26.12.3066.
A common target for therapies against human immuno-deficiency virus type 1 (HIV-1) is the viral reverse transcriptase (RT). Treatment with the widely used nucleoside analog (-)-2', 3'-deoxy-3'-thiacytidine (3TC) leads to the development of resistance-conferring mutations at residue M184 within the YMDD motif of RT. First, variants of HIV with the M184I substitution appear transiently, followed by viruses containing the M184V substitution, which persist and become the dominant variant for the duration of therapy. In the three-dimensional crystal structure of HIV-1 RT complexed with double-stranded DNA, the M184 residue lies in the vicinity of the primer terminus, near the incoming dNTP substrate. Recent studies have shown that 3TC resistance mutations, including M184I, increase the nucleotide insertion and mispair extension fidelity. Therefore, we have examined the effects of the M184I mutation on the overall polymerase fidelity of HIV-1 RT via an M13-based forward mutation assay. We found the overall error rate of the M184I variant of HIV-1 RT to be 1.7 x 10(-5) per nucleotide. This represents a 4-fold increase in fidelity over wild-type HIV-1Hxb2RT (7.0 x 10(-5) per nucleotide) and a 2.5-fold increase in fidelity over the M184V variant (4.3 x 10(-5) per nucleotide). Of the nucleoside analog resistance mutations studied using the forward assay, the M184I variant has shown the greatest increase in fidelity observed to date. Interestingly, the M184I variant RT displays significantly altered error specificity, both in terms of error rate at specific sites and in the overall ratio of substitution to frameshift mutations in the entire target.
针对人类免疫缺陷病毒1型（HIV-1）的治疗常见靶点是病毒逆转录酶（RT）。使用广泛的核苷类似物（-）-2'，3'-二脱氧-3'-硫代胞苷（3TC）进行治疗会导致RT的YMDD基序内M184残基处产生赋予抗性的突变。首先，具有M184I替代的HIV变体短暂出现，随后是含有M184V替代的病毒，这些病毒持续存在并在治疗期间成为主要变体。在与双链DNA复合的HIV-1 RT的三维晶体结构中，M184残基位于引物末端附近，靠近进入的dNTP底物。最近的研究表明，包括M184I在内的3TC抗性突变会增加核苷酸插入和错配延伸保真度。因此，我们通过基于M13的正向突变试验研究了M184I突变对HIV-1 RT整体聚合酶保真度的影响。我们发现HIV-1 RT的M184I变体的总体错误率为每核苷酸1.7×10（-5）。这表示保真度比野生型HIV-1Hxb2RT（每核苷酸7.0×10（-5））提高了4倍，比M184V变体（每核苷酸4.3×10（-5））提高了2.5倍。在使用正向试验研究的核苷类似物抗性突变中，M184I变体显示出迄今为止观察到的最大保真度增加。有趣的是，M184I变体RT在特定位点的错误率以及整个靶标中替代突变与移码突变的总体比例方面均显示出明显改变的错误特异性。
