Human Immunodeficiency Virus Reverse Transcriptase Displays Dramatically Higher Fidelity under Physiological Magnesium Conditions In Vitro
Human Immunodeficiency Virus Reverse Transcriptase Displays Dramatically Higher Fidelity under Physiological Magnesium Conditions In Vitro
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DOI：
10.1128/jvi.00752-14
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发表时间：
2014-08-01
影响因子：
5.4
通讯作者：
DeStefano, Jeffrey J.
中科院分区：
文献类型：
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作者：
Achuthan, Vasudevan;Keith, Brian J.;DeStefano, Jeffrey J.
人类免疫缺陷病毒（HIV）逆转录酶（RT）的保真度一直是深入研究的主题。体外纯化酶的突变频率变化很大，但通常在10（-4）范围内（每个核苷酸添加），使得酶比大多数聚合酶（包括其他RT）精确几倍。这经常被认为是艾滋病毒加速产生遗传多样性的一个因素。然而，细胞实验表明，HIV的保真度并不比其他逆转录病毒低，并且显示出10（-5）范围内的突变频率。在这份报告中，我们调和，至少部分地，这些差异表明，HIV RT保真度在体外是在相同的范围内的细胞结果进行实验与生理（淋巴细胞）浓度的游离Mg 2+（类似于0.25 mM），并与莫洛尼鼠白血病病毒（MuLV）RT保真度。生理条件产生的突变率比通常采用的优化RT活性的体外条件（5至10 mM Mg 2+）下获得的突变率低5至10倍。这些结果在常用的lacZ α互补和稳态保真度测定中是一致的。有趣的是，尽管HIV RT在高Mg 2+（6 mM）条件下显示出几倍较低的保真度，但MuLV RT保真度对Mg 2+不敏感。总体而言，结果表明，只要使用更多的生理条件，HIV在细胞中复制的保真度与使用纯化的HIV RT进行的体外实验的结果是一致的。重要信息人类免疫缺陷病毒通过产生和随后选择可以规避免疫反应并逃避药物治疗的突变体而迅速进化。这一过程部分是由可能高度易错的HIV聚合酶逆转录酶（RT）推动的。奇怪的是，检测细胞中HIV复制的研究结果表明，错误频率比试管中RT的错误频率低10倍，这可能是使RT在细胞中更准确的因素。这项研究使细胞和试管结果更加一致，表明HIV RT并不比其他RT更容易出错，并且当在生理镁条件下进行检测时，错误率比使用针对酶活性优化的条件进行的典型检测低得多。
The fidelity of human immunodeficiency virus (HIV) reverse transcriptase (RT) has been a subject of intensive investigation. The mutation frequencies for the purified enzyme in vitro vary widely but are typically in the 10(-4) range (per nucleotide addition), making the enzyme severalfold less accurate than most polymerases, including other RTs. This has often been cited as a factor in HIV's accelerated generation of genetic diversity. However, cellular experiments suggest that HIV does not have significantly lower fidelity than other retroviruses and shows a mutation frequency in the 10(-5) range. In this report, we reconcile, at least in part, these discrepancies by showing that HIV RT fidelity in vitro is in the same range as cellular results from experiments conducted with physiological (for lymphocytes) concentrations of free Mg2+ (similar to 0.25 mM) and is comparable to Moloney murine leukemia virus (MuLV) RT fidelity. The physiological conditions produced mutation rates that were 5 to 10 times lower than those obtained under typically employed in vitro conditions optimized for RT activity (5 to 10 mM Mg2+). These results were consistent in both commonly used lacZ alpha complementation and steady-state fidelity assays. Interestingly, although HIV RT showed severalfold-lower fidelity under high-Mg2+ (6 mM) conditions, MuLV RT fidelity was insensitive to Mg2+. Overall, the results indicate that the fidelity of HIV replication in cells is compatible with findings of experiments carried out in vitro with purified HIV RT, providing more physiological conditions are used.IMPORTANCE Human immunodeficiency virus rapidly evolves through the generation and subsequent selection of mutants that can circumvent the immune response and escape drug therapy. This process is fueled, in part, by the presumably highly error-prone HIV polymerase reverse transcriptase (RT). Paradoxically, results of studies examining HIV replication in cells indicate an error frequency that is similar to 10 times lower than the rate for RT in the test tube, which invokes the possibility of factors that make RT more accurate in cells. This study brings the cellular and test tube results in closer agreement by showing that HIV RT is not more error prone than other RTs and, when assayed under physiological magnesium conditions, has a much lower error rate than in typical assays conducted using conditions optimized for enzyme activity.