Thermostat le HIV-1 group O reverse transcriptase variants with the same fidelity as murine leukaemia virus reverse transcriptase
Thermostat le HIV-1 group O reverse transcriptase variants with the same fidelity as murine leukaemia virus reverse transcriptase
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DOI：
10.1042/bj20101852
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发表时间：
2011-06-15
影响因子：
4.1
通讯作者：
Menendez-Arias, Luis
中科院分区：
文献类型：
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作者：
Barrioluengo, Veronica;Alvarez, Mar;Menendez-Arias, Luis
与HIV-1 M组亚型B RT和MLV（鼠白血病病毒）RT相比，野生型HIV-1 O组RT（逆转录酶）显示出更高的热稳定性。然而，与肿瘤逆转录病毒RT（即MLV RT）相比，慢病毒RT的准确性降低，限制了其在RNA靶扩增中的应用。使用基于凝胶的和M13 mp2 lacZ正向突变保真度测定研究了突变K65 R、R78 A和K65 R/V751对HIV-1 O组RT的保真度的影响。正向突变试验表明，与野生型酶相比，突变型RT K65 R、R78 A和K65 R/V751的准确性提高了9倍以上，并且比MLV RT的准确性高出约2倍。与MLV RT相比，所有检测的HIV-1 O组RT变体的移码保真度均降低。然而，与其他HIV-1 O组RT变体相比，K65 RT显示出更高的引入一个核苷酸缺失的倾向。R78 A对RT具有不稳定作用，无论是在存在还是不存在V751的情况下。在高于52 ℃的温度下，K65 R和K65 R/V751保持与野生型HIV-1 O组RT相似的DNA聚合酶活性水平，但比HIV-1 M组亚型B和MLV RT更有效。K65 R，K65 R/V751和R78 A RT显示减少的错误插入和错配延伸保真度相比，野生型酶的大多数碱基对的研究。这些测定揭示，在K65 R的情况下，核苷酸选择主要由k（pol）（pol是聚合）控制，而在K65 R/V751的情况下，k（pol）和K（d）两者都影响核苷酸辨别。
Wild-type HIV-1 group O RT (reverse transcriptase) shows increased thermostability in comparison with HIV-1 group M subtype B RT and MLV (murine leukaemia virus) RT. However, its utility in the amplification of RNA targets is limited by the reduced accuracy of lentiviral RTs compared with oncoretroviral RTs (i.e. MLV RT). The effects of the mutations K65R, R78A and K65R/V751 on the fidelity of HIV-1 group O RTs were studied using gel-based and M 1 3mp2 lacZ forward-mutation fidelity assays. Forward-mutation assays demonstrated that mutant RTs K65R, R78A and K65R/V751 showed >9-fold increased accuracy in comparison with the wild-type enzyme and were approximately two times more faithful than the MLV RT. Compared with MLV RT, all of the tested HIV-1 group O RT variants showed decreased frameshift fidelity. However, K65R RT showed a higher tendency to introduce one-nucleotide deletions in comparison with other HIV-1 group O RT variants. R78A had a destabilizing effect on the RT, either in the presence or absence of V751. At temperatures above 52 C, K65R and K65R/V751 retained similar levels of DNA polymerase activity to the wild-type HIV-1 group O RT, but were more efficient than HIV-1 group M subtype B and MLV RTs. K65R, K65R/V751 and R78A RTs showed decreased misinsertion and mispair extension fidelity in comparison with the wild-type enzyme for most base pairs studied. These assays revealed that nucleotide selection is mainly governed by k(pol) (pol is polymerization) in the case of K65R, whereas both k(pol) and K(d) affect nucleotide discrimination in the case of K65R/V751.