FIDELITY OF HIV-1 REVERSE-TRANSCRIPTASE COPYING RNA INVITRO
FIDELITY OF HIV-1 REVERSE-TRANSCRIPTASE COPYING RNA INVITRO
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DOI：
10.1021/bi00119a002
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发表时间：
1992-02-04
期刊：
影响因子：
2.9
通讯作者：
LOEB, LA
中科院分区：
文献类型：
--
作者：
JI, JP;LOEB, LA
人类免疫缺陷病毒1型(HIV-1)基因组的高度变异可能是由于病毒逆转录酶的错误结合所致。我们建立了一种在体外RNA依赖和DNA依赖的DNA聚合过程中逆转录酶保真度的检测方法。用T3RNA聚合酶转录的LacZ-αRNA片段模拟第一链逆转录。使用相应的DNA模板通过逆转录酶检查第二链DNA合成过程中的错误。用这两个模板，逆转录酶引入的突变在M13 LacZ-α互补试验中根据它们的突变表型进行鉴定。我们发现，在RNA和DNA模板上，人类免疫缺陷病毒1型逆转录酶(HIV-1RT)的准确性低于Moloney小鼠白血病病毒(MLV RT)的逆转录酶或大肠杆菌DNA聚合酶I(PolI)的Klenow片段。HIV-1 RT在RNA模板上聚合的6900个核苷酸中有1个错误掺入，在DNA模板上聚合的5900个核苷酸中有1个错误掺入。MLV RT和Pol I在RNA模板上的错误率分别小于1/28000和37000。HIV-1RT复制RNA模板所产生的最常见的突变是C-T转换和G--T颠换，这是由于误掺DAMP引起的。
The genomic hypervariation of human immunodeficiency virus 1 (HIV-1) could result from misincorporations by the viral reverse transcriptase. We developed an assay for reverse transcriptase fidelity during RNA-dependent as well as DNA-dependent DNA polymerization in vitro. A lacZ-alpha RNA fragment transcribed by T3 RNA polymerase was used to mimic first-strand reverse transcription. The corresponding DNA template was used to examine errors by reverse transcriptase during second-strand DNA synthesis. With both templates, the mutations introduced by reverse transcriptase were identified by their mutant phenotypes in an M13 lacZ-alpha-complementation assay. We found that the reverse transcriptase from human immunodeficiency virus 1 (HIV-1 RT) was less accurate than the reverse transcriptase from Moloney murine leukemia virus (MLV RT) or the Klenow fragment of Escherichia coli DNA polymerase I (Pol I) on either RNA or DNA templates. The frequency of misincorporation by HIV-1 RT was 1 in 6900 nucleotides polymerized on the RNA template and 1 in 5900 on the DNA template. The error rates of MLV RT and Pol I on the RNA template were less than 1 in 28 000 and 37 000, respectively. The most frequent mutations produced by HIV-1 RT copying the RNA template were C --> T transitions and G --> T transversions resulting from misincorporation of dAMP.