Ji J P, Loeb L A
Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle 98195.
Biochemistry. 1992 Feb 4;31(4):954-8. doi: 10.1021/bi00119a002.
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.
人类免疫缺陷病毒1型（HIV-1）的基因组高度变异可能源于病毒逆转录酶的错误掺入。我们开发了一种体外检测逆转录酶在RNA依赖性以及DNA依赖性DNA聚合过程中保真度的方法。用T3 RNA聚合酶转录的lacZαRNA片段来模拟第一链逆转录。相应的DNA模板用于检测逆转录酶在第二链DNA合成过程中的错误。对于这两种模板，通过M13 lacZα互补试验中的突变表型来鉴定逆转录酶引入的突变。我们发现，在RNA或DNA模板上，来自人类免疫缺陷病毒1型的逆转录酶（HIV-1 RT）比来自莫洛尼鼠白血病病毒的逆转录酶（MLV RT）或大肠杆菌DNA聚合酶I（Pol I）的Klenow片段准确性更低。HIV-1 RT在RNA模板上聚合时错误掺入的频率为每6900个核苷酸中有1个错误，在DNA模板上为每5900个中有1个错误。MLV RT和Pol I在RNA模板上的错误率分别低于每28000个和37000个中有1个错误。HIV-1 RT复制RNA模板时产生的最常见突变是由dAMP错误掺入导致的C→T转换和G→T颠换。
