FIDELITY OF RETROVIRAL REVERSE TRANSCRIPTASES

人类免疫缺陷病毒(HIV-1)基因组序列变异 1)逆转录酶复制不准确所致。这 酶在DNA模板的体外合成过程中容易出错。 然而，依赖于RNA的DNA合成与负-相关的保真度 尚未对病毒生命周期中的链复制进行检查 之前。今年我们开发了一种检测系统来确定 转录和反转录的保真度，并用它来比较 HIV-1逆转录酶结合RNA合成DNA的保真度 和相同序列的DNA模板。总体而言，保真度是几倍的 RNA的含量高于DNA的含量。产生的突变体的序列分析 这两种底物显示，错误率的差异是 对于特定的错误来说，这是很重要的。与RNA的保真度是10倍以上 在五个不同的位置，替换和负一核苷酸误差更高 均聚位置。由于这种错误很可能是由模板引起- 引物滑移，这一结果表明错位的中间体是 使用RNA模板-DNA引物形成和/或使用的频率低于 用DNA模板--DNA引子。结果还表明，HIV-1RT 用RNA模板-DNA引物合成容易出错 前两个核苷酸的结合，可能是由于酶的异常- 当合成开始时，底物相互作用。不同的误码率 RNA和DNA模板表明，负链和正链过程中的错误 DNA合成可能不会对HIV-1的变异率做出同样的贡献。 这些数据还提供了对替换和移码错误率的估计 在转录过程中被T7RNA聚合酶转录。我们还完成了对 AZT代谢产物对正常细胞复制的影响 抗AZT病毒克隆的错配修复及RT的研究我们 将继续致力于阐明导致 HIV-1 RT的错误倾向，希望这将提供见解 酶的活性部位与其底物的相互作用 可能在设计RT靶向药物时有用。

Sequence variation in the genome of the human immunodeficiency virus (HIV- 1) results from inaccurate replication by reverse transcriptase. This enzyme is error-prone during synthesis in vitro with DNA templates. However, the fidelity of RNA-dependent DNA synthesis relevant to minus- strand replication in the virus life cycle had not been examined previously. This year we developed an assay system to determine the fidelity of transcription and reverse transcription and used it to compare the fidelity of DNA synthesis by the HIV-1 reverse transcriptase with RNA and DNA templates of the same sequence. Overall, fidelity was several-fold higher with RNA than with DNA. Sequence analysis of mutants generated with the two substrates revealed that differences in error rates were substantial for specific errors. Fidelity with RNA was more than 10-fold higher for substitution and minus-one nucleotide errors at five different homopolymeric positions. Since such errors likely result from template- primer slippage, this result suggests that misaligned intermediates are formed and/or utilized less frequently with an RNA template-DNA primer than with a DNA template-DNA primer. The results also suggest that HIV-1 RT synthesis with an RNA template-DNA primer was error-prone during incorporation of the first two nucleotides, perhaps due to aberrant enzyme- substrate interactions as synthesis initiates. The unequal error rates with RNA and DNA templates suggest that mistakes during minus- and plus-strand DNA synthesis may not contribute equally to the mutation rate of HIV-1. The data also provide estimates of substitution and frameshift error rates during transcription by T7 RNA polymerase. We also completed studies of the effects of AZT metabolites on normal cellular replication and on mismatch repair and studies of the RT from AZT-resistant virus clones. We will continue to focus on elucidating the mechanisms responsible for the error-proneness of HIV-1 RT, in the hope that this will provide insights into the interaction of the enzyme's active site with its substrates that may be useful in designing RT targeted drugs.