FIDELITY OF RETROVIRAL REVERSE TRANSCRIPTASES

逆转录病毒生命周期的一个关键特征，包括 人类免疫缺陷病毒（HIV），是它们产生 多样性 逆转录病毒有极高的突变率， 允许病毒的新形式的快速进化， 更好地逃脱宿主的防御机制 一个主要来源 这种多样性的根源可能是病毒编码的不忠实性 逆转录酶 我们的目标是阐明 遗传多样性的分子机制 艾滋病毒和其他逆转录病毒基因组。 我们已经开始 使用以下方法检查逆转录酶的致突变潜力： 我们最近开发的M13mp2诱变试验。 该测定 测量一轮天然DNA的错误频率 系统中的综合，允许分析各种各样的 突变事件，每一个都精确地定义在核苷酸 序列级。 我们对逆转录酶的初步研究结果 从禽成髓细胞瘤病毒和小鼠白血病中分离 病毒显示反向错误频率高 转录酶复制天然DNA的过程中。 这支持了 观察到的逆转录病毒的高突变率 确实反映了，至少部分， 忠诚 我们计划纯化各种形式的病毒逆转录病毒 转录酶，并检查他们的错误频率和 在合成过程中产生的突变的特异性。 然后我们将 使用回复突变试验来监测特定的突变途径， 通过逆转录酶的诱变试验模型。 最终 我们想把重点放在逆转录酶上， 艾滋病毒基因组，阐明多样性的机制， 是由这种酶产生的。

A critical feature of the life cycle of retroviruses, including the human immuno-deficiency virus (HIV), is their ability to generate diversity. Retroviruses have exceptionally high mutation rates, permitting rapid evolution of new forms of the virus that are better able to escape host defense mechanisms. A major source of this diversity may be the infidelity of the viral-encoded reverse transcriptase. Our objective is to elucidate the molecular mechanisms responsible for the genetic diversity of the HIV and other retroviral genomes. We have begun to examine the mutagenic potential of reverse transcriptase using our recently developed M13mp2 mutagenesis assay. This assay measures the error frequency of a single round of natural DNA synthesis in system that permits analysis of a wide variety of mutational events, each precisely defined at the nucleotide sequence level. Our initial results with reverse transcriptases isolated from avian myeloblastosis virus and murine leukemia virus demonstrate a high error frequency for reverse transcriptase during copying of natural DNA. This supports the concept that the observed high mutation rate of retroviruses indeed reflects, at least in part, low reverse transcription fidelity. We plan to purify various forms of viral reverse transcriptase and examine their error frequency and the specificity of mutations produced during synthesis. We will then use reversion assays to monitor specific mutational pathways to test models of mutagenesis by reverse transcriptase. Ultimately we would like to focus on the reverse transcriptase encoded by the HIV genome, to elucidate the mechanisms by which diversity is generated by this enzyme.