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（艾滋病毒），是他们的能力， 产生多样性。 逆转录病毒有很高的突变率， 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 的主要来源 这种多样性可能是病毒逆转的不忠， 转录酶（RT）。 我们的目标是检测 在体外DNA合成过程中，使用M13 mp2 监测多种碱基诱变试验 替换、移码、缺失和复杂错误。 我们开始 通过测量一轮天然DNA的错误频率， 用从禽成髓细胞瘤病毒（MLV）分离的RT合成 和鼠白血病病毒（MLV）。 两种酶都表现出低 忠诚 AMV产生的突变体的DNA序列分析 RT证明它犯了许多不同类型的错误。 两 特异性是唯一的;碱基替换错误导致 仅来自嘌呤核苷酸的错误插入和许多-1 缺失发生在非重复的碱基序列上。 更以 使用末端错配的模板-引物的测定， AMV和MLV RT表现出意想不到的扩展偏好， 与正常细胞聚合酶相比是独特的。 这些 特异性可能反映了活性物质中的结构差异， RT的位置。 总的来说，这些数据支持这样一个概念， 观察到的逆转录病毒的高突变率确实反映了低 逆转录的保真度。 我们最近获得了一个 从HIV-1制备逆转录酶。 我们最初 结果表明，这种酶甚至比 AMV和MLV RT。 我们目前正在对一大批 HIV RT产生的突变体，以确定最常见的突变 由这种酶引起的事件。 我们还打算使用reversion 检测特定突变途径的试验， 阐明艾滋病毒多样性的机制， 生成的.

A critical feature of the life cycle of retroviruses, including the human immunodeficiency virus (HIV), is their ability to generate diversity. Retroviruses have 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 reverse transcriptase (RT). Our objective is to examine the mutagenic potential of RTs during DNA synthesis in vitro, using an M13mp2 mutagenesis assay that monitors a wide variety of base substitution, frameshift, deletion and complex errors. We began by measuring the error frequency of a single round of natural DNA synthesis with RTs isolated from avian myeloblastosis virus (MLV) and murine leukemia virus (MLV). Both enzymes exhibit low fidelity. DNA sequence analysis of mutants generated by the AMV RT demonstrate that it commits many different types of errors. Two specificities are unique; base substitution errors result exclusively from misinsertion of purine nucleotides and many -1 deletions occur at non-reiterated base sequences. Furthermore, in an assay employing a terminally mismatched template-primer, both AMV and MLV RTs exhibit an unexpected extension preference that is unique compared to normal cellular polymerases. These specificities may reflect a structural differences in the active site of RTs. Overall, these data support the concept that the observed high mutation rate of retroviruses indeed reflects low fidelity of reverse transcription. We have recently obtained a preparation of reverse transcriptase from HIV-1. Our initial results indicate that this enzyme is even more error prone than the AMV and MLV RTs. We are presently sequencing a large collection of HIV RT-generated mutants to determine the most common mutational events perpetrated by this enzyme. We also intend to use reversion assays to examine specific mutational pathways in order to elucidate the mechanisms by which the diversity of the HIV is generated.