Nature, Position, and Frequency of Mutations Made in a Single Cycle of HIV-1 Replication
Nature, Position, and Frequency of Mutations Made in a Single Cycle of HIV-1 Replication
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DOI：
10.1128/jvi.00915-10
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发表时间：
2010-10-01
影响因子：
5.4
通讯作者：
Hughes, Stephen H.
中科院分区：
文献类型：
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作者：
Abram, Michael E.;Ferris, Andrea L.;Hughes, Stephen H.
患者体内存在相当大的 HIV-1 变异。变异的程度是由于病毒复制率高、病毒载量高以及病毒复制过程中出现的错误造成的。突变可能是由宿主 DNA 依赖性 RNA 聚合酶 II 或 HIV-1 逆转录酶 (RT) 产生的错误引起的，但这两种酶对突变率的相对贡献尚不清楚。此外，RT 突变会影响其保真度，但 RT 突变对体内发生的突变性质的影响知之甚少。我们基于现有技术开发了一个有效的系统，用于分析 HIV-1 载体在单个复制周期中出现的突变。 lacZ α 报告基因用于识别包含突变的病毒 DNA，并通过 DNA 测序进行分析。该系统中的正向突变率为 1.4 x 10(-5) 突变/bp/周期，相当于逆转录病毒的平均值。该比率比之前报道的体内 HIV-1 低约 3 倍，并且远低于报道的体外纯化 HIV-1 RT 的比率。尽管突变率不受 lacZ α 方向的影响，但 lacZ α 中容易发生突变（热点）的位点取决于病毒 RNA 中存在的 lacZ α 链。体内 lacZ α 中观察到的热点模式与使用纯化 RT 体外复制 lacZ α 时获得的任何已发表数据都不匹配。
There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZ alpha reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 x 10(-5) mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro. Although the mutation rate was not affected by the orientation of lacZ alpha, the sites favored for mutations (hot spots) in lacZ alpha depended on which strand of lacZ alpha was present in the viral RNA. The pattern of hot spots seen in lacZ alpha in vivo did not match any of the published data obtained when purified RT was used to copy lacZ alpha in vitro.