Relative rates of retroviral reverse transcriptase template switching during RNA- and DNA-dependent DNA synthesis
Relative rates of retroviral reverse transcriptase template switching during RNA- and DNA-dependent DNA synthesis
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DOI：
10.1128/jvi.72.6.5198-5206.1998
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发表时间：
1998-06-01
影响因子：
5.4
通讯作者：
Pathak, VK
中科院分区：
文献类型：
--
作者：
Bowman, RR;Hu, WS;Pathak, VK
逆转录病毒逆转录酶（RT）在DNA合成过程中经常转换模板，这可能导致突变和重组。RNA和DNA依赖性DNA过程中体内RT模板转换的相对速率。合成是未知的。为了确定RNA和DNA模板复制过程中RT模板转换的相对速率，我们构建了含有100 bp直接重复序列的基于脾坏死病毒的逆转录病毒载体。直接重复序列位于RB-LLP载体中多嘌呤段（PPT）的上游;相同的直接重复序列位于RB-LPL载体中PPT和附着位点（att）的侧翼，RT模板转换事件可以在RNA或DNA依赖性DNA合成期间发生，并删除一个拷贝的直接重复序列加上插入序列。在RNA和DNA依赖性DNA合成期间经历直接重复缺失的RB-LLP载体产生可以整合到宿主基因组中的病毒DNA。然而，在RNA依赖性DNA合成期间发生的RB-LPL载体中的任何同向重复序列的缺失导致必需PPT和atf位点的缺失，并产生死端病毒DNA产物。因此，只有在DNA依赖性DNA合成过程中经历直接repent缺失的RB-LPL载体才能整合形成前病毒。允许RB-LLP和RB-LPL载体经历单个复制循环，并且通过PCR和对所得前病毒的Southern分析来确定直接重复缺失的频率。RB-LLP和RB-LPL载体中的直接重复缺失频率的比较表明，在RNA和DNA依赖性DNA合成过程中RT模板的体内剪切速率几乎相同。
Retroviral reverse transcriptases (RTs) frequently switch templates during DNA synthesis, which can result in mutations and recombination. The relative rates of in vivo RT template switching during RNA- and DNA-dependent DNA. synthesis are unknown. To determine the relative rates of RT template switching during copying of RNA and DNA templates, we constructed spleen necrosis virus-based retroviral vectors containing a 100-bp direct repeat. The directly repeated sequences were upstream of the polypurine tract (PPT) in the RB-LLP vector; the same direct repeats flanked the PPT and attachment site (att) in the RB-LPL vector, RT template switching events could occur during either RNA or DNA-dependent DNA synthesis and delete one copy of the direct repeat plus the intervening sequences. RB-LLP vectors that underwent direct repeat deletions during RNA- and DNA-dependent DNA synthesis generated viral DNA that could integrate into the host genome. However, any deletion of the direct repeats in the RB-LPL vector that occurred during RNA-dependent DNA synthesis resulted in deletion of the essential PPT and atf site and generated a dead-end viral DNA product. Thus, only RB-LPL vectors that underwent direct repent deletions during DNA-dependent DNA synthesis could integrate to form proviruses. The RB-LLP and RB-LPL vectors were permitted to undergo a single replication cycle, and the frequencies of direct repeat deletions were determined by PCR and Southern analysis of the resulting proviruses. A comparison of the frequency of direct repeat deletions in the RB-LLP and RB-LPL vectors indicated that the in vivo rates of RT template snitching during RNA- and DNA-dependent DNA synthesis are nearly identical.