Klarmann G J, Schauber C A, Preston B D
Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08854.
J Biol Chem. 1993 May 5;268(13):9793-802.
Replication of human immunodeficiency virus type 1 (HIV-1) requires reverse transcriptase (RT) to synthesize double-stranded proviral DNA (9.7 kilobases) through a complex mechanism utilizing both RNA and DNA templates. We have examined DNA synthesis by HIV-1 RT on RNA and DNA templates derived from the HIV-1 genome using a primer extension assay in vitro. Analysis of polymerization products on sequencing gels revealed strong pauses in synthesis, on both RNA and DNA templates, in homopolymeric nucleotide runs, and at regions of predicted secondary structure. Polymerization pauses occurred in runs of template rGs (> or = 4 bases) and rCs (> or = 3 bases) during minus-strand synthesis on RNA templates, and in most runs (> or = 4 bases) of template dTs and dAs during plus-strand synthesis on DNA templates. Pausing also occurred on both templates within the first few nucleotides of the predicted hairpin structures of the Rev response element. The locations of pauses were dependent on template sequence and were unaffected by primer positioning, RT concentration, and ionic strength. Recombinant and virion-derived HIV-1 RTs showed similar pausing patterns. DNA products that accumulated at HIV-1 RT pause sites on RNA templates were extended by continued incubation with excess RT from Moloney murine leukemia virus, showing that the RNA templates were not broken or otherwise unable to support polymerization. Polymerizations conducted in the presence of a poly(rA) oligo(dT) trap showed that pausing results from two mechanisms: 1) RT remaining bound to the primer-template and polymerizing at a greatly reduced rate, or 2) RT dissociating from the primer-template. These results demonstrate that specific HIV-1 RNA and DNA template sequences are capable of interrupting processive DNA synthesis by HIV-1 RT in vitro. Pausing may serve specific functions in HIV-1 replication and mutagenesis. Moreover, these data suggest that one or more accessory factors are required to complete proviral DNA synthesis in vivo and that efficient HIV-1 DNA synthesis may require multiple origins.
1型人类免疫缺陷病毒（HIV-1）的复制需要逆转录酶（RT）通过一种利用RNA和DNA模板的复杂机制来合成双链前病毒DNA（9.7千碱基）。我们使用体外引物延伸试验，研究了HIV-1 RT在源自HIV-1基因组的RNA和DNA模板上的DNA合成。对测序凝胶上聚合产物的分析显示，在RNA和DNA模板上，在同聚核苷酸序列以及预测的二级结构区域，合成过程中存在强烈的停顿。在RNA模板上负链合成期间，模板rG（≥4个碱基）和rC（≥3个碱基）的序列中会出现聚合停顿；在DNA模板上正链合成期间，模板dT和dA的大多数序列（≥4个碱基）中也会出现停顿。在Rev反应元件预测的发夹结构的前几个核苷酸内，两种模板上都会出现停顿。停顿的位置取决于模板序列，不受引物定位、RT浓度和离子强度的影响。重组的和病毒体来源的HIV-1 RT显示出相似的停顿模式。通过与莫洛尼鼠白血病病毒的过量RT继续孵育，可延伸在RNA模板上HIV-1 RT停顿位点积累的DNA产物，这表明RNA模板没有断裂或以其他方式无法支持聚合反应。在存在聚（rA）寡聚（dT）陷阱的情况下进行的聚合反应表明，停顿由两种机制导致：1）RT仍然结合在引物-模板上，并以大大降低的速率聚合；或2）RT从引物-模板上解离。这些结果表明，特定的HIV-1 RNA和DNA模板序列能够在体外中断HIV-1 RT的连续DNA合成。停顿可能在HIV-1复制和诱变中发挥特定功能。此外，这些数据表明，在体内完成前病毒DNA合成需要一种或多种辅助因子，并且高效的HIV-1 DNA合成可能需要多个起始点。
