Pullen K A, Ishimoto L K, Champoux J J
Department of Microbiology, School of Medicine, University of Washington, Seattle 98195.
J Virol. 1992 Jan;66(1):367-73. doi: 10.1128/JVI.66.1.367-373.1992.
A synthetic RNA oligonucleotide (15-mer) corresponding to the 3' end of the lysine tRNA primer was hybridized to single-stranded DNA containing the human immunodeficiency virus type 1 (HIV-1) primer-binding site and extended with a DNA polymerase. The resulting structures were used to study primer removal by the RNase H activity of HIV-1 reverse transcriptase. The initial cleavage event removes the RNA primer as a 14-mer and leaves a single ribonucleotide A residue bound to the 5' end of the DNA strand. This result explains the observation by several groups that HIV-1 circle junctions contain 4 bp that are not present in the integrated provirus instead of the predicted 3 bp. Subsequent cleavage events occur at other sites internal to the RNA molecule, and the ribonucleotide A residue on the end of the DNA strand is ultimately removed. Therefore, the biologically relevant cleavage that produces the 14-mer reflects the kinetics of the reaction as well as a specificity for nucleic acid sequence. When the RNA oligonucleotide alone was hybridized to the primer-binding site and tested as a substrate for HIV-1 RNase H, the cleavage pattern near the 3' end of the RNA was altered.
将一段与赖氨酸tRNA引物3'端对应的合成RNA寡核苷酸（15聚体）与包含人类免疫缺陷病毒1型（HIV-1）引物结合位点的单链DNA杂交，并用DNA聚合酶进行延伸。所得结构用于研究HIV-1逆转录酶的RNase H活性对引物的去除作用。最初的切割事件将RNA引物切割成14聚体，并在DNA链的5'端留下一个与单核糖核苷酸A残基结合的产物。这一结果解释了几个研究小组的观察结果，即HIV-1环状连接点包含4个碱基对，而不是整合前病毒中预测的3个碱基对，这些碱基对在整合前病毒中并不存在。随后的切割事件发生在RNA分子内部的其他位点，DNA链末端的核糖核苷酸A残基最终被去除。因此，产生14聚体的生物学相关切割反映了反应动力学以及对核酸序列的特异性。当单独将RNA寡核苷酸与引物结合位点杂交并作为HIV-1 RNase H的底物进行测试时，RNA 3'端附近的切割模式发生了改变。
