Li P, Stephenson A J, Kuiper L J, Burrell C J
National Centre for HIV Virology Research, Institute of Medical and Veterinary Science, Adelaide, South Australia.
Virology. 1993 May;194(1):82-8. doi: 10.1006/viro.1993.1237.
Synthesis of unintegrated retroviral DNA via reverse transcription is thought to involve two separate template switches. Minus-strand strong-stop DNA, a major product of in vitro reactions using detergent-treated virions, is synthesized prior to the first template switch. Plus-strand strong-stop DNA, which is found base-paired to near full-length minus-strand DNA in infected cells, is believed to be synthesized before the second template switch. Using a synchronized, one-step HIV infection model, we report here the detection in acutely infected cells of a novel double-stranded strong-stop HIV DNA with a discrete length of approximately 650 base pairs, commencing at or near the left hand end of the right-hand U3 region of the HIV long terminal repeat. The plus-strand of this double-stranded strong-stop DNA possesses the primer binding site sequence and appears to be synthesized prior to the completion of the synthesis of its complementary minus-strand. In contrast, the minus-strand of the double-stranded strong-stop DNA lacks the primer binding site sequence after RNaseH digestion. We propose that a transient free plus-strand strong-stop DNA is released from its template by displacement synthesis and subsequently used as template for the synthesis of its complementary minus-strand. The proposed transient free plus-strand strong-stop DNA may also mediate the second template switch.
通过逆转录合成未整合的逆转录病毒DNA被认为涉及两个独立的模板转换。负链强终止DNA是使用去污剂处理的病毒体进行体外反应的主要产物，在第一次模板转换之前合成。正链强终止DNA在感染细胞中与接近全长的负链DNA碱基配对，据信在第二次模板转换之前合成。使用同步的一步HIV感染模型，我们在此报告在急性感染细胞中检测到一种新型的双链强终止HIV DNA，其长度约为650个碱基对，从HIV长末端重复序列右手U3区域的左手末端或其附近开始。这种双链强终止DNA的正链具有引物结合位点序列，并且似乎在其互补负链合成完成之前就已合成。相比之下，双链强终止DNA的负链在RNaseH消化后缺乏引物结合位点序列。我们提出，通过置换合成，一个短暂的游离正链强终止DNA从其模板上释放出来，随后用作合成其互补负链的模板。所提出的短暂游离正链强终止DNA也可能介导第二次模板转换。
