Double-stranded strong-stop DNA and the second template switch in human immunodeficiency virus (HIV) DNA synthesis

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.