In vitro analysis of the HIV-1 second strand-transfer reaction.

ABSTRACT

Two strand-transfer reactions are required during the retroviral reverse transcription. The second strand-transfer is believed to occur at the primer binding site (PBS). By using an in vitro model system derived from the human immunodeficiency virus type-1 (HIV-1), molecular events related to the second strand-transfer reaction were examined. The RNA-directed DNA synthesis catalyzed by the HIV-1 reverse transcriptase (RT) appears to pause twice, around the 2nd and the 11th bases downstream into the HIV-1 PBS. In either case, pausing seems to be caused by local sequence effects. From both these pause sites the nascent DNA strands can undergo transfer to PBS-homologous templates. The results confirm that pausing favors the internal strand-transfer. They also indicate that PBS-mediated transfers proceed through displacement of the DNA strands from RNA donor molecules by the acceptor template, as in one of the models proposed by DeStefano et al. (DeStefano, J.J., Bambara, R.A. and Fay, P.J. (1994) J. Biol. Chem. 269, 161-168). Whereas no hydrolysis by the ribonuclease H activity of HIV-1 RT seemed to occur within the HIV-1 PBS, specific cleavage was observed outside of this sequence and noticeably 5 bases upstream from its 3' end relative to the DNA primer growing point. This particular cleavage event remained essentially unchanged when the second pause site downstream into the PBS, located 16 bases beyond, was removed by mutation. The experiments suggest that this prominent RNA degradation event could precede the PBS-mediated strand-transfers.