Steps of the acceptor invasion mechanism for HIV-1 minus strand strong stop transfer.
Minus strand strong stop transfer is obligatory for completion of HIV-1 minus strand synthesis. We previously showed evidence for an acceptor invasion-initiated mechanism for minus strand transfer. In the present study, we examined the major acceptor invasion initiation site using a minus strand transfer system in vitro, containing the 97-nucleotide full-length R region. A series of DNA oligonucleotides complementary to different regions of the cDNA was designed to interfere with transfer. Oligomers covering the region around the base of the TAR hairpin were most effective in inhibiting transfer, suggesting that the hairpin base is a preferred site for acceptor invasion. The strong pausing of reverse transcriptase at the base of the TAR and the concomitant RNase H cleavages 10-19 nucleotides behind the pause site correlated with the location of the invasion site. Oligomers closer to the 5'-end of R also inhibited transfer, though less effectively, presumably by blocking strand exchange and branch migration. We propose that pausing of reverse transcriptase at the base of TAR increases RNase H cleavages, creating gaps for acceptor invasion and transfer initiation. Strand exchange then propagates by branch migration, displacing the fragmented donor RNA, including the fragment at the 5' terminus. The primer terminus switches to the acceptor, completing the transfer. Nucleocapsid (NC) protein stimulated transfer efficiency by 5-7-fold. NC enhanced RNase H cleavages close to the TAR base, creating more effective invasion sites for efficient transfer. Most likely, NC also stimulates transfer by promoting strand exchange invasion and branch migration.
Chen
Yan
Y
Department of Biochemistry and Biophysics, University of Rochester, New York 14642, USA.
Balakrishnan
Mini
M
Roques
Bernard P
BP
Bambara
Robert A
RA
D018076:DNA, Complementary / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology*; D008957:Models, Genetic; D019251:Nucleocapsid / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry; D012313:RNA / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D013997:Time Factors
D018076:DNA, Complementary; D004279:DNA, Viral; D009841:Oligonucleotides; D012367:RNA, Viral; D012313:RNA; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H