title: Mechanism of minus strand strong stop transfer in HIV-1 reverse transcription.
abstract: Retrovirus minus strand strong stop transfer (minus strand transfer) requires reverse transcriptase-associated RNase H, R sequence homology, and viral nucleocapsid protein. The minus strand transfer mechanism in human immunodeficiency virus-1 was examined in vitro with purified protein and substrates. Blocking donor RNA 5'-end cleavage inhibited transfers when template homology was 19 nucleotides (nt) or less. Cleavage of the donor 5'-end occurred prior to formation of transfer products. This suggests that when template homology is short, transfer occurs through a primer terminus switch-initiated mechanism, which requires cleavage of the donor 5' terminus. On templates with 26-nt and longer homology, transfer occurred before cleavage of the donor 5' terminus. Transfer was unaffected when donor 5'-end cleavages were blocked but was reduced when internal cleavages within the donor were restricted. Based on the overall data, we conclude that in human immunodeficiency virus-1, which contains a 97-nt R sequence, minus strand transfer occurs through an acceptor invasion-initiated mechanism. Transfer is initiated at internal regions of the homologous R sequence without requiring cleavage at the donor 5'-end. The acceptor invades at gaps created by reverse transcriptase-RNase H in the donor-cDNA hybrid. The fragmented donor is eventually strand-displaced by the acceptor, completing the transfer.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester, New York 14642, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.forename: Mini
authors.initials: M
authors.lastname: Balakrishnan
authors.forename: Bernard P
authors.initials: BP
authors.lastname: Roques
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bambara
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D054303:HIV Reverse Transcriptase / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D006868:Hydrolysis; D007700:Kinetics; D012367:RNA, Viral / Q000235:genetics; D016914:Ribonuclease H / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H