title: Structural features in the HIV-1 repeat region facilitate strand transfer during reverse transcription.
abstract: Two obligatory DNA strand transfers take place during reverse transcription of a retroviral RNA genome. The first strand transfer is facilitated by terminal repeat (R) elements in the viral genome. This strand-transfer reaction depends on base pairing between the cDNA of the 5'R and the 3'R. There is accumulating evidence that retroviral R regions contain features other than sequence complementarity that stimulate this critical nucleic acid hybridization step. The R region of the human immunodeficiency virus type 1 (HIV-1) is relatively extended (97 nt) and encodes two well-conserved stem-loop structures, the TAR and poly(A) hairpins. The role of these motifs was studied in an in vitro strand-transfer assay with two separate templates, the 5'R donor and the 3'R acceptor, and mutants thereof. The results indicate that the upper part of the TAR hairpin structure in the 5'R donor is critical for efficient strand transfer. This seems to pose a paradox, as the 5'R template is degraded by RNase H before strand transfer occurs. We propose that it is not the RNA hairpin motif in the 5'R donor, but rather the antisense motif in the ssDNA copy, which can also fold a hairpin structure, that is critical for strand transfer. Mutation of the loop sequence in the TAR hairpin of the donor RNA, which is copied in the loop of the cDNA hairpin, reduces the transfer efficiency more than fivefold. It is proposed that the natural strand-transfer reaction is enhanced by interaction of the anti-TAR ssDNA hairpin with the TAR hairpin in the 3'R acceptor. Base pairing can occur between the complementary loops ("loop-loop kissing"), and strand transfer is completed by the subsequent formation of an extended RNA-cDNA duplex.
authors.affiliation: Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, The Netherlands. b.berkhout@amc.uva.nl
authors.forename: B
authors.initials: B
authors.lastname: Berkhout
authors.forename: N L
authors.initials: NL
authors.lastname: Vastenhouw
authors.forename: B I
authors.initials: BI
authors.lastname: Klasens
authors.forename: H
authors.initials: H
authors.lastname: Huthoff
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D018076:DNA, Complementary / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011061:Poly A; D012313:RNA / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016914:Ribonuclease H / Q000378:metabolism; D013696:Temperature; D014158:Transcription, Genetic
chemical_list: D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D011061:Poly A; D012313:RNA; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H