title: In vitro evidence that the untranslated leader of the HIV-1 genome is an RNA checkpoint that regulates multiple functions through conformational changes.
abstract: The HIV-1 RNA genome forms dimers through base pairing of a palindromic 6-mer sequence that is exposed in the loop of the dimer initiation signal (DIS) hairpin structure (loop-loop kissing). The HIV-1 leader RNA can adopt a secondary structure conformation that is not able to dimerize because the DIS hairpin is not folded. Instead, this DIS motif is base-paired in a long distance interaction (LDI) that extends the stem of the primer-binding site domain. In this study, we show that targeting of the LDI by either antisense oligonucleotides or specific mutations can induce the conformational switch to a branched multiple hairpin (BMH) structure, and this LDI-to-BMH switch coincides with increased RNA dimerization. Another interesting finding is that the extended LDI stem can resist a certain level of destabilization, indicating that a buffer is created to prevent a premature conformational switch and early dimerization. Because the tRNA(Lys3) primer for reverse transcription anneals to multiple sequence elements of the HIV-1 leader RNA, including sequences in the LDI stem, we tested whether tRNA-annealing can destabilize the LDI stem such that RNA dimerization is triggered. Using a combination of stem-destabilizing approaches, we indeed measured a small but significant effect of tRNA-annealing on the ability of the RNA template to form dimers. This observation suggests that HIV-1 RNA can act as a checkpoint to control and coordinate different leader functions through conformational switches. This in vitro result should be verified in subsequent in vivo studies with HIV-infected cells.
authors.affiliation: Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. b.berkhout@amc.uva.nl
authors.forename: Ben
authors.initials: B
authors.lastname: Berkhout
authors.forename: Marcel
authors.initials: M
authors.lastname: Ooms
authors.forename: Nancy
authors.initials: N
authors.lastname: Beerens
authors.forename: Hendrik
authors.initials: H
authors.lastname: Huthoff
authors.forename: Edwin
authors.initials: E
authors.lastname: Southern
authors.forename: Koen
authors.initials: K
authors.lastname: Verhoef
mesh_terms: D020121:5' Untranslated Regions; D001483:Base Sequence; D019281:Dimerization; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis; D016376:Oligonucleotides, Antisense / Q000378:metabolism / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism; D012357:RNA, Transfer, Lys / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D020121:5' Untranslated Regions; D016376:Oligonucleotides, Antisense; D012357:RNA, Transfer, Lys; D012313:RNA