Role of the primer activation signal in tRNA annealing onto the HIV-1 genome studied by single-molecule FRET microscopy

Nancy Beerens, Mette D E Jepsen, Volodymyr Nechyporuk-Zloy, Asger C Krüger, Jean-Luc Darlix, Jørgen Kjems, Victoria Birkedal

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

Abstract

HIV-1 reverse transcription is primed by a cellular tRNAlys3 molecule that binds to the primer binding site (PBS) in the genomic RNA. An additional interaction between the tRNA molecule and the primer activation signal (PAS) is thought to regulate the initiation of reverse transcription. The mechanism of tRNA annealing onto the HIV-1 genome was examined using ensemble and single-molecule Förster Resonance Energy Transfer (FRET) assays, in which fluorescent donor and acceptor molecules were covalently attached to an RNA template mimicking the PBS region. The role of the viral nucleocapsid (NC) protein in tRNA annealing was studied. Both heat annealing and NC-mediated annealing of tRNAlys3 were found to change the FRET efficiency, and thus the conformation of the HIV-1 RNA template. The results are consistent with a model for tRNA annealing that involves an interaction between the tRNAlys3 molecule and the PAS sequence in the HIV-1 genome. The NC protein may stimulate the interaction of the tRNA molecule with the PAS, thereby regulating the initiation of reverse transcription.
Original languageEnglish
JournalRNA
Volume19
Issue4
Pages (from-to)517-26
Number of pages10
ISSN1355-8382
DOIs
Publication statusPublished - Apr 2013

Keywords

  • Fluorescence Resonance Energy Transfer
  • Genome, Viral
  • HIV-1
  • Host-Pathogen Interactions
  • Humans
  • Nucleic Acid Conformation
  • RNA, Transfer, Lys
  • RNA, Viral
  • Reverse Transcription

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