TRNA Annealing onto the HIV-1 Genome Studied by FRET Spectroscopy and Microscopy

Abstract

In the HIV life cycle, reverse transcription allows conversion of a single stranded genomic RNA into double stranded DNA, capable of integration. A prerequisite for the reverse transcription process is the formation of the initiation complex between the RNA genome and a host-cell tRNA that is used as a primer by reverse transcriptase. The nucleocapsid protein (NC) is involved in this process, which requires hybridization of the tRNA to the complementary primer binding site (PBS) sequence on the RNA genome. Besides the tRNA-PBS interaction, other interactions were proposed to play a role such as an 8-nucleotide motif in the U5 region of the untranslated leader RNA, named the primer activation signal (PAS). We investigate the tRNA annealing process and the presence of a tRNA-PAS interaction using fluorescence resonance energy transfer (FRET) spectroscopy and single molecule FRET microscopy. In our assay, fluorescent donor and acceptor molecules were covalently attached to an RNA template mimicking the region of the HIV-1 genome in which the PBS sequence is located. We observe better folding of the RNA molecules in the presence of NC than with heat annealing and a large change in conformation of the RNA molecule upon tRNA annealing. Our results give further evidence that tRNA also interacts with the viral RNA PAS motif.