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

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.

Heterogeneous structures formed by conserved RNA sequences within the HIV reverse transcription initiation site

“…This method has been shown to produce vRNA/tRNA complexes similar to those formed through nucleocapsid-mediated annealing (Brule et al 2002;Iwatani et al 2003;Sleiman et al 2012;Beerens et al 2013). The resulting heat-annealed reaction yielded several higher order species upon native PAGE analysis.…”

“…If an acceptor dye with an absorbance spectrum that overlaps the emission spectrum of the donor dye is in close proximity (<80 Å) to the donor dye, energy can be transferred from the donor dye to the acceptor dye through dipole-dipole interactions, leading to acceptor dye emission; the efficiency of this transfer is proportional to 1/r 6 , where r is the interdye distance. By observing FRET from a collection of single RNA molecules labeled at appropriate positions with donor and acceptor dyes, the conformational dynamics of the RNA can be delineated directly (Zhuang et al 2000;Russell et al 2002;Liu et al 2005;Solomatin et al 2010;Beerens et al 2013). We designed a labeling scheme for the 77-mer-tRNA Lys 3 complex that directly tested the presence of the potential PAS interactions.…”

“…If the intramolecular anti-PAS interaction occurs, the donor Cy3 dye will be occluded from the tRNA anti-PAS site and is at a greater distance (>40 Å) from Heterogeneous RNA structures in HIV www.rnajournal.org 1693 the acceptor Cy5 dye leading to less efficient energy transfer (low FRET). Previous work used smFRET to analyze structural rearrangements within the initiation complex; however, this labeling scheme did not allow the direct observation of the intermolecular PAS interaction (Beerens et al 2013).…”

“…Three key regions of the RNA-RNA interaction have been proposed to guide the formation of the initiation complex: the primer-binding site (PBS), A-rich loop (ARL), and primer activation site (PAS) interactions. The roles of these RNA-pairing interactions in regulating initiation kinetics are supported by mutagenesis and biochemical experiments (Aiyar et al 1992;Essink et al 1995;Puglisi and Puglisi 1998;Beerens and Berkhout 2002a,b;Rigourd et al 2003;Abbink et al 2004;Ooms et al 2007;Beerens et al 2013;Jones et al 2013;Sleiman et al 2013;Seif et al 2015).…”

The Design of RNA Binders: Targeting the HIV Replication Cycle as a Case Study

“…In these studies, the tRNA Lys 3 /PAS interaction appears to be dynamic and stimulated by the nucleocapsid protein. [16,17] The crystal structure of tRNA Lys3 has been solved by X-ray crystallography. [18] It adopts the well-known L-shape structure of tRNAs (Figure 2).…”