Fret Study of the Binding of HIV-1 RRE RNA to Rev Peptide

Abstract

In the viral life cycle of HIV-1, binding of the Rev protein to the Rev Response Element (RRE) RNA to form the Rev-RRE complex is essential for viral RNA to be exported from the cell nucleus. This interaction has been suggested as a new therapeutic target, which makes an accurate and detailed understanding of Rev-RRE interactions essential. The m6A post-transcriptional modification is known to regulate gene expression and modulate infectivity of viral RNAs. The binding region of the RRE RNA has two known, conserved m6A methylation sites, but the effect and significance of these modifications is an active area of investigation. This study measures binding of an Alexa Fluor 594-labeled RNA hairpin that contains the high-affinity Rev-binding site to an Alexa Fluor 488-labeled peptide that contains the 17 amino acid binding region of the Rev protein as a model of the primary RRE-Rev binding site. Binding of Rev peptides to immobilized RNA hairpins is measured via single molecule fluorescence resonance energy transfer (FRET) spectroscopy. Single molecule measurements can access information hidden by averaging in bulk measurements, especially heterogeneities, such as multiple conformations, short lived intermediates, or varied kinetic rates. Statistical analysis and a kinetic model enable extraction of binding and dissociation rates for the Rev protein. The eventual goal of this study is a comparison of the binding rates for methylated and unmethylated variants of the RRE RNA, which will reveal whether m6A methylation significantly impacts the Rev-RRE binding rate at the primary binding site.