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Proflavine Acts as a Rev Inhibitor by Targeting the High-Affinity Rev Binding Site of the Rev-Responsive Element of HIV-1



E S. DeJong, C Chang, M K. Gilson, John Marino


Rev is an essential regulatory HIV-1 protein that binds the Rev Responsive Element (RRE) within the env gene of the HIV-1 RNA genome, activating the switch between viral latency and active viral replication. Previously, we have shown that selective incorporation of the fluorescent probe, 2-aminopurine (2-AP), into a truncated form of the RRE sequence (RRE-IIB) allowed the binding of an arginine-rich peptide derived from Rev and aminoglycosides to be characterized directly by fluorescence methods. Using these fluorescence as well as nuclear magnetic resonance (NMR) methods, proflavine has been identified, through a limited screen of selected small heterocyclic compounds, as a specific and high-affinity RRE-IIB binder which inhibits the interaction of Rev peptide with RRE-IIB. Direct and competitive 2-AP fluorescence binding assays reveal that there are at least two classes of proflavine binding sites on RRE-IIB: a high-affinity site that competes with Rev peptide for binding to RRE-IIB (KD 0.1 0.05 mM) and a weaker binding site(s) (KD 1.1 0.05 mM). Titrations of RRE-IIB with proflavine, monitored using 1H NMR, demonstrate that proflavine binds with a 2:1 [proflavine:RRE-IIB] stoichiometry and NOEs observed in the NOESY spectrum of the 2:1 proflavineoRRE-IIB complex indicate that the two proflavine molecules bind specifically and in close proximity to each other within a single binding site. NOESY data further indicate that formation of the 2:1 proflavineoRRE-IIB complex stabilizes base pairing and stacking within the internal purine-rich bulge of RRE-IIB in a manner analogous to what has been observed in the Rev peptideoRRE-IIB complex. The observation that proflavine competes with Rev for binding to RRE-IIB by binding as a dimer to a single high-affinity site opens the possibility for rational drug design based on linking and modifying it and related compounds.
No. 26


2-aminopurine, fluorescence, HIV-1, NMR, Proflavine, RRE, structure


DeJong, E. , Chang, C. , Gilson, M. and Marino, J. (2003), Proflavine Acts as a Rev Inhibitor by Targeting the High-Affinity Rev Binding Site of the Rev-Responsive Element of HIV-1, Biochemistry (Accessed April 24, 2024)
Created July 7, 2003, Updated October 12, 2021