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Mechanism of Neomycin and Rev Peptide Binding to the Rev Responsive Element of HIV-1 as Determined by Fluorescence and NMR Spectroscopy

Published

Author(s)

K Lacourciere, J T. Stivers, John Marino

Abstract

Rev is an essential HIV-1 regulatory protein that binds the Rev Responsive Element (RRE) within the env gene of the HIV-1 RNA genome and is involved in transport of unspliced or partially spliced viral mRNA from the cell nucleus to the cytoplasm. Previous studies have shown that a short α-helical peptide derived from Rev (Rev 34-50), and a truncated form of the RRE sequence, provide a useful in vitro system to study this interaction while still preserving the essential aspects of the native complex. We have selectively incorporated the fluorescent probe, 2-aminopurine-2'-O-methylriboside (2-AP), into the RRE sequence in non-perturbing positions (A68 and U72) such that the binding of both Rev peptide and aminoglycoside ligands could be characterized directly by fluorescence methods. Rev peptide binding to the RRE-72AP variant resulted in a 2-fold fluorescence increase that provided a useful signal to monitor this binding interaction (KD = 20 7 nM). Using stopped-flow kinetic measurements, we have shown that specific Rev peptide binding occurs by a two-step process involving diffusion-controlled encounter, followed by isomerization of the RNA. Using the RRE-68AP and 72AP constructs three classes of binding sites for the aminoglycoside neomycin were detected. The first site is non-inhibitory to Rev binding (KD = 0.24 0.040 M), the second site inhibited Rev binding in a competitive fashion (KD = 1.8 0.8 M), and the third much weaker site (or sites) is attributed to nonspecific binding (KD ≥ 40 M). Complementary NMR measurements have shown that neomycin forms both a specific binary complex with RRE and a specific ternary complex with RRE and Rev. NMR data further suggest that neomycin occupies a similar high-affinity binding site in both the binary and tenary complexes, and that this site is located in the lower stem region of RRE.
Citation
Biochemistry
Volume
39
Issue
No. 19

Keywords

2-aminopurine, fluorescence assay, HIV-1 Rev responsive element, nuclear magnetic resonance spectroscopy, stopped-Flow kinetics

Citation

Lacourciere, K. , Stivers, J. and Marino, J. (2000), Mechanism of Neomycin and Rev Peptide Binding to the Rev Responsive Element of HIV-1 as Determined by Fluorescence and NMR Spectroscopy, Biochemistry (Accessed April 19, 2024)
Created May 15, 2000, Updated October 12, 2021