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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



K Lacourciere, J T. Stivers, John Marino


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.
No. 19


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


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