NOTICE: Due to a lapse in annual appropriations, most of this website is not being updated. Learn more.
Form submissions will still be accepted but will not receive responses at this time. Sections of this site for programs using non-appropriated funds (such as NVLAP) or those that are excepted from the shutdown (such as CHIPS and NVD) will continue to be updated.
An official website of the United States government
Here’s how you know
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
Secure .gov websites use HTTPS
A lock (
) or https:// means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.
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.
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 October 7, 2025)