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

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 alpha-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 nonperturbing 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 (K(D) = 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 unambiguously detected. The first site is noninhibitory to Rev binding (K(D) = 0.24 +/- 0.040 microM), the second site inhibited Rev binding in a competitive fashion (K(D) = 1. 8 +/- 0.8 microM), and the third much weaker site (or sites) is attributed to nonspecific binding (K(D) >/= 40 microM). 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 ternary complexes, and that this site is located in the lower stem region of RRE.

Stereospecificity of short Rev-derived peptide interactions with RRE IIB RNA

“…Increased cleavage at the upper stem region suggests a significant conformation change upon neomycin B binding. This result is in good accordance with previously published data (Lacourciere et al 2000).…”

“…A short ␣-helical peptide corresponding to the arginine-rich RNA-binding domain of Rev binds with high affinity to the stem-loop IIB of RRE, providing a good model for the initial process of Rev monomer recognition of the RRE stem-loop (Kjems et al 1991(Kjems et al , 1992Tan et al 1993;Tan and Frankel 1994;Frankel and Young 1998). Rev peptide binding to RRE occurs in two steps, an initial encounter, followed by isomerization of the RNA (Lacourciere et al 2000). A variety of structural and binding studies are consistent with the notion that the interactions between Rev protein, or Rev peptide, and RRE RNA exhibit elements of specificity (Kjems et al 1992).…”

“…In all of the binding experiments performed, a previously described fluorescence anisotropy methodology was utilized (Wang et al 1997;Hamasaki and Rando 1998, Lacourciere 2000, Haddad et al 2002. In this method, the increase in fluorescence anisotropy of a fluorescent ligand is measured as a function of ligand-RNA binding (Hamasaki and Rando 1998).…”

Designed Arginine-Rich RNA-Binding Peptides with Picomolar Affinity

“…Experiments were conducted following the procedures of Lacourciere et al 4 Measurements were performed at 20º C under standard buffer conditions (20 mM Tris-OAc, 50 mM KOAc, pH 7.5) using a stop-flow device from Applied Photophysics (Surray, U.K.) in twosyringe mode. Fluorescence excitation was performed at 310 nm and emmision was measured with a filter cutoff > 360 nm.…”

Synthesis and site-specific incorporation of a simple fluorescent pyrimidine

“…Many biophysical assays have taken advantage of this feature because base flipping is a rather common event in biologically relevant nucleic acids. Selected examples include: (i) 'real-time' monitoring of the hammerhead ribozyme folding, cleavage and inhibition 34,35 , (ii) evaluating the dimerization and isomerization of the HIV-1 dimerization initiation site (DIS) 36 , (iii) examining RNA-protein interactions such as the HIV RRE-Rev binding 37,38 , and (iv) studying the binding of aminoglycoside antibiotics to the bacterial decoding site, known as the A-site 39,40 .…”