Footprinting, circular dichroism and UV melting studies on neomycin B binding to the packaging region of human immunodeficiency virus type-1 RNA

Abstract

We have studied the binding of neomycin to a 171mer RNA (psi-RNA) from the packaging region of the LAI strain of human immunodeficiency virus type-1, HIV-1 (LAI). The RNase I footprinting studies reveal that the primary binding site for the drug is in stem-loop 1, which contains the dimer initiation site of HIV-1. Loading this site with neomycin causes a structural change in the RNA, allowing nucleotides in the neighboring stem-loop 2 to participate in the drug site. Drug binding to secondary sites induces structural changes in other stem-loops of the RNA. Footprinting plots, showing cutting at a site as a function of drug concentration, were analyzed using a two-state model to obtain relative site-specific binding constants. Circular dichroism measurements show that neomycin binding to psi-RNA changes the intensity of the strong negative CD band at 208 nm, confirming that neomycin induces structural changes. Melting studies of the RNA showed melting transitions in the absence of drug at 28.2, 37.2, 47.4, 55.5 and 60.8 degrees C. Only the first two were affected by drug binding, the reason for this being explained by our analysis.

HIV-1 RNA Dimerization Initiation Site Is Structurally Similar to the Ribosomal A Site and Binds Aminoglycoside Antibiotics

“…Recently, neomycin was reported to bind to a monomeric DIS-containing 176-nucleotide fragment corresponding to the encapsidation region of subtype B HIV-1 genomic RNA (36). The latter study seems to contradict our present results in which we show that neomycin does not bind either to a monomeric DIS mutant (Fig.…”

NCp7: targeting a multitask protein for next-generation anti-HIV drug development part 2. Noncovalent inhibitors and nucleic acid binders

“…Aminoglycosides, especially neomycin B (20 Fig. 4), paromomycin and tobramycin, were found to interact with the C-site of HIV-1 RNA [43][44][45]. Within the packaging site, aminoglycosides were thought to bind close to SL1 because of its striking structural similarity with the dimeric form of the prokaryotic ribosomal aminoacyl-tRNA site [46].…”

Aminoglycoside antibiotics: old drugs and new therapeutic approaches

“…Following observations of aminoglycoside binding to 1848 T. Hermann Aminoglycoside antibiotics these RNA domains, both HIV TAR and RRE have attracted efforts to discover non-glycosidic ligands as potential leads for the development of antiviral drugs [111][112][113][114][115][116][117][118]. Two other RNA motifs within the HIV genome that bind aminoglycosides are the dimerization initiation site (DIS) [119,120] and the packaging region (Y) [121]. Crystallographic structure determination and modeling studies suggest that aminoglycosides bind to the HIV DIS by docking to a metal ionbinding pocket of the RNA [120,122].…”