Abstract
The regulator of expression of virion (Rev) protein binds specifically to the Rev-responsive element (RRE) RNA in order to regulate the expression of the human immunodeficiency virus (HIV)-1 genes. Fluorescence indicator displacement assays have been used to identify ligands that can inhibit the Rev-RRE interaction; however, the small fluorescence indicators cannot fully replace the Rev peptide or protein. As a result, a single rhodamine B labeled Rev (RB-Rev) model peptide was utilized in this study to develop a direct and efficient Rev-RRE inhibitor screening model. Due to photon-induced electron transfer quenching of the tryptophan residue on the RB fluorophore, the fluorescence of RB in Rev was weakened and could be dramatically reactivated by interaction with RRE RNA in ammonium acetate buffer (approximately six times). The interaction could reduce the electron transfer between tryptophan and RB, and RRE could also increase RB fluorescence. The inhibitor screening model was evaluated using three known positive Rev-RRE inhibitors, namely, proflavin, 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine (ICR 191), and neomycin, as well as a negative drug, arginine. With the addition of the positive drugs, the fluorescence of the Rev-RRE decreased, indicating the displacement of RB-Rev. This was confirmed using atomic force microscopy (AFM) and the fluorescence was essentially unaffected by the addition of arginine. The results demonstrated that RB-Rev can be used as a fluorescent probe for recognizing small ligands that target RRE RNA. The Rev-RRE inhibitor screening model offers a novel approach to evaluating and identifying long-acting Rev inhibitors.