Structural, thermodynamic and functional investigations of the HIV and SIV frameshift site RNAs: Identification and characterization of small molecule ligands that affect (-1) translational frameshifting

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) require a (-1) translational frameshift event to properly synthesize all of the proteins required for viral replication. The frameshift mechanism is dependent upon a seven nucleotide slippery sequence (UUUUUUA) and a downstream RNA structure. Here, I report the functional, structural and thermodynamic characterization of the SW frameshift site RNA. Translational frameshift assays confirmed that this RNA sequence promoted efficient frameshifting in vitro. The structure of this 34-nucleotide RNA, as determined by NMR, was an eleven base-pair A-form helix capped by a highly-ordered 12-nucleotide loop containing a sheared G-A pair, cross-strand adenine stacking, two G-C base-pairs, and a novel CCC triloop turn. This structure and its high degree of thermostability (Tm(stem) = 84°C, T m(loop) = 66°C) preclude formation of alternate RNA tertiary structures such as pseudoknots. Sequence conservation and modeling studies suggest that the HIV type 2 (HIV-2) RNA forms the same structure.;Frameshifting in HIV type 1 (HIV-1), HIV-2 and SIV occurs with a frequency of 5-10%, and increasing or decreasing this frequency may result in a decrease in viral replication. Here, I report the results of a high-throughput screen designed to find small molecules that bind to the HIV-1 frameshift site RNA and potentially affect frameshifting. Out of 34,500 compounds screened, 202 were identified as positive hits. One of these compounds, doxorubicin, binds the HIV-1 RNA with low micromolar affinity (Kd = 2.8 microM). This binding was confirmed and localized to the RNA using NMR. Further analysis revealed that this compound increased the RNA stability by approximately 5°C, and decreased translational frameshifting by 28% (+/- 14%), as measured in vitro.;The interactions between the HIV-1 frameshift site RNA and a small molecule ligand (DB213) found to increase frameshifting were structurally and thermodynamically investivated. NMR structural studies revealed that DB213 binds the RNA in the major groove of the upper stem-loop with weak affinity (K d ∼ 300 microM). Addition of DB213 may also increase the stability of the RNA and alter the conformation of the conserved GGA bulge, as observed by NMR.