title: Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation.
abstract: The internal ribosome entry site (IRES) element located at the 5'untranslated genomic region of various RNA viruses mediates cap-independent initiation of translation. Picornavirus IRES activity is highly dependent on both its structural organization and its interaction with host factors. Small molecules able to interfere with RNA function are valuable candidates for antiviral agents. Here we show that a small molecule based on benzimidazole (IRAB) inhibited foot-and-mouth disease virus (FMDV) IRES-dependent protein synthesis in cells transfected with infectious RNA leading to a decrease of the virus titer, which was higher than that induced by a structurally related benzimidazole derivative. Interestingly, IRAB preferentially inhibited IRES-dependent translation in cell free systems in a dose-dependent manner. RNA structural analysis by SHAPE demonstrated an increased local flexibility of the IRES structure upon incubation with IRAB, which affected 3 stem-loops (SL) of domain 3. Fluorescence binding assays conducted with individual aminopurine-labeled oligoribonucleotides indicated that the SL3A binds IRAB (EC50 18 μM). Taken together, the results derived from SHAPE reactivity and fluorescence binding assays suggested that the target site of IRAB within the FMDV IRES might be a folded RNA structure that involves the entire apical region of domain 3. Our data suggest that the conformational changes induced by this compound on a specific region of the IRES structure which is essential for its activity is, at least in part, responsible for the reduced IRES efficiency observed in cell free lysates and, particularly, in RNA-transfected cells.
authors.affiliation: a Centro de Biología Molecular Severo Ochoa; CSIC-UAM; Madrid , Spain.
authors.forename: Gloria
authors.initials: G
authors.lastname: Lozano
authors.forename: Alejandro
authors.initials: A
authors.lastname: Trapote
authors.forename: Jorge
authors.initials: J
authors.lastname: Ramajo
authors.forename: Xavier
authors.initials: X
authors.lastname: Elduque
authors.forename: Anna
authors.initials: A
authors.lastname: Grandas
authors.forename: Jordi
authors.initials: J
authors.lastname: Robles
authors.forename: Enrique
authors.initials: E
authors.lastname: Pedroso
authors.forename: Encarnación
authors.initials: E
authors.lastname: Martínez-Salas
mesh_terms: D001483:Base Sequence; D001562:Benzimidazoles / Q000737:chemistry / Q000494:pharmacology; D002474:Cell-Free System; D005453:Fluorescence; D029544:Foot-and-Mouth Disease Virus / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D016679:Genome, Viral; D017665:Hydroxyl Radical / Q000378:metabolism; D000067556:Internal Ribosome Entry Sites / Q000235:genetics*; D008024:Ligands; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis / Q000187:drug effects; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics*; D012997:Solvents
chemical_list: D001562:Benzimidazoles; D000067556:Internal Ribosome Entry Sites; D008024:Ligands; D012367:RNA, Viral; D012997:Solvents; D017665:Hydroxyl Radical; C027391:2-aminobenzimidazole
keywords: 2-aminobenzimidazole; IRES elements; RNA ligands; RNA structure; SHAPE probing; antiviral molecules; fluorescence binding assay; picornavirus; translation initiation