Dataset for: G3BP1 interacts directly with the FMDV IRES and negatively regulates translation GalanAlfonso LozanoGloria PiñeiroDavid Martínez-SalasEncarnación 2017 RNA-protein interactions play a pivotal role in the function of picornavirus internal ribosome entry site (IRES) elements. Here we analysed the impact of Ras GTPase SH3 domain binding protein 1 (G3BP1) in the IRES activity of foot-and-mouth disease virus (FMDV). We found that G3BP1 interacts directly with three distinct sequences of the IRES element using RNA electrophoretic mobility-shift assays. Analysis of the interaction with domain 5 indicated that the G3BP1 binding-site is placed at the single-stranded region although it allows large sequence heterogeneity and the hairpin located upstream of this region enhances retarded complex formation. In addition, G3BP1 interacts directly with the polypyrimidine tract-binding protein (PTB) and the translation initiation factor 4B (eIF4B) through the C-terminal region. Moreover, G3BP1 is cleaved during FMDV infection yielding two fragments, Ct-G3BP1 and Nt-G3BP1. Both fragments inhibit cap- and IRES-dependent translation, but the Ct-G3BP1 fragment shows a stronger effect on IRES-dependent translation. Assembly of complexes with G3BP1 results in a significantly reduced local flexibility of the IRES element, consistent with the negative effect of this protein. Our results highlight the IRES-binding capacity of G3BP1 and illustrate its function as a translation inhibitor.