IRES interaction with translation initiation factors: Functional characterization of novel RNA contacts with eIF3, eIF4B, and eIF4GII

Translation initiation promoted by picornavirus internal ribosome entry sit e (IRES) elements is dependent on the association of specific IRES sequence s to the initiation factor eIF4G. However the RNA determinants interacting with other components of the translational machinery are still unknown. In this study, we have identified novel RNA-protein interactions between the f oot-and-mouth disease virus (FMDV) IRES and three translation initiation fa ctors. A doublet of 116/110 kDa that crosslinked to the FMDV IRES is a comp onent of eIF3. We show here that domain 5 holds the preferential binding si te for eIF3, although this complex initiation factor can establish multiple contacts with the IRES structure. We have also identified the phylogenetic ally conserved hairpin of domain 5 as the RNA motif responsible for eIF4B i nteraction. Mutation of this stem-loop structure abrogated eIF4B, but not e IF3, binding to the IRES. Remarkably, IRES mutants severely affected in the ir interaction with eIF4B showed a mild reduction in IRES activity when tes ted in the context of a bicistronic expression vector in transfected cells. Finally, we provide evidence of the interaction of eIF4GII with FMDV IRES, the RNA determinants for this interaction being shared with its functional homolog eIF4GI. The FMDV Lb protease generated a C-terminal fragment of eI F4GII that binds to the IRES as efficiently as the intact protein. Competit ion experiments showed that titration of eIF4B or p110/116 interaction with the FMDV IRES required a large excess of competitor relative to eIF4G, str ongly suggesting that eIF4G-IRES interaction is a limiting factor to titrat e the IRES. Comparative analysis of the activity of IRES mutants affected i n domains 4 and 5 regarding their pattern of RNA-protein complex formation demonstrates that while binding of eIF4B with the FMDV IRES is dispensable, interaction of eIF4G is a central feature of the activity of this element.