title: Interaction of the eIF4G initiation factor with the aphthovirus IRES is essential for internal translation initiation in vivo.
abstract: The strategies developed by internal ribosome entry site (IRES) elements to recruit the translational machinery are poorly understood. In this study we show that protein-RNA interaction of the eIF4G translation initiation factor with sequences of the foot-and-mouth disease virus (FMDV) IRES is a key determinant of internal translation initiation in living cells. Moreover, we have identified the nucleotides required for eIF4G-RNA functional interaction, using native proteins from FMDV-susceptible cell extracts. Substitutions in the conserved internal AA loop of the base of domain 4 led to strong impairment of both eIF4G-RNA interaction in vitro and IRES-dependent translation initiation in vivo. Conversely, substitutions in the vicinity of the internal AA loop that did not impair IRES activity retained their ability to interact with eIF4G. Direct UV-crosslinking as well as competition assays indicated that domains 1-2, 3, and 5 of the IRES did not contribute to this interaction. In agreement with this, binding to domain 4 alone was as efficient as to the full-length IRES. The C-terminal fragment of eIF4G, proteolytically processed by the FMDV Lb protease, was sufficient to interact with the IRES or to its domain 4 alone. Additionally, we show here that binding of the eIF4B initiation factor to the IRES required domain 5 sequences. Moreover, eIF4G-IRES interaction was detected in the absence of eIF4B-IRES binding, suggesting that both initiation factors interact with the 3' region of the IRES but use different residues. The strong correlation found between eIF4G-RNA interaction and IRES activity in transfected cells suggests that eIF4G acts as a linker to recruit the translational machinery in IRES-dependent initiation.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.
authors.forename: S
authors.initials: S
authors.lastname: López de Quinto
authors.forename: E
authors.initials: E
authors.lastname: Martínez-Salas
mesh_terms: D000818:Animals; D005537:Aphthovirus / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence; D006224:Cricetinae; D039603:Eukaryotic Initiation Factor-4G; D039642:Eukaryotic Initiation Factors; D015967:Gene Expression Regulation, Viral; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism*; D011485:Protein Binding; D023302:Protein Footprinting; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D039603:Eukaryotic Initiation Factor-4G; D039642:Eukaryotic Initiation Factors; D010448:Peptide Initiation Factors; D012367:RNA, Viral; D016601:RNA-Binding Proteins; C031799:eIF-4B