title: Analysis of the interacting partners eIF4F and 3'-CITE required for Melon necrotic spot virus cap-independent translation.
abstract: We have shown previously that the translation of Melon necrotic spot virus (MNSV, family Tombusviridae, genus Carmovirus) RNAs is controlled by a 3'-cap-independent translation enhancer (CITE), which is genetically and functionally dependent on the eukaryotic translation initiation factor (eIF) 4E. Here, we describe structural and functional analyses of the MNSV-Mα5 3'-CITE and its translation initiation factor partner. We first mapped the minimal 3'-CITE (Ma5TE) to a 45-nucleotide sequence, which consists of a stem-loop structure with two internal loops, similar to other I-shaped 3'-CITEs. UV crosslinking, followed by gel retardation assays, indicated that Ma5TE interacts in vitro with the complex formed by eIF4E + eIF4G980-1159(eIF4Fp20), but not with each subunit alone or with eIF4E + eIF4G1003-1092, suggesting binding either through interaction with eIF4E following a conformational change induced by its binding to eIF4G980-1159, or through a double interaction with eIF4E and eIF4G980-1159. Critical residues for this interaction reside in an internal bulge of Ma5TE, so that their mutation abolished binding to eIF4E + eIF4G1003-1092and cap-independent translation. We also developed an in vivo system to test the effect of mutations in eIF4E in Ma5TE-driven cap-independent translation, showing that conserved amino acids in a positively charged RNA-binding motif around amino acid position 228, implicated in eIF4E-eIF4G binding or belonging to the cap-recognition pocket, are essential for cap-independent translation controlled by Ma5TE, and thus for the multiplication of MNSV.
authors.affiliation: Centro de Edafología y Biología Aplicada del Segura (CEBAS) - CSIC, Apdo. correos 164, 30100 Espinardo, Murcia, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Miras
authors.affiliation: Centro de Edafología y Biología Aplicada del Segura (CEBAS) - CSIC, Apdo. correos 164, 30100 Espinardo, Murcia, Spain.
authors.forename: Verónica
authors.initials: V
authors.lastname: Truniger
authors.affiliation: Molecular Biology Institute of Barcelona (IBMB-CSIC), Parc Científic de Barcelona, Baldiri i Reixac 10, Barcelona, 08028, Spain.
authors.forename: Jordi
authors.initials: J
authors.lastname: Querol-Audi
authors.affiliation: Centro de Edafología y Biología Aplicada del Segura (CEBAS) - CSIC, Apdo. correos 164, 30100 Espinardo, Murcia, Spain.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Aranda
mesh_terms: D028464:Cucurbita / Q000378:metabolism / Q000821:virology; D039562:Eukaryotic Initiation Factor-4F / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000235:genetics; D019183:Tombusviridae / Q000235:genetics / Q000378:metabolism*
chemical_list: D039562:Eukaryotic Initiation Factor-4F; D012367:RNA, Viral
keywords: CITE; Carmovirus; RNA structure; Tombusviridae; cucurbit; translation enhancer; translation initiation