Analysis of the interacting partners eIF4F and 3'-CITE required for Melon necrotic spot virus cap-independent translation.

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 + eIF4G _980-1159 (eIF4F _p20 ), but not with each subunit alone or with eIF4E + eIF4G _1003-1092 , suggesting binding either through interaction with eIF4E following a conformational change induced by its binding to eIF4G _980-1159 , or through a double interaction with eIF4E and eIF4G _980-1159 . Critical residues for this interaction reside in an internal bulge of Ma5TE, so that their mutation abolished binding to eIF4E + eIF4G _1003-1092 and 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.
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