Cap-independent translation of encephalomyocarditis virus RNA: Structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein

Abstract

Translation of encephalomyocarditis virus (EMCV) mRNA occurs by ribosomal internal entry into the 5′-nontranslated region (5′ NTR) rather than by ribosomal scanning. The internal ribosomal entry site (IRES) in the EMCV 5′ NTR was determined by in vitro translation with RNAs that were generated by in vitro transcription of EMCV cDNAs containing serial deletions from either the 5′ or 3′ end of the EMCV 5′ NTR. Regions downstream of nucleotide 403 and upstream of nucleotide 811 of EMCV were required for efficient translation. Site-directed mutagenesis revealed that a stem-loop structure (400 nucleotides upstream of the initiation codon) was essential for IRES function. We discovered a 57-kD cellular protein whose specific interaction with this stem-loop appears to be prerequisite for IRES function. A pyrirnidine-rich stretch proximal to the initiation codon was also crucial for efficient translation of EMCV mRNA. We propose that ribosomes bind directly to the initiating AUG without scanning.