In vitro and in vivo identification of structural and sequence elements in the 5' untranslated region of Ectropis obliqua picorna-like virus required for internal initiation.
Translation initiation of encephalomyocarditis virus (EMCV) mRNA occurs by ribosomal entry into the 5' untranslated region. Internal ribosome binding to EMCV mRNA requires a viral cis-acting element, termed the internal ribosomal entry site (IRES), and cellular trans-acting factors. The polypyrimidine tract binding protein (PTB) has been identified as one such trans-acting factor required for EMCV IRES-dependent translation. Using a dicistronic mRNA and an in vitro translation system, we have identified cis-acting elements of the EMCV IRES required for IRES-dependent translation. The results identify several regions of the IRES that are required for efficient IRES-dependent translation, including the PTB binding site. Other regions of the IRES may act only as spacer sequences, analogous to the spacer sequences found in rhinovirus and enterovirus IRES elements, to link essential regions of the IRES together. The flexibility of one region of the EMCV IRES was demonstrated by an insertion of 125 nucleotides that had little effect on IRES function while the constraint imposed on another region was demonstrated by a 3-nucleotide deletion that nearly abolished IRES-dependent translation.