The properties of chimeric picornavirus IRESes show that discrimination between internal translation initiation sites is influenced by the identity of the IRES and not just the context of the AUG codon

The internal ribosome entry segment (IRES) of picornaviruses consists of si milar to 450 nt of 5'-untranslated region, terminating at the 3' end;with a n similar to 25 nt element consisting of an absolutely conserved UUUC motif followed by a more variable pyrimidine-rich tract and G-poor spacer, and f inally an AUG triplet, which is considered to be the actual ribosome entry site. Events following entry at this site differ among picornaviruses: in e ncephalomyocarditis virus (EMCV) virtually all ribosomes initiate translati on at this-site (AUG-11); in foot-and-mouth-disease virus (FMDV), one-third of the ribosomes initiate at this AUG (the Lab site), and the rest at the next AUG 84 nt downstream (Lb site); and in poliovirus (PV), the AUG at the 3' end of the IRES (at nt 586 in PV type 1) is considered to be a silent e ntry site, with all ribosomes initiating translation at the next AUG downst ream (nt 743). To investigate what determines this different behavior, chim eras were constructed with a crossover at the conserved UUUC motif: the bod y of the IRES, the sequences upstream of,this UUUC motif, was derived from one species, and the downstream sequences from another. When the body of th e FMDV or PV IRESes was replaced by that of EMCV, there was a marked increa se in the absolute and relative frequency of initiation at the upstream AUG , the Lab site of FMDV and (586)AUG Of PV, respectively. In contrast, when the body of the EMCV IRES was replaced by that of PV, Initiation occurred w ith no preference at three AUGs: the normal site (AUG-11), AUG-10 situated 8 nt upstream, and AUG-12, which is 12 nt downstream. Thus although the con text of the AUG at the 3' end of the IRES may influence: initiation frequen cy at this site, as was shown by improving the context, of (586)AUG of PV, the behavior of the ribosome is also highly dependent on the nature of the upstream IRES. Delivery of the ribosome to this AUG in an initiation-compet ent manner is particularly efficient and accurate with the EMCV IRES.