EFFECT OF MUTATIONS DOWNSTREAM OF THE INTERNAL RIBOSOME ENTRY SITE ONINITIATION OF POLIOVIRUS PROTEIN-SYNTHESIS

Initiation of poliovirus translation is mediated by a large, structure d segment of the 5' nontranslated region known as the internal ribosom e entry site (IRES) and normally occurs 155 nucleotides (nt) downstrea m of the IRES at AUG(743) (the AUG at nucleotide 743). Functional AUG codons introduced at nt 611 or 614 reduced initiation at AUG(743) by 1 0 to 40% in vitro but had no effect on virus phenotype. To investigate the role of the nt 586-743 spacer in greater detail, four intervening termination codons were removed, and an additional AUG triplet at nt 683 was introduced by nucleotide substitution. Initiation at AUG(743) was reduced by only 50 to 80%, depending on the number of upstream ini tiation codons. Initiation at AUG(743) was also reduced following inse rtion of a stable hairpin at nt 630, but the reduction was modest in a n ascites carcinoma cell extract. Initiation was more frequent at AUG( 743) than at AUG(683) if mRNAs contained either an upstream initiation codon or the stable hairpin. These results suggested that not all ini tiation events at AUG(743) can be accounted for by a scanning-dependen t mechanism. Translation of bicistronic mRNAs in which the intercistro nic spacer contained nt 630 to 742 of the poliovirus 5' nontranslated region indicated that these residues are not able to act as an entry p oint for ribosomes independently of the IRES. Insertion of increasingl y longer sequences immediately downstream of the stable hairpin progre ssively reduced initiation at AUG(743) without affecting initiation at AUG(683). These results are discussed in terms of a model for initiat ion of poliovirus translation in which a complex RNA superstructure up stream of nt 586 promotes ribosome binding at an entry point determine d by specific downstream cis-acting elements.