Involvement of the aphthovirus RNA region located between the two functional AUGs in start codon selection

Initiation of translation in picornavirus RNAs occurs internally, mediated by an element termed internal ribosome entry site (IRES). In the aphthoviru s RNA, the IRES element directs translation initiation at two In-frame AUGs separated by 84 nucleotides. We have found that bicistronic constructs tha t contained the IRES element followed by the fragment including the aphthov irus start codons in front of the second gene mimicked the translation init iation pattern of viral RNA observed in infected cells. In those constructs , the frequency of initiation at the first AUG was increased by a sequence context that resembled the favorable consensus for cap-dependent translatio n, although Initiation at the second site was always preferred. In addition , we have found that initiation at the second start codon was not diminishe d under conditions in which the first initiation codon was blocked by antis ense oligonucleotide interference. Interestingly, mutations that positioned the second AUG out-of-frame with the first AUG did not interfere with the frequency of initiation at the second one. On the contrary, IRES-dependent translation initiation in bicistronic constructs lacking the sequences pres ent between functional AUGs in the viral RNA was sensitive to the presence of out-of-frame initiator codons and hairpins in the spacer region. This re markable difference in start codon recognition was due to the nucleotide co mposition of the RNA that separated the IRES from the initiator codon. Thus our results indicate that the region located in the aphthovirus RNA betwee n functional AUGs is involved in start codon recognition, strongly favoring selection of the second start AUG as the main initiator codon. (C) 1999 Ac ademic Press.