INITIATION CODON SCANTHROUGH VERSUS TERMINATION CODON READTHROUGH DEMONSTRATES STRONG POTENTIAL FOR MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I-RESTRICTED CRYPTIC EPITOPE EXPRESSION

Accumulating evidence shows that the repertoire of major histocompatib ility complex class I-restricted epitopes extends beyond conventional translation reading frames. Previously, we reported that scanthrough t ranslation, where the initiating AUG of a primary open reading frame i s bypassed, is most likely to account for the presentation of cryptic epitopes from alternative reading frames within the influenza A PR/8/3 4 nucleoprotein gene. Here, we confirm and extend these findings using an epitope cassette construct that features two well-defined CD8(+) T cell (T-CD8+) epitopes in alternative reading frames, each preceded b y a single start codon. Expression of one epitope depends on scanning of the ribosome over the first AUG with translation initiation occurri ng at the second AUG. We find that scanthrough translation has great p otency in our system, with its impact being modulated, as predicted, b y the base composition surrounding the first initiation codon, the num ber of start codons preceding the point of alternate reading frame ini tiation, and the efficiency with which the epitope itself is generated . Additionally, we investigated the efficiency of eukaryotic translati on termination codons, to assess codon readthrough as a mechanism for cryptic epitope expression from 3' untranslated regions. In contrast w ith initiation codons, eukaryotic stop codons appear to be highly effi cient at preventing expression of epitopes encoded in 3' untranslated regions, suggesting that 3' untranslated regions are not a common sour ce of cryptic epitope substrate. We conclude that scanthrough is a pow erful mechanism for the expression of epitopes encoded in upstream alt ernative open reading frames that may contribute significantly to T-CD 8+ responses and to tolerance induction.