RIBOSOMAL SCANNING PAST THE PRIMARY INITIATION CODON AS A MECHANISM FOR EXPRESSION OF CTL EPITOPES ENCODED IN ALTERNATIVE READING FRAMES

An increasing amount of evidence has shown that epitopes restricted to MHC class I molecules and recognized by CTL need not be encoded in a primary open reading frame (ORF). Such epitopes have been demonstrated after stop codons, in alternative reading frames (RF) and within intr ons. We have used a series of frameshifts (FS) introduced into the Inf luenza A/PR/8/34 nucleoprotein (NP) gene to confirm the previous in vi tro observations of cryptic epitope expression, and show that they are sufficiently expressed to prime immune responses in vivo. This presen tation is not due to sub-dominant epitopes, transcription from cryptic promoters beyond the point of the FS, or internal initiation of trans lation. By introducing additional mutations to the construct exhibitin g the most potent presentation, we have identified initiation codon re adthrough (termed scanthrough here, where the scanning ribosome bypass es the conventional initiation codon, initiating translation further d ownstream) as the likely mechanism of epitope production. Further muta tional analysis demonstrated that, while it should operate during the expression of wild-type (WT) protein, scanthrough does not provide a m ajor source of processing substrate in our system. These findings sugg est (i) that the full array of self- and pathogen-derived epitopes ava ilable during thymic selection and infection has not been fully apprec iated and (ii) that cryptic epitope expression should be considered wh en the specificity of a CTL response cannot be identified or in therap eutic situations when conventional CTL targets are limited, as may be the case with latent viral infections and transformed cells. Finally, initiation codon readthrough provides a plausible explanation for the presentation of exocytic proteins by MHC class I molecules.