Abrogation of CTL epitope processing by single amino acid substitution flanking the C-terminal proteasome cleavage site

CTL directed against the Moloney murine leukemia virus (MuLV) epitope SSWDF ITV recognize Moloney MuLV-induced tumor cells, but do not recognize cells transformed by the closely related Friend MuLV, The potential Friend MuLV e pitope has strong sequence homology with Moloney MuLV and only differs in o ne amino acid within the CTL epitope and one amino acid just outside the ep itope. We now show that failure to recognize Friend MuLV-transformed tumor cells is based on a defect in proteasome-mediated processing of the Friend epitope which is due to a single amino acid substitution (N --> D) immediat ely flanking the C-terminal anchor residue of the epitope, Proteasome-media ted digestion analysis of a synthetic 26-mer peptide derived from the Frien d sequence shows that cleavage takes place predominantly C-terminal of D, i nstead of V as is the case for the Moloney MuLV sequence. Therefore, the C terminus of the epitope is not properly generated. Epitope-containing pepti de fragments extended with an additional C-terminal D are not efficiently t ranslocated by TAP and do not show significant binding affinity to MHC clas s I-K-b molecules. Thus, a potential CTL epitope present in the Friend viru s sequence is not properly processed and presented because of a natural fla nking aspartic acid that obliterates the correct C-terminal cleavage site. This constitutes a novel way to subvert proteasome-mediated generation of p roper antigenic peptide fragments.