POINT MUTATION FLANKING A CTL EPITOPE ABLATES IN-VITRO AND IN-VIVO RECOGNITION OF A FULL-LENGTH VIRAL PROTEIN

CD8(+) T cells (TCD8+) recognize viral Ags as short peptides (epitopes ) displayed at the cell surface by MHC class I molecules. Using a pane l of recombinant vaccinia viruses, we show that single-point mutations flanking either side of an H-2K(d)-restricted epitope, residues 147-1 55, within full-length influenza nucleoprotein (NP) can impact, even a blate, presentation of that epitope, while having no effect on present ation of distal epitopes. The most severe blocking mutation (Ala to Pr o at position 146) did not inhibit NP147-155 presentation in the conte xt of a truncated minigene, implying that this peptide is not a functi onal processing intermediate. An amino-terminal proline replacement al so significantly reduced presentation of NP50-57 (H-2K(k) restricted), while the same mutation did not affect a third NP epitope. Thus, whil e trends in processing specificity may exist, the epitope itself contr ibutes to flanking sequence effects. These findings were paralleled by in vivo priming experiments in which, depending on viral dose, subtle in vitro blocking effects were absolute. Proteasome/synthetic peptide coincubation studies support a role for enhanced epitope destruction in preventing presentation, as did the effect of the peptide aldehyde, LLnL, which restored presentation of NP147-155 from the mutated const ructs. This reagent did not inhibit epitope presentation, even from wi ld-type NP, suggesting that its production may be proteasome independe nt. These results support the notion that point mutation of epitope fl anking sequence can serve as a mechanism for viral immune evasion, she d light on the mechanisms involved, and suggest that in vitro assays m ay not be sensitive indicators of flanking sequence effects.