For alanine scanning, cells were stimulated ex vivo with the indicated concentration of native or variant peptide and stained for CD8 and IFN-c as described above. Data were normalized to the frequency of cells that reacted to the unmodified S598 or S598 Q600Y peptides. For functional avidity determination, mononuclear cells were harvested from the brains of rJ or rJ.S598 Q600Y -infected mice 7 days p.i. and stimulated ex vivo in the presence of EL-4 cells pulsed with tenfold dilutions of peptide corresponding to native S598 or S598 Q600Y epitopes. After 5.5 hours, cells were stained for intracellular IFN-c as described above. For each epitope-specific population, data were normalized to the frequency of antigenspecific CTL detected using the highest titration of peptide (1 mM).