Next, we used the consensus H-2K b binding motif [24] to engineer a novel, high-avidity S598 CTL epitope. Importantly, Gln-3 diverges from the consensus H-2K b -restricted ligand binding motif, in which a tyrosine is often present at position 3 [24, 25] . Therefore, we substituted a glutamine residue for tyrosine (Q600Y, CAA to TAT, RCYIFANI) at position 3 of the determinant with the aim of creating a peptide that bound more tightly to H-2K b . Stability of the H-2K b /S598 and H-2K b /S598 Q600Y complexes was assessed by circular dichroism (CD). As shown in Figure 1 , H-2K b /S598 Q600Y was considerably more thermostable than the native complex (Tm 54uC vs 64uC). To probe the biological properties of the Q600Y substitution, we used reverse genetics to engineer a recombinant version of JHMV expressing the S598 Q600Y epitope (Figure 2A) . Recombinant viruses encoding this substitution replicated as efficiently as wild type JHMV (rJ) in vitro during one-step growth kinetics analyses and in vivo virus competition assays ( Figure 2B , C). The immunogenicity of S598 Q600Y was assessed by intracellular expression of IFN-c by central nervous system (CNS)-derived lymphocytes. Since Cys-containing peptides are often diminished in