In mice infected with wild type JHMV, the CTL response is functionally focused on S510 [21] ; the Q600Y substitution effectively prevents CTL escape at either S510 or S598 Q600Y by the induction of a second high avidity CTL response. Mutations in S598 Q600Y were not detected even when the CTL response was directed primarily at this determinant (e.g. mice infected with rJ.S W513R + Q600Y ). Consistent with this inability to readily tolerate mutations, we were unable to generate recombinant virus mutated in position Ile-4 (I601D,E,K,R,T) and recombinant virus mutated at Phe-5 (F602A) was highly attenuated (data not shown). The combination of induction of high avidity CTL and inability to tolerate mutation without adversely affecting virus fitness make S598 Q600Y an ideal target for the anti-JHMV CTL response. Further, the ala scanning results suggest that S598 Q600Y -specific CTLs may more readily tolerate changes in the H-2K b /peptide complex, and this plasticity would also minimize the likelihood of CTL escape. In contrast, we have previously shown that introduction of the LCMV-specific GP33 determinant, which also elicits CTL with high functional avidity, into JHMV greatly diminished clinical disease but did not prevent CTL escape [22] . The GP33 determinant was introduced at a site in the genome that tolerated mutation and deletion and intact determinant was no longer present in virus by day 20 p.i. Collectively these results suggest that a response to a second determinant that elicits CTL exhibiting high functional avidity at early times p.i. results in enhanced suppression of virus replication, but its presence throughout the infection is required to protect against CTL escape.