CTL ESCAPE VIRAL VARIANTS .1. GENERATION AND MOLECULAR CHARACTERIZATION

Cytotoxic T lymphocytes (CTL) play a pivotal role in preventing persis tent viral infections and aborting acute infections. H-2D(b)-restricte d CTL optimally recognize a specific peptide of 9 to 11 amino acids (a a) derived from a viral protein and held in place (restricted) by a MH C class I glycoprotein on the surfaces of infected cells. Only three p eptide sequences with the appropriate Db motif from lymphocytic chorio meningitis virus Armstrong strain (LCMV) are known to be presented to CTL by H-2D(b) molecules; they are from the glycoproteins (GP), residu es 33-41 KAVYFATC (GP1) and 276-286 SGVENPGGYCL (GP2), and the nucleop rotein (NP), 396-404 FQPQNGQFI. Incubation of virally infected H-2(b) cells with CTL clones that recognize only GP1,GP2, or NP leads to the selection of viral Variants which upon infecting cells bearing H-2(b) molecules, escape recognition by CTL of the appropriate specificity. N ucleic acid sequencing showed a single mutation in GP1 (aa 38 F-->L), GP2 (aa 282 G-->D), or NP (aa 403 F-->L) in the variant viruses. When wild-type (wt) LCMV peptides and the three variant peptides (GP1, GP2, NP) were synthesized and subjected to a competitive inhibition bindin g assay, no differences in binding affinity for H-2D(b) were found bet ween the wt and variant peptides. Uninfected cells coated with the wt peptide were recognized and lysed by the appropriate CTL clone or by i n vivo-primed bulk CTL, but similar targets coated with the GP1, GP2, or NP variant peptides were not. This result, coupled with computer gr aphic analysis of these variant peptides with the recently solved thre e-dimensional structure for the Db MHC class I molecule, placed the si de chain of the mutated residues on the outer surface of the MHC-pepti de complex and accessible to the T cell receptor. Ala substitution at GP residue 38 or 282 or at NP 403 also abrogated CTL recognition and l ysis. Inoculation of any one of the mutated Viral variants into mice p roduced an effective CTL response to the other two nonmutated GP or NP peptides, suggesting that production of biologically relevant CTL esc ape virus variants in vivo requires selection of mutations in more tha n one and likely all the CTL epitopes, a low probability event. (C) 19 95 Academic Press, Inc.