FUNCTIONAL-ANALYSIS OF AMINO-ACID-RESIDUES ENCOMPASSING AND SURROUNDING 2 NEIGHBORING H-2D(B)-RESTRICTED CYTOTOXIC T-LYMPHOCYTE EPITOPES INSIMIAN-VIRUS-40 TUMOR-ANTIGEN

Simian virus 40 tumor (T) antigen contains three H-2D(b)- and one H-2K (b)-restricted cytotoxic T. lymphocyte (CTL) epitopes (sites). Two of the H-2D(b)-restricted CTL epitopes, I and II/III, are separated by 7 amino acids in the amino-terminal one third of T antigen. In this stud y, we determine if the amino acids separating these two H-2D(b) restri cted CTL epitopes are dispensable for efficient processing and present ation. In addition, the importance of amino acid residues lying within and flanking the H-2D(b)-restricted epitopes I and II/III for efficie nt processing, presentation, and recognition by site-specific CTL clon es was determined by using T-antigen mutants containing single-amino-a cid substitutions between residues 200 and 239. Using synthetic peptid es in CTL lysis and major histocompatibility complex class I stabiliza tion assays, CTL recognition site I has been redefined to include resi dues 206 to 215. Substitutions in amino acids flanking either site I o r site II/III did not affect recognition by any of the T-antigen-speci fic CTL clones. Additionally, the removal of the 7 residues separating site I and site II/III did not affect CTL recognition, thus demonstra ting that these two epitopes when arranged in tandem in the native T a ntigen can be efficiently processed and presented to CTL clones. Diffe rences in fine specificities of two CTL clones which recognize the sam e epitope (Y-1 and K-11 for site I and Y-2 and Y-3 for site II/III) ha ve been used in conjunction with synthetic peptide variants to assign roles for residues within epitopes I and II/III with respect to TCR re cognition and/or peptide major histocompatibility complex association.