CTL recognize peptides derived from protein Ags bound to MHC-class I molecules. Proteasomes probably participate in the generation of these peptide epitopes. We investigated the role of proteasomes in the presentation of endogenously synthesized short viral proteins. To this end, we employed proteasome and cysteine protease inhibitors and two closely related recombinant vaccinia viruses that code for 17- and 19-amino acid-long products encompassing murine CMV 9pp89 epitope. Presentation of both minigene products required processing to shorter peptides and was independent of ubiquitination. Proteasomes were necessary for processing the 17-mer product, and cysteine proteases were not required. In contrast, the 19-mer product could be processed in parallel either by proteasomes or by cysteine proteases independently. These results highlight the diversity of alternative processing pathways even for short peptidic Ags, provide evidence for the involvement of cysteine proteases in MHC class I presentation, and show that cleavage by cysteine proteases is governed by sequences flanking the epitope.S
