Cytotoxic T lymphocyte epitopes of HIV-1 Nef: Generation of multiple definitive major histocompatibility complex class I ligands by proteasomes

Although a pivotal role of proteasomes in the proteolytic generation of epi topes for major histocompatibility complex (MHC) class I presentation is un disputed, their precise function is currently the subject of an active deba te: do proteasomes generate many epitopes in definitive form, or do they me rely generate the COOH termini, whereas the definitive NH, termini are clea ved by aminopeptidases? We determined five naturally processed MHC class I ligands derived from HIV-1 Nef. Unexpectedly, the five ligands correspond t o only three cytotoxic T lymphocyte (CTL) epitopes, two of which occur in t wo COOH-terminal length variants. Parallel analyses of proteasomal digests of a Nef fragment encompassing the epitopes revealed that all five ligands are direct products of proteasomes. Moreover, in four of the five ligands, the NH, termini correspond to major proteasome cleavage sites, and putative NH2-terminally extended precursor fragments were detected for only one of the five ligands. AU ligands are transported by the transporter associated with antigen processing (TAP). The combined results from these five ligands provide strong evidence that many definitive MHC class I ligands are preci sely cleaved at both ends by proteasomes. Additional evidence supporting th is conclusion is discussed, along with contrasting results of others who pr opose a strong role for NH2-terminal trimming with direct proteasomal epito pe generation being a rare event.