Generation of the vesicular stomatitis virus nucleoprotein cytotoxic T lymphocyte epitope requires proteasome-dependent and -independent proteolytic activities

The proteasome is involved in the generation of most of the MHC class I ant igenic epitopes. However, it is not known if the proteasome generates the e xact cytotoxic T lymphocyte (CTL) epitope or only epitope precursors which require further modification by additional proteases. Digestion of the exte nded vesicular stomatitis virus nucleoprotein epitope 52-59 (RGYVYQGL) by t he 20S proteasome in vitro shows that the proteasome is capable of generati ng the correct C terminus but not the exact N terminus of the CTL epitope. This finding suggests that proteolytic activity in addition to the proteaso me is required for generation of the CTL epitope. By using the proteasome i nhibitor lactacystin we were able to confirm this finding in vivo. Lactacys tin prevented the processing of N- and C-terminally extended epitopes, wher eas the processing of only N-terminally extended epitopes was unaffected. T hus, the proteasome is necessary and sufficient for the generation of the e xact C terminus of this CTL epitope, whereas the exact N terminus seems to be generated by a different protease.