Targeting of EBNA1 for rapid intracellular degradation overrides the inhibitory effects of the Gly-Ala repeat domain and restores CD8+T cell recognition

Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) includes a uniqu e glycine-alanine repeat domain that inhibits the endogenous presentation o f cytotoxic T lymphocyte (CTL) epitopes through the class I pathway by bloc king proteasome-dependent degradation of this antigen. This immune evasion mechanism has been implicated in the pathogenesis of EBV-associated disease s. Here, we show that cotranslational ubiquitination combined with N-end ru le targeting enhances the intracellular degradation of EBNA1, thus resultin g in a dramatic reduction in the half-life of the antigen. Using DNA expres sion vectors encoding different forms of ubiquitinated EBNA1 for in vivo st udies revealed that this rapid degradation, remarkably, leads to induction of a very strong CTL response to an EBNA1-specific CTL epitope. Furthermore , this targeting also restored the endogenous processing of HLA class I-res tricted CTL epitopes within EBNA1 for immune recognition by human EBV-speci fic CTLs. These observations provide, for the first time, evidence that the glycine-alanine repeat-mediated proteasomal block on EBNA1 can be reversed by specifically targeting this antigen for rapid degradation resulting in enhanced CD8+ T cell-mediated recognition in vitro and in vivo.