Epstein-Barr virus (EBV)–encoded nuclear antigen (EBNA)1 is thought to escape cytotoxic T lymphocyte (CTL) recognition through either self-inhibition of synthesis or by blockade of proteasomal degradation by the glycine-alanine repeat (GAr) domain. Here we show that EBNA1 has a remarkably varied cell type–dependent stability. However, these different degradation rates do not correspond to the level of major histocompatibility complex class I–restricted presentation of EBNA1 epitopes. In spite of the highly stable expression of EBNA1 in B cells, CTL epitopes derived from this protein are efficiently processed and presented to CD8+ T cells. Furthermore, we show that EBV-infected B cells can readily activate EBNA1-specific memory T cell responses from healthy virus carriers. Functional assays revealed that processing of these EBNA1 epitopes is proteasome and transporter associated with antigen processing dependent. We also show that the endogenous presentation of these epitopes is dependent on the newly synthesized protein rather than the long-lived stable EBNA1. Based on these observations, we propose that defective ribosomal products, not the full-length antigen, are the primary source of endogenously processed CD8+ T cell epitopes from EBNA1.

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Many viruses avoid immune surveillance during latent infection through reduction in the synthesis of virally encoded proteins.Although antigen presentation critically depends on the level of viral protein synthesis, the precise mechanism used to regulate the generation of antigenic peptide precursors remains elusive. Here, we demonstrate that a purine overloaded virally encoded mRNA lacking secondary structure significantly impacts the efficiency of protein translation and prevents endogenous antigen presentation. Reducing this purine bias through the generation of constructs expressing codon-modified sequences, while maintaining the encoded protein sequence, increased the stem-loop structure of the corresponding mRNA and dramatically enhanced self-synthesis of the viral protein. As a consequence, a higher number of HLA-peptide complexes were detected on the surface of cells expressing this viral protein. Furthermore, these cells were more efficiently recognized by virus-specific T cells compared with those expressing the same antigen expressed by a purine-biased mRNA. These findings delineate a mechanism by which viruses regulate self-synthesis of proteins and offer an effective strategy to evade CD8 ؉ T cell-mediated immune regulation.antigen processing ͉ EBV-encoded nuclear antigen 1 ͉ immune evasion ͉ protein synthesis V iruses that establish persistent infections or are involved in malignant processes have evolved unique mechanisms to evade the potent antiviral cytotoxic T cell response in the immunocompetent host (1-4). These evasion mechanisms include downregulated gene expression during latent infection, virus replication in immune-privileged tissues, loss of HLA and adhesion protein expression, and sequence variation affecting peptide binding to HLA class I molecules or recognition by the T cell receptor on CD8 ϩ T cells (5-7). It is now firmly established that activation of CD8 ϩ T cells after viral infection critically depends on the efficient presentation of virally encoded epitopes in complex with HLA class I molecules (reviewed in refs. 8 and 9).Although the human immune system is highly efficient in rapidly processing and presenting peptide epitopes from foreign proteins, many pathogens have adopted strategies to evade this rapid immune scanning by interfering with the HLA class I processing pathway or limiting the HLA-peptide complexes on the cell surface through cis-acting translational inhibition (10-12). Indeed, the EBV-encoded nuclear antigen, EBNA1, which is ubiquitously expressed in all EBV-associated malignancies, is an example of one such protein, which inhibits its self-synthesis and blocks proteasomal degradation, thereby restricting immune recognition by CD8 ϩ T lymphocytes (11, 12). These effects have been accredited to a glycine-alanine repeat domain (GAr) within EBNA1, and observations that removal of this GAr domain led to increased translational efficiency and enhanced immune recognition (11, 13) suggested that the GAr sequence may be contributing to the inhibition of EBNA1 levels...