The Epstein-Barr virus (EBV)-encoded nuclear antigen (EBNA1) is expressed in latently EBV-infected B lymphocytes that persist for life in healthy virus carriers, and is the only viral protein regularly detected in all malignancies associated with EBV. Major histocompatibility complex (MHC) class I-restricted, EBNA1-specific cytotoxic T lymphocyte (CTL) responses have not been demonstrated. Using recombinant vaccinia viruses encoding chimaeric proteins containing an immunodominant human leukocyte antigen A11-restricted CTL epitope, amino acids 416-424 of the EBNA4 protein, inserted within the intact EBNA1, or within an EBNA1 deletion mutant devoid of the internal Gly-Ala repetitive sequence, we demonstrate that the Gly-Ala repeats generate a cis-acting inhibitory signal that interferes with antigen processing and MHC class I-restricted presentation. Insertion of the Gly-Ala repeats downstream of the 416-424 epitope inhibited CTL recognition of a chimaeric EBNA4 protein. The results highlight a previously unknown mechanism of viral escape from CTL surveillance, and support the view that the resistance of cells expressing EBNA1 to rejection mediated by CTL is a critical requirement for EBV persistence and pathogenesis.
Defects of antigen processing/presentation have been suggested to play a role in the escape of Burkitt's lymphoma (BL) from cytotoxic T lymphocyte (CTL)-mediated rejection. Impaired presentation of an immunodominant HLA A11-restricted epitope from the resident or recombinant vaccinia virus-expressed Epstein-Barr virus nuclear antigen (EBNA)4 was demonstrated in the EBV-positive E95B-BL28 and its EBV-negative parental BL28 cell lines. We have investigated whether this was due to (i) impaired production of the antigenic peptide, (ii) poor peptide translocation into the ER lumen or (iii) inefficient maturation and transport of the MHC-peptide complexes at the cell surface. The defect was not overcome by cytosolic expression of a pre-formed epitope, suggesting that presentation of EBNA4 is not limited by inefficient production of the antigenic peptide. BL28 expressed 5- to 10-fold lower levels of the transporter associated with antigen presentation (TAP) 1 and TAP2 proteins and behaved poorly in a streptolysin-O-mediated peptide translocation assay, whereas E95B-BL28 showed higher TAP expression and virtually normal transporter function. Up-regulation of HLA A11 and reconstitution of TAP activity by treatment with IFN-gamma did not restore presentation of the resident EBNA4 in E95SB-BL28 and did not enhance presentation of the vaccinia virus-expressed intact protein or preformed epitope. Efficient maturation of class I molecules to Endo H-resistant species was demonstrated in pulse-chase experiments. Taken together, our findings identify a previously uncharacterized defect of antigen presentation which appears to affect events occurring after proteasomal degradation but before TAP-dependent peptide transport and MHC class I assembly and maturation.
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