Developing a novel approach to follow the processing of viral antigens for MHC class I presentation. (93.7)

Abstract

Abstract Cytotoxic T cells recognize their targets by the presence of unique peptides displayed by MHC class I molecules on the surface of antigen presenting cells. The peptides are generated by the antigen processing pathway and some proteins are known to inhibit this processing to escape the immune response. This is the case of EBNA1 (EBV nuclear antigen), a viral antigen detected in EBV-associated tumors. It is now established that the glycine-alanine repeat sequence present in EBNA1 is responsible for the absence of cell surface antigen presentation. Nevertheless how the antigen processing is modified remains unknown. Indeed, using conventional T cell assays, only the final peptide can be efficiently detected on the cell surface. We developed a novel assay, based on HPLC fractionation, allowing the detection of antigenic precursors in vivo. So far, we have successfully used this system to isolate, quantify and characterize antigenic intermediates derived from EBNA1 proteolysis. Using inhibitors as well as RNA interference, we can determine for the first time how EBNA1 interacts intracellularly with key steps of its antigen processing: degradation of newly synthezed proteins (DRiPs), proteasome proteolysis, TAP transport, and the role of chaperonin molecules.