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Journal of Virology, January 2006, p. 883-890, Vol. 80, No. 2
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.2.883-890.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Three Immunoproteasome-Associated Subunits Cooperatively Generate a Cytotoxic T-Lymphocyte Epitope of Epstein-Barr Virus LMP2A by Overcoming Specific Structures Resistant to Epitope Liberation

Division of Immunology, Aichi Cancer Center Research Institute, Nagoya, Japan,¹ Department of Internal Medicine II, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan,² Department of Cell Therapy, Aichi Cancer Center Hospital, Nagoya, Japan³

Received 17 June 2005/ Accepted 19 October 2005

The precise roles of gamma interferon-inducible immunoproteasome-associated molecules in generation of cytotoxic T-lymphocyte (CTL) epitopes have yet to be fully elucidated. We describe here a unique epitope derived from the Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) presented by HLA-A*2402 molecules. Generation of the epitope, designated LMP2A_222-230, from the full-length protein requires the immunoproteasome subunit low-molecular-weight protein 7 (ip-LMP7) and the proteasome activator 28- subunit and is accelerated by ip-LMP2, as revealed by gene expression experiments using an LMP2A_222-230-specific CTL clone as a responder in enzyme-linked immunospot assays. The unequivocal involvement of all three components was confirmed by RNA interference gene silencing. Interestingly, the LMP2A_222-230 epitope could be efficiently generated from incomplete EBV-LMP2A fragments that were produced by puromycin treatment or gene-engineered shortened EBV-LMP2A lacking some of its hydrophobic domains. In addition, epitope generation was increased by a single amino acid substitution from leucine to alanine immediately flanking the C terminus, this being predicted by a web-accessible program to increase the cleavage strength. Taken together, the data indicate that the generation of LMP2A_222-230 is influenced not only by extrinsic factors such as immunoproteasomes but also by intrinsic factors such as the length of the EBV-LMP2A protein and proteasomal cleavage strength at specific positions in the source antigen.