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 unique glycine-alanine repeat domain that inhibits the endogenous presentation of cytotoxic T lymphocyte (CTL) epitopes through the class I pathway by blocking proteasome-dependent degradation of this antigen. This immune evasion mechanism has been implicated in the pathogenesis of EBV-associated diseases. Here, we show that cotranslational ubiquitination combined with N-end rule targeting enhances the intracellular degradation of EBNA1, thus resulting in a dramatic reduction in the half-life of the antigen. Using DNA expression vectors encoding different forms of ubiquitinated EBNA1 for in vivo studies 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-restricted CTL epitopes within EBNA1 for immune recognition by human EBV-specific 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.
Tellam
J
J
Tumour Immunology Laboratory, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, Bancroft Centre and Joint Oncology Program, University of Queensland, Brisbane, Queensland 4006, Australia.
Sherritt
M
M
Thomson
S
S
Tellam
R
R
Moss
D J
DJ
Burrows
S R
SR
Wiertz
E
E
Khanna
R
R
D000409:Alanine / Q000737:chemistry; D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism*; D002460:Cell Line; D002469:Cell Separation; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D003720:Densitometry; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism*; D005434:Flow Cytometry; D005998:Glycine / Q000737:chemistry; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013154:Spleen / Q000166:cytology; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014452:Ubiquitins / Q000378:metabolism
D019309:Epstein-Barr Virus Nuclear Antigens; D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D049452:Green Fluorescent Proteins; D004247:DNA; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C417029:EBV-encoded nuclear antigen 1; D000409:Alanine; D005998:Glycine