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Endogenous presentation of CD8+ T cell epitopes from Epstein-Barr virus-encoded nuclear antigen 1.

Tellam J, Connolly G, Green KJ, Miles JJ, Moss DJ, Burrows SR, Khanna R - J. Exp. Med. (2004)

Bottom Line: However, these different degradation rates do not correspond to the level of major histocompatibility complex class I-restricted presentation of EBNA1 epitopes.Functional assays revealed that processing of these EBNA1 epitopes is proteasome and transporter associated with antigen processing dependent.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.

View Article: PubMed Central - PubMed

Affiliation: EBV Unit, Tumour Immunology Laboratory, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, 300 Herston Road, Brisbane (Qld) 4006, Australia.

ABSTRACT
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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(A) Schematic description of EBNA1 and EBNA1ΔGA expression constructs showing localization of FLR and HPV epitopes. (B) Intracellular degradation of EBNA1-GFP in different cell types. DG75 B cells, HEK293 epithelial cells, SVMR6 keratinocytes, and HaCaT keratinocytes were transfected with expression constructs EBNA1-GFP, EBNA1ΔGA-GFP, or the control plasmid pEGFP-N1. At 36 h after transfection, the cells were degraded over a 30-h time course in the presence of 50 μg/ml cycloheximide as described in Materials and Methods. Molecular weight standards are indicated at the side of each panel. (C) Densitometric analysis of EBNA1-GFP, EBNA1ΔGA-GFP, and GFP expression. Band intensities were quantified by analysis of the imaging data and plotted as a relative percentage of the signal at time 0 for EBNA1-GFP, EBNA1ΔGA-GFP, and GFP.
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fig1: (A) Schematic description of EBNA1 and EBNA1ΔGA expression constructs showing localization of FLR and HPV epitopes. (B) Intracellular degradation of EBNA1-GFP in different cell types. DG75 B cells, HEK293 epithelial cells, SVMR6 keratinocytes, and HaCaT keratinocytes were transfected with expression constructs EBNA1-GFP, EBNA1ΔGA-GFP, or the control plasmid pEGFP-N1. At 36 h after transfection, the cells were degraded over a 30-h time course in the presence of 50 μg/ml cycloheximide as described in Materials and Methods. Molecular weight standards are indicated at the side of each panel. (C) Densitometric analysis of EBNA1-GFP, EBNA1ΔGA-GFP, and GFP expression. Band intensities were quantified by analysis of the imaging data and plotted as a relative percentage of the signal at time 0 for EBNA1-GFP, EBNA1ΔGA-GFP, and GFP.

Mentions: Full-length EBNA1 was cloned into the expression vector pcDNA3.1 (Invitrogen) to generate the expression construct EBNA1 (11). In addition, a modified EBNA1 construct in which the (GAr) domain was deleted was generated to give EBNA1ΔGA (11). The above constructs included insertion of a well-defined, HLA-B8–restricted EBNA-3 CTL epitope, FLRGRAYGL (referred to as FLR; reference 23), into the SacII site at nucleotide position 1853 of EBNA1, to allow assessment of endogenous processing of EBNA1 by FLR-specific CTL clones (see Fig. 1 A). To assess expression of EBNA1 and EBNA1ΔGA, the inserts from the above pcDNA3.1 constructs were subcloned in frame with a sequence coding for green fluorescent protein (GFP; pEGFP-N1; CLONTECH Laboratories, Inc.): EBNA1-GFP and EBNA1ΔGA-GFP.


Endogenous presentation of CD8+ T cell epitopes from Epstein-Barr virus-encoded nuclear antigen 1.

Tellam J, Connolly G, Green KJ, Miles JJ, Moss DJ, Burrows SR, Khanna R - J. Exp. Med. (2004)

(A) Schematic description of EBNA1 and EBNA1ΔGA expression constructs showing localization of FLR and HPV epitopes. (B) Intracellular degradation of EBNA1-GFP in different cell types. DG75 B cells, HEK293 epithelial cells, SVMR6 keratinocytes, and HaCaT keratinocytes were transfected with expression constructs EBNA1-GFP, EBNA1ΔGA-GFP, or the control plasmid pEGFP-N1. At 36 h after transfection, the cells were degraded over a 30-h time course in the presence of 50 μg/ml cycloheximide as described in Materials and Methods. Molecular weight standards are indicated at the side of each panel. (C) Densitometric analysis of EBNA1-GFP, EBNA1ΔGA-GFP, and GFP expression. Band intensities were quantified by analysis of the imaging data and plotted as a relative percentage of the signal at time 0 for EBNA1-GFP, EBNA1ΔGA-GFP, and GFP.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211806&req=5

fig1: (A) Schematic description of EBNA1 and EBNA1ΔGA expression constructs showing localization of FLR and HPV epitopes. (B) Intracellular degradation of EBNA1-GFP in different cell types. DG75 B cells, HEK293 epithelial cells, SVMR6 keratinocytes, and HaCaT keratinocytes were transfected with expression constructs EBNA1-GFP, EBNA1ΔGA-GFP, or the control plasmid pEGFP-N1. At 36 h after transfection, the cells were degraded over a 30-h time course in the presence of 50 μg/ml cycloheximide as described in Materials and Methods. Molecular weight standards are indicated at the side of each panel. (C) Densitometric analysis of EBNA1-GFP, EBNA1ΔGA-GFP, and GFP expression. Band intensities were quantified by analysis of the imaging data and plotted as a relative percentage of the signal at time 0 for EBNA1-GFP, EBNA1ΔGA-GFP, and GFP.
Mentions: Full-length EBNA1 was cloned into the expression vector pcDNA3.1 (Invitrogen) to generate the expression construct EBNA1 (11). In addition, a modified EBNA1 construct in which the (GAr) domain was deleted was generated to give EBNA1ΔGA (11). The above constructs included insertion of a well-defined, HLA-B8–restricted EBNA-3 CTL epitope, FLRGRAYGL (referred to as FLR; reference 23), into the SacII site at nucleotide position 1853 of EBNA1, to allow assessment of endogenous processing of EBNA1 by FLR-specific CTL clones (see Fig. 1 A). To assess expression of EBNA1 and EBNA1ΔGA, the inserts from the above pcDNA3.1 constructs were subcloned in frame with a sequence coding for green fluorescent protein (GFP; pEGFP-N1; CLONTECH Laboratories, Inc.): EBNA1-GFP and EBNA1ΔGA-GFP.

Bottom Line: However, these different degradation rates do not correspond to the level of major histocompatibility complex class I-restricted presentation of EBNA1 epitopes.Functional assays revealed that processing of these EBNA1 epitopes is proteasome and transporter associated with antigen processing dependent.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.

View Article: PubMed Central - PubMed

Affiliation: EBV Unit, Tumour Immunology Laboratory, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, 300 Herston Road, Brisbane (Qld) 4006, Australia.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus