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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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Ubiquitination analysis of EBNA1-GFP and EBNA1ΔGA-GFP in vitro. (A) SVMR6 keratinocytes were transiently cotransfected with expression vectors encoding HA-tagged 8xUb and LMP1-GFP, EBNA1-GFP, or EBNA1ΔGA-GFP. Ubiquitinated complexes were immunoprecipitated with an anti-HA–specific mAb and immunoblotted with anti-GFP. The ubiquitinated LMP1+ control is indicated. (B) Effect of the proteasomal inhibitor lactacystin on the stability of EBNA1-GFP and LMP1-GFP in epithelial cells. Duplicate aliquots of HaCaT cells were transfected with the expression construct EBNA1-GFP or LMP1-GFP. At 36 h after transfection, the proteasome inhibitor lactacystin was added at a final concentration of 10 μg/ml for 12 h to one of the duplicates. Both duplicates were then subjected to treatment with 50 μg/ml cycloheximide over a 6–8-h time course. Cell lysates at the indicated time points were separated by SDS-PAGE for immunoblotting with a GFP-specific antibody. The absence (−) or presence (+) of lactacystin is indicated. Densitometric analysis of the EBNA1-GFP, LMP1-GFP, EBNA1-GFP plus lactacystin, and LMP1-GFP plus lactacystin expression products are shown.
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fig2: Ubiquitination analysis of EBNA1-GFP and EBNA1ΔGA-GFP in vitro. (A) SVMR6 keratinocytes were transiently cotransfected with expression vectors encoding HA-tagged 8xUb and LMP1-GFP, EBNA1-GFP, or EBNA1ΔGA-GFP. Ubiquitinated complexes were immunoprecipitated with an anti-HA–specific mAb and immunoblotted with anti-GFP. The ubiquitinated LMP1+ control is indicated. (B) Effect of the proteasomal inhibitor lactacystin on the stability of EBNA1-GFP and LMP1-GFP in epithelial cells. Duplicate aliquots of HaCaT cells were transfected with the expression construct EBNA1-GFP or LMP1-GFP. At 36 h after transfection, the proteasome inhibitor lactacystin was added at a final concentration of 10 μg/ml for 12 h to one of the duplicates. Both duplicates were then subjected to treatment with 50 μg/ml cycloheximide over a 6–8-h time course. Cell lysates at the indicated time points were separated by SDS-PAGE for immunoblotting with a GFP-specific antibody. The absence (−) or presence (+) of lactacystin is indicated. Densitometric analysis of the EBNA1-GFP, LMP1-GFP, EBNA1-GFP plus lactacystin, and LMP1-GFP plus lactacystin expression products are shown.

Mentions: To delineate the pathway for the rapid degradation of EBNA1 in epithelial cells, we first tested the possibility that this protein is targeted through the Ub-dependent pathway. SVMR6 cells were transiently cotransfected with expression vectors encoding HA-tagged 8xUb and EBNA1-GFP, EBNA1ΔGA-GFP, or LMP1-GFP. Previous studies have shown that the EBV-LMP1 is degraded through the Ub/proteasome-dependent pathway (27) and thus LMP1-GFP was included as a positive control. 36 h after transfection, these cells were lysed and Ub–EBNA1 or Ub–LMP1 complexes were immunoprecipitated with an anti-HA–specific mAb. These complexes were then resolved by SDS-PAGE, followed by immunoblotting with a GFP-specific antibody. Data presented in Fig. 2 A clearly shows that although LMP1-GFP was ubiquitinated, no ubiquitination was observed for EBNA1-GFP or EBNA1ΔGA-GFP. These results are supported by recent studies by Holowaty et al. (28) who showed that EBNA1 interacts with a Ub-specific protease, HAUSP/USP7, which is demonstrated to deubiquitinate EBNA1. Furthermore, the pattern of intracellular degradation of EBNA1 in epithelial cells remained unaffected even after the addition of the proteasome inhibitor, lactacystin. This was not the case when the same concentration of lactacystin (10 μM) was used to inhibit proteasome-dependent degradation of another EBV protein, LMP1 (Fig. 2 B). Surprisingly, these inhibitory effects do not prevent EBNA1 degradation in epithelial cells, suggesting that the rapid degradation of EBNA1 in epithelial cells does not proceed through the classical Ub/proteasome-dependent pathway.


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)

Ubiquitination analysis of EBNA1-GFP and EBNA1ΔGA-GFP in vitro. (A) SVMR6 keratinocytes were transiently cotransfected with expression vectors encoding HA-tagged 8xUb and LMP1-GFP, EBNA1-GFP, or EBNA1ΔGA-GFP. Ubiquitinated complexes were immunoprecipitated with an anti-HA–specific mAb and immunoblotted with anti-GFP. The ubiquitinated LMP1+ control is indicated. (B) Effect of the proteasomal inhibitor lactacystin on the stability of EBNA1-GFP and LMP1-GFP in epithelial cells. Duplicate aliquots of HaCaT cells were transfected with the expression construct EBNA1-GFP or LMP1-GFP. At 36 h after transfection, the proteasome inhibitor lactacystin was added at a final concentration of 10 μg/ml for 12 h to one of the duplicates. Both duplicates were then subjected to treatment with 50 μg/ml cycloheximide over a 6–8-h time course. Cell lysates at the indicated time points were separated by SDS-PAGE for immunoblotting with a GFP-specific antibody. The absence (−) or presence (+) of lactacystin is indicated. Densitometric analysis of the EBNA1-GFP, LMP1-GFP, EBNA1-GFP plus lactacystin, and LMP1-GFP plus lactacystin expression products are shown.
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Related In: Results  -  Collection

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fig2: Ubiquitination analysis of EBNA1-GFP and EBNA1ΔGA-GFP in vitro. (A) SVMR6 keratinocytes were transiently cotransfected with expression vectors encoding HA-tagged 8xUb and LMP1-GFP, EBNA1-GFP, or EBNA1ΔGA-GFP. Ubiquitinated complexes were immunoprecipitated with an anti-HA–specific mAb and immunoblotted with anti-GFP. The ubiquitinated LMP1+ control is indicated. (B) Effect of the proteasomal inhibitor lactacystin on the stability of EBNA1-GFP and LMP1-GFP in epithelial cells. Duplicate aliquots of HaCaT cells were transfected with the expression construct EBNA1-GFP or LMP1-GFP. At 36 h after transfection, the proteasome inhibitor lactacystin was added at a final concentration of 10 μg/ml for 12 h to one of the duplicates. Both duplicates were then subjected to treatment with 50 μg/ml cycloheximide over a 6–8-h time course. Cell lysates at the indicated time points were separated by SDS-PAGE for immunoblotting with a GFP-specific antibody. The absence (−) or presence (+) of lactacystin is indicated. Densitometric analysis of the EBNA1-GFP, LMP1-GFP, EBNA1-GFP plus lactacystin, and LMP1-GFP plus lactacystin expression products are shown.
Mentions: To delineate the pathway for the rapid degradation of EBNA1 in epithelial cells, we first tested the possibility that this protein is targeted through the Ub-dependent pathway. SVMR6 cells were transiently cotransfected with expression vectors encoding HA-tagged 8xUb and EBNA1-GFP, EBNA1ΔGA-GFP, or LMP1-GFP. Previous studies have shown that the EBV-LMP1 is degraded through the Ub/proteasome-dependent pathway (27) and thus LMP1-GFP was included as a positive control. 36 h after transfection, these cells were lysed and Ub–EBNA1 or Ub–LMP1 complexes were immunoprecipitated with an anti-HA–specific mAb. These complexes were then resolved by SDS-PAGE, followed by immunoblotting with a GFP-specific antibody. Data presented in Fig. 2 A clearly shows that although LMP1-GFP was ubiquitinated, no ubiquitination was observed for EBNA1-GFP or EBNA1ΔGA-GFP. These results are supported by recent studies by Holowaty et al. (28) who showed that EBNA1 interacts with a Ub-specific protease, HAUSP/USP7, which is demonstrated to deubiquitinate EBNA1. Furthermore, the pattern of intracellular degradation of EBNA1 in epithelial cells remained unaffected even after the addition of the proteasome inhibitor, lactacystin. This was not the case when the same concentration of lactacystin (10 μM) was used to inhibit proteasome-dependent degradation of another EBV protein, LMP1 (Fig. 2 B). Surprisingly, these inhibitory effects do not prevent EBNA1 degradation in epithelial cells, suggesting that the rapid degradation of EBNA1 in epithelial cells does not proceed through the classical Ub/proteasome-dependent pathway.

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