Endogenous Presentation of CD8+ T Cell Epitopes from Epstein-Barr Virus–encoded Nuclear Antigen 1

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

Epstein-Barr virus-associated Hodgkin's lymphoma

Survivors of Hodgkin's lymphoma (HL) frequently have many years to experience the long-term toxicities of combined modality therapies. Also, a significant proportion of HL patients will relapse or have refractory disease, and less than half of these patients will respond to current salvage strategies. 30–50% of HL cases are Epstein–Barr virus associated (EBV-positive HL). The virus is localized to the malignant cells and is clonal. EBV-positive HL is more frequent in childhood, in older adults (>45 years) and in mixed cellularity cases. The survival of EBV-positive HL in the elderly and the immunosuppressed is particularly poor. Despite improvements in our understanding of EBV-positive HL, the true contribution of EBV to the pathogenesis of HL remains unknown. Increased knowledge of the virus’ role in the basic biology of HL may generate novel therapeutic strategies for EBV-positive HL and the presence of EBV-latent antigens in the malignant HL cells may represent a target for cellular immunotherapy

Messenger RNA Sequence Rather than Protein Sequence Determines the Level of Self-synthesis and Antigen Presentation of the EBV-encoded Antigen, EBNA1

peer-reviewedViruses establishing persistent latent infections have evolved various mechanisms to avoid immune surveillance. The Epstein-Barr virus-encoded nuclear antigen, EBNA1, expressed in all EBV-associated malignancies, modulates its own protein levels at quantities sufficient to maintain viral infection but low enough so as to minimize an immune response by the infected host cell. This evasion mechanism is regulated through an internal purine-rich mRNA repeat sequence encoding glycine and alanine residues. In this study we assess the impact of the repeat's nucleotide versus peptide sequence on inhibiting EBNA1 self-synthesis and antigen presentation. We demonstrate that altered peptide sequences resulting from frameshift mutations within the repeat do not alleviate the immune-evasive function of EBNA1, suggesting that the repetitive purine-rich mRNA sequence itself is responsible for inhibiting EBNA1 synthesis and subsequent poor immunogenicity. Our comparative analysis of the mRNA sequences of the corresponding repeat regions of different gammaherpesvirus maintenance homologues to EBNA1 highlights the high degree of identity between the nucleotide sequences despite very little homology in the encoded amino acid sequences. These studies demonstrate the importance of gammaherpesvirus purine-rich mRNA repeat sequences on antigenic epitope generation and evasion from T-cell mediated immune control, suggesting novel approaches to prevention and treatment of latent infection by this class of virus.National Health & Medical Research Council (NH&MRC) Canberra, Australia (#496684 APP1005091); NH&MRC Career Development Award Research Fellowship (#496712

Characterization of Munc-18c and syntaxin-4 in 3T3-L1 adipocytes: Putative role in insulin-dependent movement of GLUT-4

We have previously identified three mammalian Sec1/Munc-18 homologues in adipocytes (Tellam, J. T., McIntosh, S., and James, D. E. (1995) J. Biol. Chem. 270, 5857-5863). These proteins are thought to modulate the interaction between vesicle membrane and target membrane soluble N. ethylmaleimide- sensitive factor attachment protein receptors (SNAREs) and thus regulate intracellular vesicular transport. This study aimed to further characterize these Munc-18 isoforms and to define their potential role in the trafficking of GLUT-4 in adipocytes, a process reported to involve the vesicle membrane SNARE, VAMP-2. Using an in vitro binding assay with recombinant fusion proteins, we show that Munc-18a and Munc-18b bind to syntaxin. 1A, -2, and - 3, while Munc-18c binds only to syntaxin-2 and -4. The specific interaction between Munc-18c and syntaxin-4 is of interest because aside from syntaxin- 1A, which is not expressed in adipocytes, syntaxin-4 is the only syntaxin that binds to VAMP-2. Using a three-way binding assay, it was shown that Munc-18c inhibits the binding of syntaxin-4 to VAMP-2. The subcellular distribution of syntaxin-4 and Munc-18c was almost identical, both being enriched in the plasma membrane, and both exhibiting an insulin-dependent movement out of an intracellular membrane fraction similar to that observed for GLUT-4. Munc-18b had a similar distribution to Mune-18c and so may also be involved in vesicle transport to the cell surface, whereas Munc-18a was undetectable by immunoblotting in adipocytes. Microinjection of a syntaxin-4 antibody into 3T3-L1 adipocytes blocked the insulin-dependent recruitment of GLUT-4 to the cell surface. These data suggest that syntaxin-4/Munc-18c/VAMP- 2 may play a role in the docking/fusion of intracellular GLUT-4-containing vesicles with the cell surface in adipocytes

Detection of H-2K^b–SIIN complexes on the surface of 293KbC2 cells expressing EBNA1-SIIN-GFP frameshift variants.

<p>H-2K^b–SIIN expression was assessed by flow cytometry of 293KbC2 cells transfected with E1-GA(wild-type)-SIIN-GFP, E1ΔGA-SIIN-GFP, E1-GQE(frameshift 1)-SIIN-GFP or E1-GRS(frameshift 2)-SIIN-GFP following staining with the monoclonal antibody 25D1.16 conjugated to Allophycocyanin <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003112#ppat.1003112-Porgador1" target="_blank">[38]</a>. Values shown in each FACS plot are the percentage of GFP⁺H-2K^b–SIIN⁺ cells as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003112#s4" target="_blank">Materials and Methods</a>. These data are representative of three separate experiments.</p

T-cell recognition of 293KbC2 cells transfected with EBNA1-SIIN-GFP frameshift variants.

<p>Panels, A and B: 293kbC2 cells expressing EBNA1-SIIN-GFP encoding either alternative repeat peptide sequences or no repeat were exposed to SIIN-specific OT-1 T-cells and then incubated for 3 hours in the presence of brefeldin A at a responder to stimulator ratio of 5∶1 (panel A) and ratios of 2.5∶1–40∶1 (panel B). Following incubation, IFN-γ production by OT-1 T-cells was determined by intracellular cytokine staining. The top right hand corner of the FACS plot in panel A indicates the percentage of OT-1-specific CD8⁺ lymphocytes producing IFN-γ. Panels, C and D: EBV-negative DG75 cells were co-transfected with EBNA1-GFP expression constructs encoding alternative repeat peptide sequences or no repeat and a HLA B*3508-GFP expression vector. The transfected cells were exposed to HPV-specific T-cells and incubated in the presence of brefeldin A overnight at a responder to stimulator ratio of 5∶1 (panel C) and ratios of 2.5∶1–20∶1 (panel D). Following incubation, IFN-γ production by HPV-specific T-cells was determined by intracellular cytokine staining and shown in the top right hand corner of the FACS plot in panel C as the percentage of HPV-specific CD8⁺ lymphocytes producing IFN-γ. These data are representative of three separate experiments.</p

Localization and expression of EBNA1-SIIN-GFP frameshift constructs.

<p>Panel A, GFP fluorescence of EGFP-N1, E1-GA(wild-type)-SIIN-GFP, E1ΔGA-SIIN-GFP, E1-GQE(frameshift 1)-SIIN-GFP or E1-GRS(frameshift 2)-SIIN-GFP expression constructs in 293KbC2 cells. The cells were examined using a laser-scanning Bio-Rad (Hercules, CA) MRC600 confocal microscope with original magnification ×63. Panel B, DAPI staining, and; Panel C, phase contrast images of the EBNA1-SIIN-GFP transfected cells. Panel D, Flow cytometric analysis of GFP expression in 293KbC2 cells following transfection with EGFP-N1 or EBNA1-SIIN-GFP frameshift variants. The Mean Fluorescence Intensity (MFI) of the EBNA1-GFP positive cells is indicated in the top right hand corner of each plot.</p

Intracellular degradation kinetics and expression of alternative EBNA1 repeat peptide sequences.

<p>A, 293KbC2 cells were transfected with the following expression constructs E1ΔGA-SIIN-GFP; E1-GA(wild-type)-SIIN-GFP; E1-GQE(frameshift 1)-SIIN-GFP or E1-GRS(frameshift 2)-SIIN-GFP in the presence or absence of the proteasome inhibitor MG132 (10 µM). At 24 h post-transfection, the cells were incubated with cycloheximide (50 µg/ml) and then monitored over a 30-h time course as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003112#s4" target="_blank">Materials and Methods</a>. EBNA1-SIIN-GFP expression of the cells at each time point was monitored by flow cytometry and plotted as the relative change in the levels of EBNA1-GFP expression following the addition of cycloheximide at time point 0. B, <i>In vitro</i> translation (IVT) assay of pcDNA3 expression constructs encoding E1ΔGA (lane 1), E1-GA(wild-type) (lane 2), E1-GQE(frameshift 1) (lane 3) or E1-GRS(frameshift 2) (lane 4).The constructs were transcribed and translated <i>in vitro</i> with T7 RNA polymerase by using a coupled transcription/translation reticulocyte lysate system. ³⁵S-methionine-labeled proteins were visualized by autoradiography (upper panel). An asterisk indicates the full-length translation product of each EBNA1 frameshift variant. Band intensities from the IVT assay were quantified by densitometric analysis using Imagequant software (Molecular Dynamics) and graphed to demonstrate absolute intensities (lower panel). These data are representative of three separate experiments.</p

Schematic description of EBNA1 expression constructs containing identical mRNA sequences whilst encoding alternative repeat reading frames.

<p>A, The EBNA1 frameshift constructs were generated in either pcDNA3 for <i>in vitro</i> translation studies or in pEGFP-N1 for EBNA1-GFP expression and immunological studies. The overlapping DNA binding and dimerization domain, nuclear localization signal (NLS) and Glycine/Arginine (GR) repeat flanking sequences essential for genome maintenance functions are shown. The localizations of a model SIINFEKL epitope and the endogenous EBNA1 HPVGEADYFEY epitope used in the presentation assays are highlighted. B, Alignment of amino acid sequences of the EBNA1 (E1) repeat frameshift variants E1-GA(wild-type), E1-GQE(frameshift 1) and E1-GRS(frameshift 2). An asterisk indicates identical residues in all three proteins. Arrows denote nucleotide deletion positions at the start of the internal repeat and nucleotide insertion positions at the ends of the repeat in the E1-GA(wild-type) sequence to generate alternative repeat reading frames whilst maintaining wild-type EBNA1 sequence at both the N- and C-terminal domains flanking the internal repeat sequence.</p

Localization, expression and T-cell recognition of 293KbC2 cells transfected with an EBNA1-Ateline-SIIN-GFP expression construct.

<p>A, GFP fluorescence, DAPI staining and Flow cytometric analysis of GFP expression in 293KbC2 cells following transfection with an EBNA1-Ateline-SIIN-GFP expression construct. The cells were examined using a laser-scanning Bio-Rad (Hercules, CA) MRC600 confocal microscope with original magnification ×63. The Mean Fluorescence Intensity (MFI) of the EBNA1-GFP positive cells is indicated in the top right hand corner of the FACS plot. B, T-cell recognition of 293KbC2 cells transfected with EBNA1-SIIN-GFP variants. 293kbC2 cells expressing EBNA1-SIIN-GFP encoding either wildtype or Ateline repeat peptide sequences or no repeat were exposed to SIIN-specific OT-1 T-cells and incubated for 3 hours in the presence of brefeldin A at responder to stimulator ratios of 2.5∶1–20∶1. Following incubation, IFN-γ production by OT-1 T-cells was determined by intracellular cytokine staining. These data are representative of three separate experiments.</p