Inhibition of Antigen Presentation by the Glycine/Alanine Repeat Domain Is Not Conserved in Simian Homologues of Epstein-Barr Virus Nuclear Antigen 1

ABSTRACT Most humans and Old World nonhuman primates are infected for life with Epstein-Barr virus (EBV) or closely related gammaherpesviruses in the same lymphocryptovirus (LCV) subgroup. Several potential strategies for immune evasion and persistence have been proposed based on studies of EBV infection in humans, but it has been difficult to test their actual contribution experimentally. Interest has focused on the EBV nuclear antigen 1 (EBNA1) because of its essential role in the maintenance and replication of the episomal viral genome in latently infected cells and because EBNA1 endogenously expressed in these cells is protected from presentation to the major histocompatibility complex class-I restricted cytotoxic T-lymphocyte (CTL) response through the action of an internal glycine-alanine repeat (GAR). Given the high degree of biologic conservation among LCVs which infect humans and Old World primates, we hypothesized that strategies essential for viral persistence would be well conserved among viruses of this subgroup. We show that the rhesus LCV EBNA1 shares sequence homology with the EBV and baboon LCV EBNA1 and that the rhesus LCV EBNA1 is a functional homologue for EBV EBNA1-dependent plasmid maintenance and replication. Interestingly, all three LCVs possess a GAR domain, but the baboon and rhesus LCV EBNA1 GARs fail to inhibit antigen processing and presentation as determined by using three different in vitro CTL assays. These studies suggest that inhibition of antigen processing and presentation by the EBNA1 GAR may not be an essential mechanism for persistent infection by all LCV and that other mechanisms may be important for immune evasion during LCV infection.

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T cell responses and virus evolution: loss of HLA A11-restricted CTL epitopes in Epstein-Barr virus isolates from highly A11-positive populations by selective mutation of anchor residues.

Journal of Experimental Medicine ◽

10.1084/jem.179.4.1297 ◽

1994 ◽

Vol 179 (4) ◽

pp. 1297-1305 ◽

Cited By ~ 103

Author(s):

P O de Campos-Lima ◽

V Levitsky ◽

J Brooks ◽

S P Lee ◽

L F Hu ◽

...

Keyword(s):

Epstein Barr Virus ◽

Cytotoxic T Lymphocyte ◽

Virus Evolution ◽

Nuclear Antigen ◽

Ctl Epitopes ◽

Barr Virus ◽

Southern Chinese ◽

Epstein Barr ◽

Ctl Responses

Epstein-Barr virus (EBV) is a B lymphotropic herpesvirus of humans that elicits strong HLA class I-restricted cytotoxic T lymphocyte (CTL) responses. An influence of such responses on virus evolution was first suggested by our finding that EBV isolates from the highly HLA A11-positive Papua New Guinea (PNG) population carried a lys-thr mutation at residue 424 of the nuclear antigen EBV-encoded nuclear antigen (EBNA4) that destroyed the immunodominant target epitope for A11-restricted CTL recognition. Here we turn to a much larger population, Southern Chinese, where the A11 allele is again present in over 50% of the individuals. Each of 23 EBV isolates analyzed from this population were also mutated in the EBNA4 416-424 epitope, the mutations selectively involving one of the two anchor residues in positions 2 (417 val-leu) or 9 (424 lys-asp, -arg or -thr) that are critical for A11-peptide interaction. The majority of the Chinese isolates and all 10 PNG isolates also carried mutations affecting positions 1 and 2 of the next most immunodominant A11-restricted epitope, EBNA4 residues 399-408. These changes clearly affected antigenicity since A11-positive lymphoblastoid cell lines (LCLs) carrying these mutant EBV strains were not recognized by A11-restricted CTLs raised against the prototype B95.8 virus. Furthermore, Chinese donors naturally infected with these mutant viruses did not mount detectable A11-restricted CTL responses on in vitro stimulation with autologous LCL cells carrying either the B95.8 or their endogenous EBV strain. In two different highly A11-positive populations, therefore, immune pressure appears to have selected for resident EBV strains lacking immunodominant A11-restricted CTL epitopes.

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A possible function of Epstein-Barr virus-determined nuclear antigen (EBNA): Stimulation of chromatin template activity in vitro

Virology ◽

10.1016/0042-6822(79)90419-7 ◽

1979 ◽

Vol 95 (1) ◽

pp. 222-226 ◽

Cited By ~ 5

Author(s):

Tohru Kamata ◽

Shigeaki Tanaka ◽

Shogo Aikawa ◽

Yorio Hinuma ◽

Yasushi Watanabe

Keyword(s):

Epstein Barr Virus ◽

Nuclear Antigen ◽

Template Activity ◽

Barr Virus ◽

Chromatin Template ◽

Epstein Barr ◽

Stimulation Of

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Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 Mimics Epstein-Barr Virus EBNA1 Immune Evasion through Central Repeat Domain Effects on Protein Processing

Journal of Virology ◽

10.1128/jvi.00411-07 ◽

2007 ◽

Vol 81 (15) ◽

pp. 8225-8235 ◽

Cited By ~ 70

Author(s):

Hyun Jin Kwun ◽

Suzane Ramos da Silva ◽

Ishita M. Shah ◽

Neil Blake ◽

Patrick S. Moore ◽

...

Keyword(s):

Antigen Processing ◽

Kaposi's Sarcoma ◽

Epstein Barr Virus ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Nuclear Antigen ◽

Barr Virus ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Epstein Barr ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV8]) and Epstein-Barr virus (EBV/HHV4) are distantly related gammaherpesviruses causing tumors in humans. KSHV latency-associated nuclear antigen 1 (LANA1) is functionally similar to the EBV nuclear antigen-1 (EBNA1) protein expressed during viral latency, although they have no amino acid similarities. EBNA1 escapes cytotoxic lymphocyte (CTL) antigen processing by inhibiting its own proteosomal degradation and retarding its own synthesis to reduce defective ribosomal product processing. We show here that the LANA1 QED-rich central repeat (CR) region, particularly the CR2CR3 subdomain, also retards LANA1 synthesis and markedly enhances LANA1 stability in vitro and in vivo. LANA1 isoforms have half-lives greater than 24 h, and fusion of the LANA1 CR2CR3 domain to a destabilized heterologous protein markedly decreases protein turnover. Unlike EBNA1, the LANA1 CR2CR3 subdomain retards translation regardless of whether it is fused to the 5′ or 3′ end of a heterologous gene construct. Manipulation of sequence order, orientation, and composition of the CR2 and CR3 subdomains suggests that specific peptide sequences rather than RNA structures are responsible for synthesis retardation. Although mechanistic differences exist between LANA1 and EBNA1, the primary structures of both proteins have evolved to minimize provoking CTL immune responses. Simple strategies to eliminate these viral inhibitory regions may markedly improve vaccine effectiveness by maximizing CTL responses.

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Establishment and characterization of an Epstein-Barr virus transformed cell line with strong phagocytic activity

Blood ◽

10.1182/blood.v74.1.430.430 ◽

1989 ◽

Vol 74 (1) ◽

pp. 430-436 ◽

Cited By ~ 4

Author(s):

P von den Driesch ◽

R Bhardwaj ◽

HD Flad ◽

DC Neugebauer ◽

HJ Pielken ◽

...

Keyword(s):

Cell Line ◽

Phagocytic Activity ◽

Antigen Processing ◽

Epstein Barr Virus ◽

Interleukin 1 ◽

Immunoglobulin M ◽

Nuclear Antigen ◽

Barr Virus ◽

Transformed Cell Line ◽

Epstein Barr

Abstract An immunoglobulin M (IgM)-positive cell line, Ms 28, apparently spontaneously transformed by Epstein-Barr virus (EBV) was established from peripheral blood cells of a patient with immature myeloblastic leukemia. It has been characterized according to phenotype, cytochemistry, and membrane antigen pattern. The cell line expresses lymphoid markers like CD 19, CD 22, and CD 30 and synthesizes and secretes IgM. Monocyte markers CD 11c, CD 14, and CD 15 are absent. Neither interleukin-1 (IL-1), nor tumor necrosis factor (TNF-alpha) are produced. But Ms 28 cells show strong phagocytic activity and engulf Latex particles and sheep RBCs (SRBCs) that need not to be opsonized. The phagocytic activity can be inhibited by chloroquine. Both phagocytosis and EBV nuclear-antigen (EBNA) expression can be observed in one and the same cell. Ms 28 cells might be useful to study immunologic activities like antigen processing and presentation.

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The ATM/ATR Signaling Effector Chk2 Is Targeted by Epstein-Barr Virus Nuclear Antigen 3C To Release the G2/M Cell Cycle Block

Journal of Virology ◽

10.1128/jvi.00053-07 ◽

2007 ◽

Vol 81 (12) ◽

pp. 6718-6730 ◽

Cited By ~ 56

Author(s):

Tathagata Choudhuri ◽

Subhash C. Verma ◽

Ke Lan ◽

Masanao Murakami ◽

Erle S. Robertson

Keyword(s):

Cell Cycle ◽

B Lymphocytes ◽

Epstein Barr Virus ◽

Cell Cycle Checkpoint ◽

Nuclear Antigen ◽

M Cell ◽

Barr Virus ◽

Cycle Checkpoint ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) infects most of the human population and persists in B lymphocytes for the lifetime of the host. The establishment of latent infection by EBV requires the expression of a unique repertoire of genes. The product of one of these viral genes, the EBV nuclear antigen 3C (EBNA3C), is essential for the growth transformation of primary B lymphocytes in vitro and can regulate the transcription of a number of viral and cellular genes important for the immortalization process. This study demonstrates an associated function of EBNA3C which involves the disruption of the G2/M cell cycle checkpoint. We show that EBNA3C-expressing lymphoblastoid cell lines treated with the drug nocodazole, which is known to block cells at the G2/M transition, did not show a G2/M-specific checkpoint arrest. Analyses of the cell cycles of cells expressing EBNA3C demonstrated that the expression of this essential EBV nuclear antigen is capable of releasing the G2/M checkpoint arrest induced by nocodazole. This G2/M arrest in response to nocodazole was also abolished by caffeine, suggesting an involvement of the ATM/ATR signaling pathway in the regulation of this cell cycle checkpoint. Importantly, we show that the direct interaction of EBNA3C with Chk2, the ATM/ATR signaling effector, is responsible for the release of this nocodazole-induced G2/M arrest and that this interaction leads to the serine 216 phosphorylation of Cdc25c, which is sequestered in the cytoplasm by 14-3-3. Overall, our data suggest that EBNA3C can directly regulate the G2/M component of the host cell cycle machinery, allowing for the release of the checkpoint block.

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Retinoids irreversibly inhibit in vitro growth of Epstein-Barr virus- immortalized B lymphocytes

Blood ◽

10.1182/blood.v88.8.3147.bloodjournal8883147 ◽

1996 ◽

Vol 88 (8) ◽

pp. 3147-3159 ◽

Cited By ~ 24

Author(s):

F Pomponi ◽

R Cariati ◽

P Zancai ◽

P De Paoli ◽

S Rizzo ◽

...

Keyword(s):

Cell Cycle ◽

Epstein Barr Virus ◽

Antigen Expression ◽

Inhibitory Effect ◽

Lymphoproliferative Disorders ◽

Nuclear Antigen ◽

Barr Virus ◽

Drug Concentrations ◽

Epstein Barr

Natural and synthetic retinoids have proved to be effective in the treatment and prevention of various human cancers. In the present study, we investigated the effect of retinoids on Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines (LCLs), since these cells closely resemble those that give rise to EBV-related lymphoproliferative disorders in the immunosuppressed host. All six compounds tested inhibited LCL proliferation with no significant direct cytotoxicity, but 9-cis-retinoic acid (RA), 13-cis-RA, and all-trans-RA (ATRA) were markedly more efficacious than Ro40–8757, Ro13–6298, and etretinate. The antiproliferative action of the three most effective compounds was confirmed in a large panel of LCLs, thus appearing as a generalized phenomenon in these cells. LCL growth was irreversibly inhibited even after 2 days of treatment at drug concentrations corresponding to therapeutically achievable plasma levels. Retinoid-treated cells showed a marked downregulation of CD71 and a decreased S-phase compartment with a parallel accumulation in Gzero/ G1 phases. These cell cycle perturbations were associated with the upregulation of p27 Kip1, a nuclear protein that controls entrance and progression through the cell cycle by inhibiting several cyclin/cyclin-dependent kinase complexes. Unlike what is observed in other systems, the antiproliferative effect exerted by retinoids on LCLs was not due to the acquisition of a terminally differentiated status. In fact, retinoid-induced modifications of cell morphology, phenotype (downregulation of CD19, HLA-DR, and s-Ig, and increased expression of CD38 and c-Ig), and IgM production were late events, highly heterogeneous, and often slightly relevant, being therefore only partially indicative of a drug-related differentiative process. Moreover, EBV-encoded EBV nuclear antigen-2 and latent membrane protein-1 proteins were inconstantly downregulated by retinoids, indicating that their growth-inhibitory effect is not mediated by a direct modulation of viral latent antigen expression. The strong antiproliferative activity exerted by retinoids in our experimental model indicates that these compounds may represent a useful tool in the medical management of EBV-related lymphoproliferative disorders of immunosuppressed patients.

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Epstein–Barr virus nuclear antigen 3A contains six nuclear-localization signals

Journal of General Virology ◽

10.1099/vir.0.81927-0 ◽

2006 ◽

Vol 87 (10) ◽

pp. 2879-2884 ◽

Cited By ~ 7

Author(s):

Marion Buck ◽

Anita Burgess ◽

Roslynn Stirzaker ◽

Kenia Krauer ◽

Tom Sculley

Keyword(s):

Nuclear Localization ◽

Epstein Barr Virus ◽

Fluorescent Protein ◽

Viral Proteins ◽

Site Directed Mutagenesis ◽

Nuclear Antigen ◽

Nuclear Localization Signals ◽

Barr Virus ◽

Epstein Barr

The Epstein–Barr nuclear antigen 3A (EBNA3A) is one of only six viral proteins essential for Epstein–Barr virus-induced transformation of primary human B cells in vitro. Viral proteins such as EBNA3A are able to interact with cellular proteins, manipulating various biochemical and signalling pathways to initiate and maintain the transformed state of infected cells. EBNA3A has been reported to have one nuclear-localization signal and is targeted to the nucleus during transformation, where it associates with components of the nuclear matrix. By using enhanced green fluorescent protein-tagged deletion mutants of EBNA3A in combination with site-directed mutagenesis, an additional five functional nuclear-localization signals have been identified in the EBNA3A protein. Two of these (aa 63–66 and 375–381) were computer-predicted, whilst the remaining three (aa 394–398, 573–578 and 598–603) were defined functionally in this study.

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Characterization of an human leucocyte antigen A2-restricted Epstein-Barr virus nuclear antigen-1-derived cytotoxic T-lymphocyte epitope

Immunology ◽

10.1111/j.1365-2567.2009.03190.x ◽

2010 ◽

Vol 129 (3) ◽

pp. 386-395 ◽

Cited By ~ 8

Author(s):

Diego Marescotti ◽

Federica Destro ◽

Anna Baldisserotto ◽

Mauro Marastoni ◽

Giuseppe Coppotelli ◽

...

Keyword(s):

T Lymphocyte ◽

Epstein Barr Virus ◽

Human Leucocyte Antigen ◽

Cytotoxic T Lymphocyte ◽

Nuclear Antigen ◽

Barr Virus ◽

Epstein Barr

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

Journal of Experimental Medicine ◽

10.1084/jem.20040191 ◽

2004 ◽

Vol 199 (10) ◽

pp. 1421-1431 ◽

Cited By ~ 104

Author(s):

Judy Tellam ◽

Geoff Connolly ◽

Katherine J. Green ◽

John J. Miles ◽

Denis J. Moss ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

Antigen Processing ◽

Epstein Barr Virus ◽

Cd8 T Cell ◽

Nuclear Antigen ◽

T Cell Epitopes ◽

Barr Virus ◽

Degradation Rates ◽

Epstein Barr

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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Mitosis-Specific Hyperphosphorylation of Epstein-Barr Virus Nuclear Antigen 2 Suppresses Its Function

Journal of Virology ◽

10.1128/jvi.78.7.3542-3552.2004 ◽

2004 ◽

Vol 78 (7) ◽

pp. 3542-3552 ◽

Cited By ~ 15

Author(s):

Wei Yue ◽

Matthew G. Davenport ◽

Julia Shackelford ◽

Joseph S. Pagano

Keyword(s):

Epstein Barr Virus ◽

Regulation Of Gene Expression ◽

Cyclin B1 ◽

Nuclear Antigen ◽

Mrna Levels ◽

Type Iii ◽

Barr Virus ◽

M Phase ◽

Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is a key gene expressed in EBV type III latent infection that can transactivate numerous promoters, including those for all the other type III viral latency genes as well as cellular genes responsible for cell proliferation. EBNA-2 is essential for EBV-mediated immortalization of primary B lymphocytes. We now report that EBNA-2, a phosphoprotein, is hyperphosphorylated specifically in mitosis. Evidence that the cyclin-dependent kinase p34cdc2 may be involved in this hyperphosphorylation includes (i) coimmunoprecipitation of EBNA-2 and p34cdc2, suggesting physical association; (ii) temporal correlation between hyperphosphorylation of EBNA-2 and an increase in p34cdc2 kinase activity; and (iii) ability of purified p34cdc2/cyclin B1 kinase to phosphorylate EBNA-2 in vitro. Hyperphosphorylation of EBNA-2 appears to suppress its ability to transactivate the latent membrane protein 1 (LMP-1) promoter by about 50%. The association between EBNA-2 and PU.1 is also decreased by about 50% in M-phase-arrested cells, as shown by coimmunoprecipitation from cell lysates, suggesting that hyperphosphorylation of EBNA-2 impairs its affinity for PU.1. Finally, endogenous LMP-1 mRNA levels in M phase are around 55% of those in asynchronously growing cells. These results suggest that regulation of gene expression during type III latency may be regulated in a cell-cycle-related manner.

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