Epstein-Barr virus-mediated protection against etoposide-induced apoptosis in BJA-B B cell lymphoma cells: role of Bcl-2 and caspase proteins.

Epstein-Barr virus (EBV)-infected B cell lymphomas are resistant to apoptosis during cancer development and treatment with therapies. The molecular controls that determine why EBV infection causes apoptosis resistance need further definition. EBV-positive and EBV-negative BJA-B B cell lymphoma cell lines were used to compare the expression of selected apoptosis-regulating Bcl-2 and caspase proteins in EBV-related apoptosis resistance, after 8 hr or 18-24 hr etoposide treatment (80 microM). Apoptosis was quantified using morphology and verified with Hoechst 33258 nuclear stain and electron microscopy. Fluorescence activated cell sorting (FACS) was used to analyse effects on cell cycle of the EBV infection as well as etoposide treatment. Anti-apoptotic Bcl-2 and Bcl-XL, pro-apoptotic Bax, caspase-3 and caspase-9 expression and activation were analysed using Western immunoblots and densitometry. EBV-positive cultures had significantly lower levels of apoptosis in untreated and etoposide-treated cultures in comparison with EBV-negative cultures (p < 0.05). FACS analysis indicated a strong G2/M block in both cell sublines after etoposide treatment. Endogenous Bcl-2 was minimal in the EBV-negative cells in comparison with strong expression in EBV-positive cells. These levels did not alter with etoposide treatment. Bcl-XL was expressed endogenously in both cell lines and had reduced expression in EBV-negative cells after etoposide treatment. Bax showed no etoposide-induced alterations in expression. Pro-caspase-9 and -3 were seen in both EBV-positive and -negative cells. Etoposide induced cleavage of caspase-9 in both cell lines, with the EBV-positive cells having proportionally less cleavage product, in agreement with their lower levels of apoptosis. Caspase-3 cleavage occurred in the EBV-negative etoposide-treated cells but not in the EBV-positive cells. The results indicate that apoptosis resistance in EBV-infected B cell lymphomas is promoted by an inactive caspase-3 pathway and elevated expression of Bcl-2 that is not altered by etoposide drug treatment.

Antiserum raised against the Epstein-Barr virus BARF0 protein reacts to HLA-DR beta chain.

The role that Epstein-Barr virus plays in nasopharyngeal carcinoma and Burkitt's lymphoma has been under intense study for many years. With only a limited set of viral genes being expressed in these tumours it has been difficult to understand how the virus could cause/aid in the generation of the tumours. In 1997 a paper was published by Fries et al. [Fries et al. (1997) Identification of a novel protein encoded by the BamH1 A region of the Epstein-Barr virus. J Virol 71: 2765-2771.] in which a rabbit serum was generated and used to identify protein products (RK-BARF0) encoded from the BamH1 A region of EBV. In this paper we have isolated these proteins from two-dimensional gels and identified them, using mass spectrometry, as components of HLA DR.

Epstein-Barr virus-encoded RK-BARF0 protein expression.

The cellular localization of the Epstein-Barr virus-encoded RK-BARF0 protein was analyzed by fluorescence microscopy and immunoblotting. The recombinant RK-BARF0 protein was found to be tightly bound to nuclear structures, whereas 16- to 20-kDa RK-BARF0 derivatives, generated by differential splicing of the RK-BARF0 transcript, were present throughout the cell. Moreover, a previously generated anti-RK-BARF0 rabbit serum was found to cross-react with cellular proteins, showing that the previously identified 30- to 35-kDa membrane-associated proteins do not represent RK-BARF0.

Cutting edge: silencing virus-specific cytotoxic T cell-mediated immune recognition by differential splicing: a novel implication of RNA processing for antigen presentation.

Persistent viruses have developed potent strategies to overcome host immune defenses. In particular, viral interference with Ag presentation by HLA class I molecules can effectively impair the host's CTL function. Here we provide evidence for a novel aspect of differential splicing on endogenous processing of a latent viral transcript resulting in dominant protein isoforms from which the CTL determinant has been deleted. Consequently, virus-infected cells expressing these isoforms were poorly recognized by CTLs. Molecular analysis revealed that this splicing significantly reduced expression of the viral transcript encoding the relevant epitope to levels below the threshold required for CTL recognition. The importance of splicing was further reinforced by the observation of efficient CTL recognition of target cells expressing a truncated viral transcript that abolished differential splicing. Thus, differential splicing, which is a common mechanism of gene regulation in many pathogens, may unexpectedly interfere with immune recognition.

A-type and B-type Epstein-Barr virus differ in their ability to spontaneously enter the lytic cycle.

In this study replication of A-type and B-type Epstein-Barr virus (EBV) strains has been assessed. A-type and B-type type lymphoblastoid cell lines (LCLs) were established by infecting B lymphocytes, isolated from five EBV-seropositive donors, with different A-type and B-type virus isolates. The presence of viral capsid antigens (VCA) in these LCLs was determined by immunofluoresence assay and by immunoblotting. All of the B-type EBV strains were capable of spontaneously generating virus regardless of the origin of the donor cells. In contrast the A-type strains, other than strain IARC-BL36, did not readily produce VCA in any of the different donor lymphocytes used. This study demonstrates another biological difference between the two virus types: their ability to spontaneously enter the lytic cycle.

Intron retention may regulate expression of Epstein-Barr virus nuclear antigen 3 family genes.

The nuclear antigen 3 family genes (EBNA-3, EBNA-4, and EBNA-6) of Epstein-Barr virus (EBV) are important for EBV-induced immortalization and survival of B lymphocytes. However, little is known about how the expression of these genes is regulated. Each of the EBNA-3, EBNA-4, and EBNA-6 genes consists of two exons separated by a small intron. Reverse transcriptase PCR assays revealed that the vast majority of the EBNA-3, EBNA-4, and EBNA-6 mRNA, expressed in transfected and EBV-infected B cells, retained intron sequences. Northern blot and S1 protection assays confirmed that most of the EBNA-3 mRNA contained intron. Examination of deletion mutants of EBNA-3 indicated that the EBNA-3 protein was not necessary for intron retention and that there was no splicing silencing element encoded in the EBNA-3 mRNA. Cell fractionation and RNA gradient analysis revealed that the unspliced EBNA 3 family mRNAs were transported into the cytoplasm and associated with the polysomes. However, Western blot analysis of FLAG-epitope tagged EBNA-3 gave no indication of the presence of splice variant protein forms of EBNA-3. In contrast, transiently transfected cells expressing EBNA-3 revealed a sixfold increase in EBNA-3 protein expression from the genomic EBNA-3 gene compared to EBNA-3 cDNA. These data show that the intronic sequences can influence EBNA-3 protein expression and suggest that intron retention may provide a means for the fine-tuning of expression of the individual EBNA 3 family genes.

Identification of a cytotoxic T-lymphocyte response to the novel BARF0 protein of Epstein-Barr virus: a critical role for antigen expression.

The Epstein-Barr virus (EBV)-encoded BARF0 open reading frame gene products are consistently expressed in EBV-positive Burkitt's lymphoma (BL) cell lines, nasopharyngeal carcinoma cell lines, and lymphoblastoid cell lines (LCLs). Here we show that the BARF0 sequence includes an HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitope. By using theoretically predicted HLA A2 binding motifs and peptide-loaded antigen presentation-deficient T2 cells, polyclonal BARF0-specific CD8(+) CTLs were isolated from four different healthy EBV-seropositive donors but not from two seronegative donors. These CTL lines recognized the peptide epitope LLWAARPRL, which was found to be conserved in 33 of 34 virus strains originating from Caucasian, African, and Asian individuals. The BARF0-specific CTL lines could lyse EBV-negative BL cells stably transfected with the BARF0 gene but did not kill HLA A2-matched EBV-positive BL cells and LCLs in a standard 51Cr release assay. Reverse transcriptase PCR analysis demonstrated that these EBV-positive cell lines expressed significantly lower levels of BARF0 mRNA than transfected cells. This data indicated that the BARF0 epitope could be endogenously processed; however, antigen levels in the target cell were a limiting factor for the effective interaction between BARF0-expressing cells and CTLs. The limited expression of BARF0 antigen in EBV-infected BL cells and LCLs might contribute to the escape of immune recognition from virus-specific CTLs present in the host.

Regulation of interleukin-1beta transcription by Epstein-Barr virus involves a number of latent proteins via their interaction with RBP.

Epstein-Barr virus (EBV) infects B cells, resulting in the outgrowth of immortalised lymphoblastoid cell lines (LCLs). Here, we demonstrate through the use of intracellular staining that interleukin-1beta (IL-1beta) is expressed in LCLs and investigate the influence of the individual latent proteins on the expression of IL-1beta. Using RT-PCR, IL-1beta was shown to be up-regulated in EBV-transformed LCLs as well as in group III Burkitt's lymphoma (BL) cell lines, compared with group I BL cell lines. The up-regulation of IL-1beta message could be mediated by the latent membrane protein-1, EBV nuclear proteins 2, 3, 4, and 6 genes. Electrophoretic mobility shift assays (EMSAs) demonstrated that the -300 region of the IL-1beta promoter, which contains a nuclear factor-kappaB (NF-kappaB) binding site, contained a functional RBP binding site. Binding of RBP to this site could be inhibited by addition of EBV nuclear proteins 3 and 6, suggesting that these proteins displace RBP from its recognition sequence, removing transcriptional repression and allowing gene transcription to occur. In group I BL cells, containing low levels of NF-kappaB, only RBP binding was observed in EMSAs, whereas NF-kappaB binding could be demonstrated in EBV-transformed B cell lines containing high levels of activated NF-kappaB. In addition, the expression of latent membrane protein-1 led to activation of NF-kappaB that was capable of binding the IL-1beta promoter. The study demonstrates that EBV can up-regulate IL-1beta expression, possibly by using RBP, NF-kappaB, or both.

Both A type and B type Epstein-Barr virus nuclear antigen 6 interact with RBP-2N.

Using the yeast two-hybrid system, Epstein-Barr virus nuclear antigen 6A (EBNA6A) was found to interact with the RBP-2N isoform of RBP-J kappa. The interaction of EBNA6A and EBNA6B with RBP-2N was compared and the results indicated that EBNA6B was less efficient at interacting with RBP-2N than was EBNA6A. Deletion mutation analysis of EBNA6A identified a region involved in the interaction with RBP-2N, while analysis of RBP-2N identified a domain which interacts with EBNA6A. The region of RBP-2N to which EBNA6A binds has previously been shown to interact with EBNA2.

Identification of a novel protein encoded by the BamHI A region of the Epstein-Barr virus.

An unusual set of 3' coterminal, spliced mRNAs transcribed through the BamHI A fragment have been previously identified in nasopharyngeal carcinoma (NPC) tissues. These RNAs have also been detected at low levels in Burkitt's lymphoma (BL) cell lines and Epstein-Barr virus (EBV)-transformed lymphocytes. Sequence analyses of clones from a cDNA library derived from the C15 NPC tumor indicated that the primary transcripts are differentially spliced, giving rise to a family of related transcripts, all of which encompass the BARF0 open reading frame (ORF) at the 3' end of the transcripts. One cDNA was identified that extended the BARF0 ORF at the 5' end, forming the RK-BARF0 ORF. In this study, a rabbit antiserum to a synthetic peptide representing an amino acid sequence encoded by the BARF0 ORF was prepared. This antiserum detected a glutathione S-transferase-BARF0 fusion protein and both BARF0 and RK-BARF0 proteins expressed from transfected constructs in H1299 cells. The serum also immunoprecipitated the 20-kDa BARF0 and 30-kDa RK-BARF0 in vitro-translated proteins. Immunoblot analyses identified a protein doublet of 30 and 35 kDa in all of the EBV-infected cell lines tested. Cellular fractionation studies revealed that the proteins were membrane associated. The sizes of the proteins detected in cell lines and their association with membranes suggests that they are likely encoded by the RK-BARF0 transcript, which is predicted to contain a membrane localization signal. The proteins were also detected in protein extracts prepared from NPC biopsies and a BL biopsy but not from hairy leukoplakia, a permissive EBV infection. These results reveal that the rightward RNA transcripts from the BamHI A region of EBV encode one or more proteins that are expressed in latently infected cells and in tumor tissue.

Identification of type B-specific and cross-reactive cytotoxic T-lymphocyte responses to Epstein-Barr virus.

Persistent Epstein-Barr virus (EBV) infection is primarily controlled by HLA class I-restricted memory cytotoxic T-cell (CTL) responses which can be reactivated in vitro by stimulation of peripheral blood lymphocytes with autologous lymphoblastoid cell lines. During an investigation of a donor infected by both type A and type B EBV, CTL specific for type B EBV were isolated. The CTL were found to recognize an epitope encoded by the EBNA-6B gene. The minimal epitope sequence was identified as QNGALAINTF, corresponding to residues 213 to 222 in the EBNA-6B protein, and presentation of this epitope was shown to be via HLA B62 (B15). This is the first report of the characterization of an epitope that is EBV type B specific. CTL recognizing sequences common to type A and type B EBV were identified as well. A cross-reactive epitope recognized by these CTL was encoded within the EBNA-6 gene of both type A and type B. This minimal sequence for this epitope was LLDFVRFMGV (residues 284 to 293 in both types), and the epitope was restricted through HLA A*0201. This second epitope sequence overlaps with a published EBV B44-restricted epitope (EENLLDFVRF). The implications of these findings are discussed with respect to the design and efficacy of epitope-based vaccines.

Epstein-Barr nuclear antigen-3 and -4 interact with RBP-2N, a major isoform of RBP-J kappa in B lymphocytes.

The Epstein-Barr nuclear antigen (EBNA)-3 and EBNA-4 proteins are thought to act as transcriptional transactivators. The yeast two-hybrid system and coimmunoprecipitation were used to demonstrate that EBNA-3 and -4 associate with the DNA-binding protein RBP-2N, an isoform of RBP-J kappa. A comparison between EBNA-3, EBNA-4, and EBNA-6 binding to RBP-2N indicated that EBNA-3 enhanced beta-galactosidase activity 4-fold more than EBNA-6 and 30-fold more than EBNA-4. Assay of RBP-2N deletion mutants demonstrated that EBNA-3 binds to regions of RBP-2N which are distinct from those to which EBNA-2 and -6 interact, whereas EBNA-4 binds to the same region of RBP-2N as EBNA-2 and -6 (amino acids 159-331 of RBP-2N). Interaction of both A- and B-type EBNA-3 with RBP-2N was also demonstrated by immunoprecipitation. RT-PCR analysis of a panel of B cell lymphomas and lymphoblastoid cell lines demonstrated that higher levels of RBP-2N were expressed, in comparison to RBP-J kappa, indicating that RBP-2N is a major isoform expressed in B cells. These results suggest that all the EBNA-3 family proteins lead to transcriptional regulation via interaction with RBP-2N.

Induction of pleckstrin by the Epstein-Barr virus nuclear antigen 3 family.

The Epstein-Barr virus (EBV) encoded nuclear antigens, EBNA-3, -4, and -6 (EBNA 3 family) are expressed in latently infected human B-cells and are involved in the transformation of lymphocytes by EBV. Human Burkitt's lymphoma (BL) dG75 cells which stably expressed either the complete EBNA 3 gene family or the vector alone were generated and changes in gene activity in these transfectants were assayed using the differential display of mRNA technique. For the first time, the human gene pleckstrin, which is thought to be involved in signalling and differentiation of hemopoietic cells, was found to be upregulated in the presence of the EBNA 3 protein family, but not in cells expressing the individual EBNA-3, -4, or -6 gene. Pleckstrin was increased up to sevenfold in different cell clones and the bulk culture of EBNA 3 gene family expressing cells as demonstrated by Northern blot. RT-PCR, and immunoblot in contrast to EBV-negative BL cells, pleckstrin RNA and protein were highly expressed in EBV growth transformed lymphoblastoid cell lines which are thought to play an important role in EBV persistence in vivo. These data suggest that induction of pleckstrin might be important for the EBV-controlled activation of cells and offers a unique biological system for analyzing pleckstrin function.

Identification of a naturally occurring recombinant Epstein-Barr virus isolate from New Guinea that encodes both type 1 and type 2 nuclear antigen sequences.

In this report we describe an Epstein-Barr virus isolate, derived from the peripheral blood lymphocytes of a healthy adult from Papua New Guinea, that is a recombinant of the two major Epstein-Barr virus types, encoding type 1 Epstein-Barr nuclear antigen 2 (EBNA2) sequences, and type 2 EBNA3, EBNA4, and EBNA6 sequences.

Unusually high frequency of Epstein-Barr virus genetic variants in Papua New Guinea that can escape cytotoxic T-cell recognition: implications for virus evolution.

Cytotoxic T lymphocytes (CTLs) which recognize viral antigens in association with human leukocyte antigens (HLAs) play an important role in controlling persistent virus infections. These viruses use several mechanisms to evade the immune response, including mutations that affect either T-cell receptor recognition or binding of viral epitopes to the HLA. It has recently been proposed that the distribution of HLA frequencies and the specific CTL response may influence the long-term evolution of Epstein-Barr virus (EBV) by selecting variants which lack immunodominant CTL epitopes. To test this hypothesis, we have studied EBV isolates from two genetically distinct Papua New Guinea (PNG) populations, residing in coastal and highland regions, for polymorphism within seven viral CTL epitope sequences restricted through several class I HLAs. Surprisingly, all EBV isolates analyzed displayed identical amino acid substitutions within HLA A11-, B35- and B8-restricted CTL epitope sequences which completely abrogated CTL recognition and binding of synthetic peptides to HLA molecules. Furthermore, these substitutions revealed no correlation with the contemporary distribution of HLAs in the different PNG populations, which argues for a minimal influence of immune pressure. The sequence homology between EBV isolates from coastal and highland PNG suggests that the virus may have had a single origin and, more importantly, that these isolates are genetically distinct from those present in a Caucasian population.

Burkitt's lymphoma cells are resistant to programmed cell death in the presence of the Epstein-Barr virus latent antigen EBNA-4.

Group I Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cells display a surface phenotype characteristic of germinal centre B cells and readily undergo apoptosis in response to a variety of stimuli, including serum deprivation. Activation of EBV latent gene expression has been shown to increase the survival of these tumour cells by blocking programmed cell death. To investigate the nature of this protection, we assessed the function of the EBV latent EBNA-4 gene in a group I lymphoma line, dG75. Group I BL cells induced to undergo apoptosis in response to serum starvation were protected in the presence of EBNA-4 protein. A possible factor underlying this EBNA-4-associated survival was increased expression of the oncoprotein bcl-2, a known repressor of cell death. Together these data suggest that EBNA-4 plays an important role in the regulation of programmed cell death in BL tumour cells.

Dominant selection of an invariant T cell antigen receptor in response to persistent infection by Epstein-Barr virus.

To examine T cell receptor (TCR) diversity involved in the memory response to a persistent human pathogen, we determined nucleotide sequences encoding TCR-alpha and -beta chains from HLA-B8-restricted, CD8+ cytotoxic T cell clones specific for an immunodominant epitope (FLRGRAYGL) in Epstein-Barr virus (EBV) nuclear antigen 3. Herein, we show that identical TCR protein sequences are used by clones from each of four healthy unrelated virus carriers; a clone from a fifth varied conservatively at only two residues. This dominant selection of alpha and beta chain rearrangements suggest that a persistent viral infection can select for a highly focused memory response and indicates a strong bias in gene segment usage and recombination. A novel double-step semiquantitative polymerase chain reaction (PCR) procedure and direct sequencing of amplified TCR cDNA from fresh lymphocytes derived from three HLA-B8 individuals detected transcripts specific for the conserved beta chain in an EBV-seropositive donor but not in two seronegative donors. This report describes an unprecedented degree of conservation in TCR selected in response to a natural persistent infection.