Andrew I. Bell

Epstein-Barr virus-associated Burkitt lymphomagenesis selects for downregulation of the nuclear antigen EBNA2

Epstein–Barr virus (EBV) is etiologically linked to endemic Burkitt lymphoma (BL), but its contribution to lymphomagenesis, versus that of the chromosomal translocation leading to c-myc gene deregulation, remains unclear. The viruss growth-transforming (Latency III) program of gene expression is extinguished in tumor cells, and only a single viral protein, the EBV nuclear antigen (EBNA)1, is expressed via the alternative Latency I program. It is not known if BL arises from a B-cell subset in which EBV naturally adopts a Latency I infection or if a clone with limited antigen expression has been selected from an EBV-transformed Latency III progenitor pool. Here we identify a subset of BL tumors in which the Latency III-associated EBNA promoter Wp is active and most EBNAs are expressed, but where a gene deletion has specifically abrogated the expression of EBNA2. This implies that BL can be selected from a Latency III progenitor and that the principal selection pressure is for downregulation of the c-Myc antagonist EBNA2.

Stringent spacing requirements for transcription activation by CRP.

The cyclic AMP receptor protein-cAMP complex (CRP-cAMP) binds at a variety of distances upstream of several E. coli promoters and activates transcription. We have constructed a model system in which a consensus CRP binding site is placed at different distances upstream of the melR promoter. CRP-cAMP activates transcription from melR when bound at a number of positions, all of which lie on the same face of the DNA helix. The two distances at which transcription is strongly activated correspond exactly to those at which CRP-cAMP binds upstream of the well-studied galP1 and lac promoters. Footprinting of the synthetic promoters reveals that RNA polymerase makes identical contacts with their -10 regions even though CRP-cAMP binds at a different distance in each case. Kinetic analysis in vitro indicates that CRP-cAMP activates transcription from these promoters in similar but distinct ways. A model is proposed to explain this two-position activation.

Target cells of Epstein-Barr-virus (EBV)-positive post-transplant lymphoproliferative disease: similarities to EBV-positive Hodgkin's lymphoma.

BACKGROUND Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) encompasses a histologically broad range of lesions, arising from the expanded pool of EBV-infected B cells in the immunocompromised host. Identification of the precise cellular origin of these tumours could clarify their pathogenesis. METHODS Of 13 cases of EBV-positive cases of PTLD characterised by histological analysis, pattern of EBV gene expression, and clinical course, 11 had monoclonal or biclonal lesions in which we determined the progenitor B cell by immunoglobulin heavy chain (IgH) genotyping. RESULTS Two tumours had a naive B cell genotype and two showed patterns of IgH somatic mutation typical of antigen-selected (post-germinal-centre) memory cells. All four arose early post-transplant and expressed the markers of EBV transformation--Epstein-Barr nuclear antigen (EBNA) 2 and latent membrane protein (LMP) 1. However, seven tumours, either of early or late onset and including some with downregulated EBNA 2 and LMP 1, arose from post-germinal cells with randomly mutated or sterile IgH genotypes usually incompatible with B-cell survival in vivo. INTERPRETATION PTLD can arise from a broad range of target B cells and not only from the pool of antigen-selected memory cells that EBV generally colonises in immunocompetent individuals. Tumour development seems frequently associated with the EBV-induced rescue and expansion of B cells that have failed the physiological process of germinal centre selection into memory. This finding shows an unexpected connection between pathogenesis of PTLD and that of EBV-positive Hodgkins lymphoma, another B-cell malignancy of atypical post-germinal-centre cell origin.

An Epstein-Barr virus anti-apoptotic protein constitutively expressed in transformed cells and implicated in burkitt lymphomagenesis: the Wp/BHRF1 link.

Two factors contribute to Burkitt lymphoma (BL) pathogenesis, a chromosomal translocation leading to c-myc oncogene deregulation and infection with Epstein-Barr virus (EBV). Although the virus has B cell growth–transforming ability, this may not relate to its role in BL since many of the transforming proteins are not expressed in the tumor. Mounting evidence supports an alternative role, whereby EBV counteracts the high apoptotic sensitivity inherent to the c-myc–driven growth program. In that regard, a subset of BLs carry virus mutants in a novel form of latent infection that provides unusually strong resistance to apoptosis. Uniquely, these virus mutants use Wp (a viral promoter normally activated early in B cell transformation) and express a broader-than-usual range of latent antigens. Here, using an inducible system to express the candidate antigens, we show that this marked apoptosis resistance is mediated not by one of the extended range of EBNAs seen in Wp-restricted latency but by Wp-driven expression of the viral bcl2 homologue, BHRF1, a protein usually associated with the virus lytic cycle. Interestingly, this Wp/BHRF1 connection is not confined to Wp-restricted BLs but appears integral to normal B cell transformation by EBV. We find that the BHRF1 gene expression recently reported in newly infected B cells is temporally linked to Wp activation and the presence of W/BHRF1-spliced transcripts. Furthermore, just as Wp activity is never completely eclipsed in in vitro–transformed lines, low-level BHRF1 transcripts remain detectable in these cells long-term. Most importantly, recognition by BHRF1-specific T cells confirms that such lines continue to express the protein independently of any lytic cycle entry. This work therefore provides the first evidence that BHRF1, the EBV bcl2 homologue, is constitutively expressed as a latent protein in growth-transformed cells in vitro and, in the context of Wp-restricted BL, may contribute to virus-associated lymphomagenesis in vivo.

Three restricted forms of Epstein–Barr virus latency counteracting apoptosis in c-myc-expressing Burkitt lymphoma cells

Epstein–Barr virus (EBV), a human herpesvirus, transforms B cell growth in vitro through expressing six virus-coded Epstein–Barr nuclear antigens (EBNAs) and two latent membrane proteins (LMPs). In many EBV-associated tumors, however, viral antigen expression is more restricted, and the aetiological role of the virus is unclear. For example, endemic Burkitt lymphoma (BL) classically presents as a monoclonal, c-myc-translocation-positive tumor in which every cell carries EBV as an EBNA1-only (Latency I) infection; such homogeneity among EBV-positive cells, and the lack of EBV-negative comparators, hampers attempts to understand EBVs role in BL pathogenesis. Here, we describe an endemic BL that was unusually heterogeneous at the single-cell level and, in early passage culture, yielded a range of cellular clones, all with the same c-myc translocation but differing in EBV status. Rare EBV-negative cells were isolated alongside EBV-positive cells displaying one of three forms of restricted latency: (i) conventional Latency I expressing EBNA1 only from a WT virus genome, (ii) Wp-restricted latency expressing EBNAs 1, 3A, 3B, 3C, and -LP only from an EBNA2-deleted genome, and (iii) a previously undescribed EBNA2+/LMP1− latency in which all six EBNAs are expressed again in the absence of the LMPs. Interclonal comparisons showed that each form of EBV infection was associated with a specific degree of protection from apoptosis. Our work suggests that EBV acts as an antiapoptotic rather than a growth-promoting agent in BL by selecting among three transcriptional programs, all of which, unlike the full virus growth-transforming program, remain compatible with high c-myc expression.

Epstein-Barr Virus Nuclear Antigen 2 (EBNA2) Gene Deletion Is Consistently Linked with EBNA3A, -3B, and -3C Expression in Burkitt's Lymphoma Cells and with Increased Resistance to Apoptosis

ABSTRACT Most Epstein-Barr virus (EBV)-positive Burkitts lymphomas (BLs) carry a wild-type EBV genome and express EBV nuclear antigen 1 (EBNA1) selectively from the BamHI Q promoter (latency I). Recently we identified a distinct subset of BLs carrying both wild-type and EBNA2 gene-deleted (transformation-defective) viral genomes. The cells displayed an atypical “BamHI W promoter (Wp)-restricted” form of latency where Wp (rather than Qp) was active and EBNA1, -3A, -3B, -3C, and -LP were expressed in the absence of EBNA2 or latent membrane proteins 1 and 2. Here we present data strongly supporting the view that the EBNA2-deleted genome is transcriptionally active in these cells and the wild-type genome is silent. Single-cell cloning of three parental Wp-restricted BL lines generated clones carrying either both viral genomes or the EBNA2-deleted genome only, never clones with the wild-type genome only. All rescued clones displayed the Wp-restricted form of latency characteristic of the parent line and retained the original parent cell phenotype. Interestingly, Wp-restricted parent lines and derived clones were markedly more resistant to inducers of apoptosis than standard latency I BL lines. Furthermore, in vitro infection of EBV-negative BL lines with an EBNA2 gene-deleted virus generated EBV-positive converts with Wp-restricted latency and a similarly marked apoptosis resistance. We postulate that, in the subset of BLs displaying Wp-restricted latency, infection of a tumor progenitor cell with an EBNA2 gene-deleted virus has provided that cell with a survival advantage through broadening antigen expression to include the EBNA3 proteins.

Features Distinguishing Epstein-Barr Virus Infections of Epithelial Cells and B Cells: Viral Genome Expression, Genome Maintenance, and Genome Amplification

ABSTRACT Epstein-Barr virus (EBV) is associated with malignant diseases of lymphoid and epithelial cell origin. The tropism of EBV is due to B-cell-restricted expression of CD21, the major receptor molecule for the virus. However, efficient infection of CD21− epithelial cells can be achieved via transfer from EBV-coated B cells. We compare and contrast here the early events following in vitro infection of primary B cells and epithelial cells. Using sensitive, quantitative reverse transcription-PCR assays for several latent and lytic transcripts and two-color immunofluorescence staining to analyze expression at the single cell level, we confirmed and extended previous reports indicating that the two cell types support different patterns of transcription. Furthermore, whereas infection of B cells with one or two copies of EBV resulted in rapid amplification of the viral genome to >20 copies per cell, such amplification was not normally observed after infection of primary epithelial cells or undifferentiated epithelial lines. In epithelial cells, EBNA1 expression was detected in only ca. 40% of EBER+ cells, and the EBV genome was subsequently lost during prolonged culture. One exception was that infection of AGS, a gastric carcinoma line, resulted in maintenance of EBNA1 expression and amplification of the EBV episome. In contrast to B cells, where amplification of the EBV episome occurred even with a replication-defective BZLF1-knockout virus, amplification in AGS cells was dependent upon early lytic cycle gene expression. These data highlight the influence of the host cell on the outcome of EBV infection with regard to genome expression, amplification, and maintenance.

Tonsillar homing of Epstein-Barr virus–specific CD8+ T cells and the virus-host balance

Patients with infectious mononucleosis (IM) undergoing primary EBV infection show large expansions of EBV-specific CD8+ T cells in the blood. While latent infection of the B cell pool is quickly controlled, virus shedding from lytically infected cells in the oropharynx remains high for several months. We therefore studied how responses localize to the tonsil, a major target site for EBV, during primary infection and persistence. In acute IM, EBV-specific effectors were poorly represented among CD8+ T cells in tonsil compared with blood, coincident with absence of the CCR7 lymphoid homing marker on these highly activated cells. In patients who had recently recovered from IM, latent epitope reactivities were quicker than lytic reactivities both to acquire CCR7 and to accumulate in the tonsil, with some of these cells now expressing the CD103 integrin, which mediates retention at mucosal sites. By contrast, in long-term virus carriers in whom both lytic and latent infections had been controlled, there was 2- to 5-fold enrichment of lytic epitope reactivities and 10- to 20-fold enrichment of latent epitope reactivities in tonsil compared with blood; up to 20% of tonsillar CD8+ T cells were EBV specific, and many now expressed CD103. We suggest that efficient control of EBV infection requires appropriate CD8+ T cell homing to oropharyngeal sites.

Quantitative Studies of Epstein-Barr Virus-Encoded MicroRNAs Provide Novel Insights into Their Regulation

ABSTRACT Epstein-Barr virus (EBV) has been shown to encode at least 40 microRNAs (miRNAs), an important class of molecules that negatively regulate the expression of many genes through posttranscriptional mechanisms. Here, we have used real-time PCR assays to quantify the levels of EBV-encoded BHRF1 and BART miRNAs in latently infected cells and in cells induced into the lytic cycle. During latency, BHRF1 miRNAs were seen only in cells with detectable Cp- and/or Wp-initiated EBNA transcripts, while the BART miRNAs were expressed in all forms of latent infection. Surprisingly, levels of different BART miRNAs were found to vary up to 50-fold within a cell line. However, this variation could not be explained by differential miRNA turnover, as all EBV miRNAs appeared to be remarkably stable. Following entry into the virus lytic cycle, miR-BHRF1-2 and -1-3 were rapidly induced, coincident with the onset of lytic BHRF1 transcripts, while miR-BHRF1-1 expression was delayed until 48 h and correlated with the appearance of Cp/Wp-initiated EBNA transcripts. In contrast, levels of BART miRNAs were relatively unchanged during virus replication, despite dramatic increases in BART transcription. Finally, we show that BHRF1 and BART miRNAs were delayed relative to the induction of BHRF1 and BART transcripts in freshly infected primary B cell cultures. In summary, our data show that changes in BHRF1 and BART transcription are not necessarily reflected in altered miRNA levels, suggesting that miRNA maturation is a key step in regulating steady-state levels of EBV miRNAs.

The functional subunit of a dimeric transcription activator protein depends on promoter architecture.

In Class I CAP‐dependent promoters, the DNA site for CAP is located upstream of the DNA site for RNA polymerase. In Class II CAP‐dependent promoters, the DNA site for CAP overlaps the DNA site for RNA polymerase, replacing the ‐35 site. We have used an ‘oriented heterodimers’ approach to identify the functional subunit of CAP at two Class I promoters having different distances between the DNA sites for CAP and RNA polymerase [CC(‐61.5) and CC(‐72.5)] and at one Class II promoter [CC(‐41.5)]. Our results indicate that transcription activation at Class I promoters, irrespective of the distance between the DNA sites for CAP and RNA polymerase, requires the activating region of the promoter‐proximal subunit of CAP. In striking contrast, our results indicate that transcription activation at Class II promoters requires the activating region of the promoter‐distal subunit of CAP.