Epstein-Barr virus.

Epstein–Barr virus (EBV) was discovered 40 years ago from examining electron micrographs of cells cultured from Burkitt's lymphoma, a childhood tumour that is common in sub-Saharan Africa, where its unusual geographical distribution — which matches that of holoendemic malaria —indicated a viral aetiology. However, far from showing a restricted distribution, EBV — a γ-herpesvirus — was found to be widespread in all human populations and to persist in the vast majority of individuals as a lifelong, asymptomatic infection of the B-lymphocyte pool. Despite such ubiquity, the link between EBV and 'endemic' Burkitt's lymphoma proved consistent and became the first of an unexpectedly wide range of associations discovered between this virus and tumours.

Epstein–Barr virus: 40 years on

RNA silencing processes are guided by small RNAs that are derived from double-stranded RNA. To probe for function of RNA silencing during infection of human cells by a DNA virus, we recorded the small RNA profile of cells infected by Epstein-Barr virus (EBV). We show that EBV expresses several microRNA (miRNA) genes. Given that miRNAs function in RNA silencing pathways either by targeting messenger RNAs for degradation or by repressing translation, we identified viral regulators of host and/or viral gene expression.

https://science.sciencemag.org/content/sci/304/5671/734.full.pdf

Identification of Virus-Encoded MicroRNAs

CD40 is an integral membrane protein found on the surface of B lymphocytes, dendritic cells, follicular dendritic cells, hematopoietic progenitor cells, epithelial cells, and carcinomas. It is a 45-50 kDa glycoprotein of 277 aa, which is a member of the tumor necrosis factor receptor superfamily. The CD40 gene maps to human chromosome 20q11-2-q13-2. CD40 binds to a ligand (CD40-L) which is an approximately 35 kDa glycoprotein of 261 aa, a member of the tumor necrosis factor superfamily. The CD40-L gene maps to human chromosome Xq24. This CD40-L is expressed on activated T cells, mostly CD4+ but also some CD8+ as well as basophils/mast cells. The CD40-L is defective in the X-linked hyper-IgM syndrome. Cross-linking of CD40 with immobilized anti-CD40 or cells expressing CD40-L induces B cells to proliferate strongly, and addition of IL-4 or IL-13 allows the generation of factor-dependent long-term normal human B cell lines and the secretion of IgE following isotype switching. Addition of IL-10 results in very high immunoglobulin production with limited cell proliferation. IL-10 induces naive B cells to produce IgG3, IgG1, and IgA1, and further addition of TGF beta permits the secretion of IgA2. Several evidences suggest that CD40-dependent activation of B cells is important for the generation of memory B cells within the germinal centers: (i) CD40 activated germinal center B cells cultured in the presence of IL-4 acquire a memory B cell phenotype, (ii) CD40 activated B cells can undergo isotype switching, (iii) the deficit of CD40-L results in the hyper-IgM syndrome characterized by lack of germinal centers in secondary lymphoid organ follicles and lack of IgG, IgA, and IgE, and (iv) CD40-L positive T cells are present in secondary follicles. Thymic epithelial cells, activated monocytes, and dendritic cells express CD40 antigen which may be involved in an enhanced cytokine production by these cells, allowing an amplification of T cell proliferation. Finally, as other members of the tumor necrosis factor receptor family have been shown to bind several ligands, it is possible that CD40 may bind other ligands that may trigger CD40 on different cell types such as hematopoietic cells or epithelial cells.

The CD40 Antigen and its Ligand

An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells

We have previously described the derivation of a monoclonal antibody, S2C6, to a novel 50 Kdalton antigen associated with human urinary bladder carcinoma. No reactions were obtained with carcinomas of unrelated origin or with normal urothelial cells. However, the antibody also reacted with a similar antigen on some cell lines of B lymphocyte origin. Using large panels of target cells we have now shown that this reactivity was entirely restricted to cells of the B lineage within the haematopoietic system. As opposed to its apparent restriction to malignant cells of the urothelium, the S2C6 antigen was expressed by normal B lymphocytes as well as by many malignant B cells (chronic lymphocytic leukaemia, hairy cell leukaemia and immunocytoma). Pre-B cells derived from acute lymphocytic leukaemia and plasma cells from multiple myeloma lacked the antigen. Expression was significantly enhanced on cultured B cells from Burkitt lymphomas and on Epstein-Barr virus-transformed lymphoblastoid cell lines including those of the pre-B phenotype derived from fetal bone marrow. As judged from the molecular size and the distribution pattern displayed by the S2C6 antigen it appears to be distinct from other B cell antigens previously described. A possible relation of the S2C6 antigen to a receptor for B cell growth factors is discussed.

A p50 surface antigen restricted to human urinary bladder carcinomas and B lymphocytes

The human B lymphocyte and carcinoma-associated Ag, CDw40, (p50, Bp50) is a receptor candidate for normal growth regulation. Interaction of mAb with this pan-B Ag, together with preactivating agents such as 12-O-tetradecanoylphorbol-13-acetate or anti-mu, deliver strong growth-promoting signals to the cells. We here demonstrate that signaling through this Ag is dependent on its aggregation on the cell surface. Thus, monovalent antibody fragments were relatively inefficient in this respect but effectively blocked stimulation by intact antibody. By using affinity purified CDw40 protein we have also demonstrated that it is antigenically distinct from other B cell-associated Ag, including the six differentiation clusters CD19 to CD24. The mAb S2C6 and G28.5, prepared by immunizing mice with human bladder carcinoma cells or tonsillar B-cells, respectively, were the only antibodies giving detectable binding. Either of these antibodies could also completely block the binding of the other, suggesting an identity or structural proximity of the epitopes recognized. The CDw40 Ag was shown to be a phosphoprotein lacking intrinsic protein kinase activity. The results provide further evidence for CDw40 being an important B cell growth factor receptor which may also have growth regulatory functions in the development of certain human carcinomas.

The human B lymphocyte and carcinoma antigen, CDw40, is a phosphoprotein involved in growth signal transduction.

The developmental stage from which stems the malignant B cell population in Burkitt’s lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rearranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells and their descendents. Rearranged Vκ region genes from 10 κ-expressing sporadic and endemic BL-derived cell lines (9 IgM and 1 IgG positive) and three κ-expressing endemic BL biopsy specimens were amplified by polymerase chain reaction and sequenced. In addition, VH region gene sequences from these cell lines were determined. All BL cell lines and the three biopsy specimens carried somatically mutated V region genes. The average mutation frequency of rearranged Vκ genes from eight BL cell lines established from sporadic BL was 1.8%. A higher frequency (6%) was found in five endemic cases (three biopsy specimens and two BL cell lines). The detection of somatic mutations in the rearranged V region genes suggests that both sporadic and endemic BL represent a B-cell malignancy originating from germinal center B cells or their descendants. Interestingly, the mutation frequency detected in sporadic BL is in a range similar to that characteristic for IgM-expressing B cells in the human peripheral blood and for μ chain-expressing germinal center B cells, whereas the mutation frequency found in endemic BL is significantly higher.

/pdf/burkitt-s-lymphoma-is-a-malignancy-of-mature-b-cells-35c4mj3r17.pdf

Burkitt’s Lymphoma Is a Malignancy of Mature B Cells Expressing Somatically Mutated V Region Genes

Five peptides corresponding to amino acid sequences predicted from all three reading frames of the nucleotide sequence of the third internal repeat array (IR3) of the Epstein-Barr virus (EBV) genome were synthesized chemically. All five peptides elicited antipeptide antibodies in rabbits. The antiserum raised against a 14-residue copolymer of glycine and alanine gave brilliant EBV-specific nuclear staining in the anticomplement immunofluorescence (ACIF) assay, in line with the original definition of the EBV-determined nuclear antigen (EBNA) [Reedman, B. M. & Klein, G. (1973) Int. J. Cancer 11, 499-520]. Eight EBNA and EBV DNA-carrying lines showed nuclear staining with the antipeptide antibody, whereas five EBV DNA negative lines failed to stain. The staining pattern was more discretely punctate than the finely dispersed diffuse EBNA staining obtained with human antisera. Human EBV antibody-positive but not EBV-negative sera reacted with the synthetic peptide in an ELISA test. The peptide-specific antibodies were purified from the sera of healthy EBV-seropositive persons by affinity chromatography with the peptide. They gave an EBV-specific, brilliant punctate nuclear ACIF staining similar to that of the rabbit antipeptide antibodies. It was concluded that the glycine-alanine structure encoded by the IR3 region contains a native determinant of EBNA, detected by the ACIF test. Immunoblotting with the rabbit and human peptide-specific antibodies identified poly-peptides that varied between 70 and 92 kilodaltons in size in different EBV-positive cell lines, corresponding closely to a previously identified variation pattern in the size of EBNA. In addition, rabbit antipeptide antibodies identified two cellular polypeptides, 44 and 49 kilodaltons in size.

Antibodies against a synthetic peptide identify the Epstein-Barr virus-determined nuclear antigen.

The expression of Epstein-Barr virus (EBV)-determined antigens associated with growth-transformation of B cells was studied by immunoblotting with human sera from healthy donors. Four antigens were detected in EBV-carrying cell lines and in B lymphocytes early after infection with the transforming B95-8 substrain of virus. They were not found in uninfected cells, nor could they be demonstrated with sera lacking antibodies to EBV antigens. All four antigens were nuclear. Each of them varied in size in the different cell lines. The two antigens with the lowest molecular weight were identified as EBV-determined nuclear antigens (EBNAs) 1 and 2. The two high molecular weight antigens (140-160 kDa and 150-180 kDa, respectively) were detected with 6 of 16 EBV antibody-positive sera. These proteins appeared to be antigenically unrelated to each other and to EBNAs 1 and 2 and were designated EBNAs 3 and 4. Like EBNAs 1 and 2, they bound to double- and single-stranded DNA in vitro.

Barbro Ehlin-Henriksson

Papers

A p50 surface antigen restricted to human urinary bladder carcinomas and B lymphocytes

The human B lymphocyte and carcinoma antigen, CDw40, is a phosphoprotein involved in growth signal transduction.

Burkitt’s Lymphoma Is a Malignancy of Mature B Cells Expressing Somatically Mutated V Region Genes

Antibodies against a synthetic peptide identify the Epstein-Barr virus-determined nuclear antigen.

Four virally determined nuclear antigens are expressed in Epstein-Barr virus-transformed cells.