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

The complete (172,282 base pairs) nucleotide sequence of the B95-8 strain of Epstein-Barr virus has been established using the dideoxynucleotide/M13 sequencing procedure. Many RNA polymerase II promoters have been mapped and the mRNAs from these promoters have been assigned to the latent or early/late productive virus cycles. Likely protein-coding regions have been identified and three of these have been shown to encode a ribonucleotide reductase, a DNA polymerase and two surface glycoproteins.

DNA sequence and expression of the B95-8 Epstein—Barr virus genome

Epstein-Barr virus (EBV) infects human B lymphocytes, transforming the infected cells into dividing blasts that can proliferate indefinitely. The viral genome of 172 kilobase pairs (kbp) is a plasmid in most transformed cells. We have identified a region of EBV DNA, termed oriP (nucleotides 7,333-9,109 of strain B95-8), which acts in cis to permit linked DNAs to replicate as plasmids in cells containing EBV DNA. We have postulated the existence of a trans-acting gene allowing oriP function. Here we report that this gene lies in a 2.6-kbp region of the viral genome (nucleotides 107, 567-110, 176) which encodes the EBNA-1 antigen. We show that circular DNAs containing oriP, the EBNA-1 gene and a selectable marker replicate autonomously in cells derived from at least four developmental lineages and from at least three species. We also find that the one-third of the EBNA-1 gene repetitive in sequence is not essential for the trans-acting function that EBNA-1 gives oriP.

Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells

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

Many tumors express tumor-specific antigens capable of being presented to CD8+ T cells by major histocompatibility complex (MHC) class I molecules. Antigen presentation models predict that the tumor cell itself should present these antigens to T cells. However, when conditions for the priming of tumor-specific responses were examined in mice, no detectable presentation of MHC class I-restricted tumor antigens by the tumor itself was found. Rather, tumor antigens were exclusively presented by host bone marrow-derived cells. Thus, MHC class I-restricted antigens are efficiently transferred in vivo to bone marrow-derived antigen-presenting cells, which suggests that human leukocyte antigen matching may be less critical in the application of tumor vaccines than previously thought.

https://science.sciencemag.org/content/sci/264/5161/961.full.pdf

Role of bone marrow-derived cells in presenting MHC class I-restricted tumor antigens

Epstein-Barr virus (EBV) has been associated with serious or fatal lymphoproliferative disease in immunocompromised patients. EBV nuclear protein 2 and latent membrane protein are characteristically expressed in B lymphocytes proliferating in vitro in response to growth transformation by EBV. These two proteins are thought to be effectors of lymphocyte growth since they increase the expression of B-lymphocyte activation (CD23) and cell-adhesion (LFA 3 and ICAM 1) molecules in vitro. Using monoclonal antibody-immune microscopy, we have demonstrated that these two EBV proteins and their associated B-lymphocyte activation or adhesion molecules are expressed in the infiltrating B lymphocytes in immunocompromised patients with EBV lymphoproliferative disease. These monoclonal antibodies should be useful in the early diagnosis of EBV lymphoproliferative disease and in distinguishing it from other B-lymphocyte cancers associated with EBV, such as Burkitt's lymphoma. The finding of EBV nuclear protein 2 and latent membrane protein and their associated activation or adhesion molecules provides a further pathophysiologic link between EBV and the proliferation of B lymphocytes in immunocompromised patients.

Expression of Epstein-Barr virus transformation-associated genes in tissues of patients with EBV lymphoproliferative disease.

Sequence analysis of the U2 regions of the B95-8 and AG876 Epstein-Barr virus (EBV) isolates reveals divergence within a long open reading frame previously identified as encoding 1.5 kilobases of the 3' end of a viral RNA expressed in latently infected, growth-transformed, B-lymphocyte cell lines. Differences among EBV isolates within the U2 open reading frame are shown to correlate with differences in an EBV nuclear antigen, EBNA2. B95-8, W91, Raji, Cherry, and Lamont EBV isolates have similar U2 domains and encode similar-size EBNA2 proteins, while AG876, Jijoye, and P3HR-1 have variant or absent U2 domains and variant or absent EBNA2 proteins. The AG876 U2 open reading frame and EBNA2 protein are both shorter than those of B95-8. These data indicate that the U2 open reading frame encodes EBNA2.

https://www.pnas.org/content/pnas/81/23/7632.full.pdf

U2 region of Epstein-Barr virus DNA may encode Epstein-Barr nuclear antigen 2

The nucleotide sequence of an Epstein-Barr virus gene expressed in latently infected growth-transformed cells is known to include a long open reading frame containing a 33-base-pair repeat element. A bacterial fusion protein constructed from a portion of the reading frame and Escherichia coli beta-galactosidase was used to produce sera in rabbits against the previously unidentified gene product. The viral protein detected with these sera in latently infected cells varies in size with the number of copies of the DNA repeat element. Translation of the RNA in vitro yields a protein of similar size. As expected from its primary sequence, the protein is a membrane protein. Immunofluorescence studies with the rabbit antisera suggest that the protein is in the plasma membrane. Thus, this protein could be the lymphocyte-determined membrane antigen (LYDMA) responsible for the generation of T-cell immunity to latently infected cells.

A membrane protein encoded by Epstein-Barr virus in latent growth-transforming infection.

A gene fusion between an Epstein-Barr virus (EBV) triplet nucleotide repeat array (IR3), which has homology to host DNA, and lacZ was used to demonstrate that this EBV sequence encodes part of the Epstein-Barr nuclear antigen (EBNA). The IR3 sequence is translated into a glycine-alanine copolymer that reacts with anti-EBNA human sera. Some EBV-immune human antisera recognize a second intranuclear protein that is also specific for latently infected cells and is designated EBNA2. EBNA2 is not related to EBNA1 because the molecular mass of EBNA2 is 82 kilodaltons, whereas that of EBNA1 varies from 68 to 85 kilodaltons among cells transformed by different EBV isolates; also EBNA2 does not contain the copolymer domain of EBNA1.

One of two Epstein-Barr virus nuclear antigens contains a glycine-alanine copolymer domain.

A region of the Epstein-Barr virus (EBV) genome that is important in inducing cell proliferation includes a single long open reading frame. Part of this open reading frame has been fused to the lacZ gene and expressed in Escherichia coli. Antisera to the fusion protein identify a protein in the nuclei of latently infected growth-transformed lymphocytes and in Burkitt tumor cells grown in vitro. This nuclear protein is encoded by a different virus-gene than that which encodes the previously described EBV nuclear antigen, EBNA.

https://science.sciencemag.org/content/sci/227/4691/1238.full.pdf

Kevin Hennessy

Papers

Expression of Epstein-Barr virus transformation-associated genes in tissues of patients with EBV lymphoproliferative disease.

U2 region of Epstein-Barr virus DNA may encode Epstein-Barr nuclear antigen 2

A membrane protein encoded by Epstein-Barr virus in latent growth-transforming infection.

One of two Epstein-Barr virus nuclear antigens contains a glycine-alanine copolymer domain.

A second nuclear protein is encoded by Epstein-Barr virus in latent infection.