Host cell-dependent regulation of growth transformation-associated Epstein-Barr virus antigens in somatic cell hybrids.

We have analyzed the expression of the three major known growth transformation-associated Epstein-Barr virus (EBV) proteins, EBNA-1, EBNA-2, and latent membrane protein (LMP), in a series of somatic cell hybrids derived from the fusion of EBV-carrying Burkitt lymphoma (BL) lines with EBV-positive or EBV-negative B-cell lines. Independently of the cell phenotype, EBNA-1 was invariably coexpressed in all EBV-carrying hybrids. In hybrids between EBV-carrying, LMP-positive and LMP-negative Burkitt lymphoma lines, LMP was expressed, indicating positive control. Two EBV-negative lymphoma lines, Ramos and BJAB, differed in their ability to express LMP after B95-8 virus-induced conversion and after hybridization with Raji cells. BJAB was permissive while Ramos was nonpermissive for LMP, although both expressed EBNA-2. The EBNA-2-deleted P3HR-1 virus gave the same pattern of LMP expression in these two cells. Our findings indicate that the expression of EBNA-1, EBNA-2, and LMP is regulated by independent mechanisms.     

Epstein-barr virus regulates c-MYC, apoptosis, and tumorigenicity in Burkitt lymphoma

Loss of the Epstein-Barr virus (EBV) genome from Akata Burkitt lymphoma (BL) cells is coincident with a loss of malignant phenotype, despite the fact that Akata and other EBV-positive BL cells express a restricted set of EBV gene products (type I latency) that are not known to overtly affect cell growth. Here we demonstrate that reestablishment of type I latency in EBV-negative Akata cells restores tumorigenicity and that tumorigenic potential correlates with an increased resistance to apoptosis under growth-limiting conditions. The antiapoptotic effect of EBV was associated with a higher level of Bcl-2 expression and an EBV-dependent decrease in steady-state levels of c-MYC protein. Although the EBV EBNA-1 protein is expressed in all EBV-associated tumors and is reported to have oncogenic potential, enforced expression of EBNA-1 alone in EBV-negative Akata cells failed to restore tumorigenicity or EBV-dependent down-regulation of c-MYC. These data provide direct evidence that EBV contributes to the tumorigenic potential of Burkitt lymphoma and suggest a novel model whereby a restricted latency program of EBV promotes B-cell survival, and thus virus persistence within an immune host, by selectively targeting the expression of c-MYC.     

Alterations of the p53 gene in Epstein-Barr virus-associated immunodeficiency-related lymphomas.

Mutations of the p53 tumor suppressor gene are among the most common genetic alterations found in many different human malignancies, including those of the colon, lung, and breast. Alterations in wild-type p53 lead to loss of the suppressor function and thus contribute to tumorigenesis. The potential role of p53 mutations in a sampling of B-cell lymphomas, the majority of which were associated with Epstein-Barr virus (EBV), was investigated. Twenty-six biopsy specimens from immunocompromised patients, including allograft recipients and patients with AIDS, Wiscott-Aldrich syndrome, and human T-cell leukemia virus type 1 infection, in comparison with three Burkitt lymphomas and four Burkitt lymphoma cell lines were analyzed. Mutation in p53 was detected in all four Burkitt lymphoma cell lines as well as the three Burkitt lymphoma biopsy specimens. In patients with AIDS, 5 of 10 lymphomas were EBV positive, and 1 had a mutation in p53. Mutation in p53 was not detected in 14 EBV-positive lymphomas which arose in transplant recipients. These data indicate that with the exception of Burkitt lymphomas, p53 mutations are not involved in the majority EBV-positive B-cell lymphomas which develop in immunocompromised patients.     

Epstein-Barr virus nuclear protein EBNA-3C interacts with the human metastatic suppressor Nm23-H1: a molecular link to cancer metastasis

Epstein-Barr virus (EBV) is an oncogenic virus associated with a number of human malignancies including Burkitt lymphoma, nasopharyngeal carcinoma, lymphoproliferative disease and, though still debated, breast carcinoma. A subset of latent EBV antigens is required for mediating immortalization of primary B-lymphocytes. Here we demonstrate that the carboxy-terminal region of the essential latent antigen, EBNA-3C, interacts specifically with the human metastatic suppressor protein Nm23-H1. Moreover, EBNA-3C reverses the ability of Nm23-H1 to suppress the migration of Burkitt lymphoma cells and breast carcinoma cells. We propose that EBNA-3C contributes to EBV-associated human cancers by targeting and altering the role of the metastasis suppressor Nm23-H1.     

Differences in B cell growth phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt''s lymphoma cells.

Recently established Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cell lines, carrying chromosomal translocations indicative of their malignant origin, have been monitored for their degree of in vitro progression towards a more 'lymphoblastoid' cell surface phenotype and growth pattern, and for their expression of three EBV latent gene products which are constitutively present in all virus-transformed normal lymphoblastoid cell lines (LCLs). BL cell lines which stably retained the original tumour biopsy phenotype on serial passage were all positive for the nuclear antigen EBNA 1 but did not express detectable amounts of two other 'transforming' proteins, EBNA 2 and the latent membrane protein (LMP). This novel pattern of EBV gene expression was also observed on direct analysis of BL biopsy tissue. All three viral proteins became detectable, however, in BL cell lines which had progressed towards a more LCL-like phenotype in vitro. This work establishes a link between B cell phenotype and the accompanying pattern of EBV latent gene expression, and identifies a novel type of EBV:cell interaction which may be unique to BL cells.     

Structural basis for apoptosis inhibition by Epstein-Barr virus BHRF1

Epstein-Barr virus (EBV) is associated with human malignancies, especially those affecting the B cell compartment such as Burkitt lymphoma. The virally encoded homolog of the mammalian pro-survival protein Bcl-2, BHRF1 contributes to viral infectivity and lymphomagenesis. In addition to the pro-apoptotic BH3-only protein Bim, its key target in lymphoid cells, BHRF1 also binds a selective sub-set of pro-apoptotic proteins (Bid, Puma, Bak) expressed by host cells. A consequence of BHRF1 expression is marked resistance to a range of cytotoxic agents and in particular, we show that its expression renders a mouse model of Burkitt lymphoma untreatable. As current small organic antagonists of Bcl-2 do not target BHRF1, the structures of it in complex with Bim or Bak shown here will be useful to guide efforts to target BHRF1 in EBV-associated malignancies, which are usually associated with poor clinical outcomes.     

Epstein‒Barr virus–associated cellular immunotherapy

Epstein‒Barr virus (EBV) is a human herpes virus that is saliva-transmissible and universally asymptomatic. It has been confirmed that more than 90% of the population is latently infected with EBV for life. EBV can cause a variety of related cancers, such as nasopharyngeal carcinoma, diffuse large B-cell lymphoma, and Burkitt lymphoma. Currently, many clinical studies have demonstrated that EBV-specific cytotoxic T lymphocytes and other cell therapies can be safely and effectively transfused to prevent and treat some diseases caused by EBV. This review will mainly focus on discussing EBV-specific cytotoxic T lymphocytes and will touch on therapeutic EBV vaccines and chimeric antigen receptor T-cell therapy briefly.     

Latent membrane protein 1 is dispensable for Epstein-Barr virus replication in human embryonic kidney 293 cells

Epstein Barr Virus (EBV) replicates in oral epithelial cells and gains entry to B-lymphocytes. In B-lymphocytes, EBV expresses a restricted subset of genes, the Latency III program, which converts B-lymphocytes to proliferating lymphoblasts. Latent Membrane Protein 1 (LMP1) and the other Latency III associated proteins are also expressed during virus replication. LMP1 is essential for virus replication and egress from Akata Burkitt Lymphoma cells, but a role in epithelial cell replication has not been established. Therefore, we have investigated whether LMP1 enhances EBV replication and egress from HEK293 cells, a model epithelial cell line used for EBV recombinant molecular genetics. We compared wild type (wt) and LMP1-deleted (LMP1Δ) EBV bacterial artificial chromosome (BAC) based virus replication and egress from HEK293. Following EBV immediate early Zta protein induction of EBV replication in HEK293 cells, similar levels of EBV proteins were expressed in wt- and LMP1Δ-infected HEK293 cells. LMP1 deletion did not impair EBV replication associated DNA replication, DNA encapsidation, or mature virus release. Indeed, virus from LMP1Δ-infected HEK293 cells was as infectious as EBV from wt EBV infected HEK cells. Trans-complementation with LMP1 reduced Rta expression and subsequent virus production. These data indicate that LMP1 is not required for EBV replication and egress from HEK293 cells.     

Biology and disease associations of Epstein-Barr virus

Epstein-Barr virus (EBV) is a human herpesvirus which infects almost all of the world's population subclinically during childhood and thereafter remains in the body for life. The virus colonizes antibody-producing (B) cells, which, as relatively long-lived resting cells, are an ideal site for long-term residence. Here EBV evades recognition and destruction by cytotoxic T cells. EBV is passed to naive hosts in saliva, but how the virus gains access to this route of transmission is not entirely clear. EBV carries a set of latent genes that, when expressed in resting B cells, induce cell proliferation and thereby increase the chances of successful virus colonization of the B-cell system during primary infection and the establishment of persistence. However, if this cell proliferation is not controlled, or if it is accompanied by additional genetic events within the infected cell, it can lead to malignancy. Thus EBV acts as a step in the evolution of an ever-increasing list of malignancies which are broadly of lymphoid or epithelial cell origin. In some of these, such as B-lymphoproliferative disease in the immunocompromised host, the role of the virus is central and well defined; in others, such as Burkitt's lymphoma, essential cofactors have been identified which act in concert with EBV in the evolution of the malignant clone. However, in several diseases in which the presence of EBV has more recently been discovered, the role of the virus is unclear. This review describes recent views on the EBV life cycle and its interlinks with normal B-cell biology, and discusses how this interrelationship may be upset and result in EBV-associated disease.     

Monoclonal and polyclonal antibodies against Epstein-Barr virus nuclear antigen 5 (EBNA-5) detect multiple protein species in Burkitt''s lymphoma and lymphoblastoid cell lines.

The Epstein-Barr virus nuclear antigen 5 (EBNA-5) is encoded by highly spliced mRNA from the major IR1 (BamHI-W) repeat region of the virus genome. A mouse monoclonal antibody, JF186, has been raised against a synthetic 18-amino-acid peptide deduced from the EBNA-5 message of B95-8 and Raji cells. The antibody showed characteristic coarse nuclear granules by indirect immunofluorescence and revealed multiple EBNA-5 species by immunoblotting and immunoprecipitation. The B95-8 line itself and all B95-8 virus-carrying cells, whether lymphoblastoid cell lines or in vitro-converted sublines of Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL) lines, were EBNA-5 positive. Among 36 cell lines carrying different EBV strains, only 10 expressed the B95-8-Raji-prototype EBNA-5 recognized by JF186; this was probably due to genetic variation in the epitope recognized by JF186, as shown for P3HR-1. Human antibodies, affinity purified against EBNA-5-JF186 immunoprecipitates, detected EBNA-5 in the majority of EBV-positive BL lines and in all lymphoblastoid cell lines containing the BL-derived viruses. Thus, EBNA-5 can be expressed by all virus isolates examined, but is down-regulated, together with other latent gene products, in a minority of BL lines which have a particular cellular phenotype. EBNA-5 was detected as a ladder of protein species of 20 to 130 kilodaltons (kDa), with a regular spacing of 6 to 8 kDa, consistent with the coding capacity of the combined BamHI-W 66- and 132-base-pair exons, together with shifts of 2 to 4 kDa, consistent with the size of the separate 66- and 132-base-pair exons. Multiple EBNA-5 proteins can be expressed by the single cell as shown by cloning of newly infected cells.     

An Epstein-Barr virus immortalization associated gene segment interferes specifically with the IFN-induced anti-proliferative response in human B-lymphoid cell lines

Immortalization of human B-lymphocytes by Epstein-Barr virus (EBV) is associated with a decreased anti-proliferative response to interferon (IFN). In the present investigation we show that the resistance to the anti-proliferative effect of IFN class I on certain EBV-carrying Burkitt lymphoma cell lines is connected to the presence of the EBNA-2 gene and parts of the EBNA-5 gene of the EBV genome. Transfection of the genomic segment comprising these open reading frames into an IFN-sensitive lymphoma cell line demonstrated that it is sufficient to make cells resistant towards the antiproliferative effect of IFN class I. Expression of the EBNA-2 gene seems to be correlated with the IFN-resistant phenotype. The antiviral function of IFN, as tested by inhibition by vesicular stomatitis virus (VSV) infection, and the IFN-receptor binding are not suppressed. The present results suggest that the neutralization of the anti-proliferative effect of IFN-alpha is involved in the EBV-mediated immortalization of B-cells and that the anti-proliferative action of IFN class I does not necessarily recruit the same mechanism as the antiviral effect.     

CD4+ T-cell effectors inhibit Epstein-Barr virus-induced B-cell proliferation

In immunodeficient hosts, Epstein-Barr virus (EBV) often induces extensive B-cell lymphoproliferative disease and lymphoma. Without effective in vitro immune surveillance, B cells infected by the virus readily form immortalized cell lines. In the regression assay, memory T cells inhibit the formation of foci of EBV-transformed B cells that follows recent in vitro infection by EBV. No one has yet addressed which T cell regulates the early proliferative phase of B cells newly infected by EBV. Using new quantitative methods, we analyzed T-cell surveillance of EBV-mediated B-cell proliferation. We found that CD4+ T cells play a significant role in limiting proliferation of newly infected, activated CD23+ B cells. In the absence of T cells, EBV-infected CD23+ B cells divided rapidly during the first 3 weeks after infection. Removal of CD4+ but not CD8+ T cells also abrogated immune control. Purified CD4+ T cells eliminated outgrowth when added to EBV-infected B cells. Thus, unlike the killing of EBV-infected lymphoblastoid cell lines, in which CD8+ cytolytic T cells play an essential role, prevention of early-phase EBV-induced B-cell proliferation requires CD4+ effector T cells.     

Epstein-Barr virus latent infection membrane protein alters the human B-lymphocyte phenotype: deletion of the amino terminus abolishes activity.

A latent infection membrane protein (LMP) encoded by the Epstein-Barr virus (EBV) genome in latently infected, growth-transformed lymphocytes alters the phenotype of a human EBV-negative B-lymphoma cell line (Louckes) when introduced by gene transfer. These LMP-expressing cells exhibit increased homotypic adhesion due to increased expression of the adhesion molecules LFA-1 and ICAM-1. Increased homotypic adhesion could foster B-cell growth by facilitating autocrine growth factor effects. LFA-3 expression is also induced. The induction of LFA-3 and ICAM-1 results in increased heterotypic adhesion to T lymphocytes. This could result in more effective T-cell immune surveillance. Since LMP is expressed in EBV-transformed lymphocytes and has been demonstrated to transform rodent fibroblasts in vitro, a wide range of possible effects on B-lymphoma cell growth were assayed. In the Louckes B-lymphoma cell line, EBV LMP causes increased cell size, acid production, plasma membrane ruffling, and villous projections. Although cell proliferation rate was not greatly affected, the steady-state intracellular free calcium level, transforming growth factor beta responsiveness, and expression of the lymphocyte activation markers (CD23 and transferrin receptor) were increased. Thus, LMP appears to be a mediator of EBV effects on B-cell transformation. In transfected lymphoma cells, LMP localizes to patches at the cell periphery and associates with the cytoskeleton as it does in EBV-transformed B lymphocytes or in rodent fibroblasts. A partially deleted form of LMP (D1LMP) does not aggregate in patches or associate with the cytoskeleton and had little effect on B-cell growth. Thus, cytoskeletal association may be integral to LMP activity.     

A molecular link between malaria and Epstein-Barr virus reactivation

Although malaria and Epstein-Barr (EBV) infection are recognized cofactors in the genesis of endemic Burkitt lymphoma (BL), their relative contribution is not understood. BL, the most common paediatric cancer in equatorial Africa, is a high-grade B cell lymphoma characterized by c-myc translocation. EBV is a ubiquitous B lymphotropic virus that persists in a latent state after primary infection, and in Africa, most children have sero-converted by 3 y of age. Malaria infection profoundly affects the B cell compartment, inducing polyclonal activation and hyper-gammaglobulinemia. We recently identified the cystein-rich inter-domain region 1alpha (CIDR1alpha) of the Plasmodium falciparum membrane protein 1 as a polyclonal B cell activator that preferentially activates the memory compartment, where EBV is known to persist. Here, we have addressed the mechanisms of interaction between CIDR1alpha and EBV in the context of B cells. We show that CIDR1alpha binds to the EBV-positive B cell line Akata and increases the number of cells switching to the viral lytic cycle as measured by green fluorescent protein (GFP) expression driven by a lytic promoter. The virus production in CIDR1alpha-exposed cultures was directly proportional to the number of GFP-positive Akata cells (lytic EBV) and to the increased expression of the EBV lytic promoter BZLF1. Furthermore, CIDR1alpha stimulated the production of EBV in peripheral blood mononuclear cells derived from healthy donors and children with BL. Our results suggest that P. falciparum antigens such as CIDR1alpha can directly induce EBV reactivation during malaria infection that may increase the risk of BL development for children living in malaria-endemic areas. To our knowledge, this is the first report to show that a microbial protein can drive a latently infected B cell into EBV replication.     

Epstein-Barr virus suppresses a G(2)/M checkpoint activated by genotoxins

Several Epstein-Barr virus (EBV)-negative Burkitt lymphoma-derived cell lines (for example, BL41 and Ramos) are extremely sensitive to genotoxic drugs despite being functionally null for the tumor suppressor p53. They rapidly undergo apoptosis, largely from G(2)/M of the cell cycle. 5-bromo-2'-deoxyuridine labeling experiments showed that although the treated cells can pass through S phase, they are unable to complete cell division, suggesting that a G(2)/M checkpoint is activated. Surprisingly, latent infection of these genotoxin-sensitive cells with EBV protects them from both apoptosis and cell cycle arrest, allowing them to complete the division cycle. However, a comparison with EBV-immortalized B-lymphoblastoid cell lines (which have functional p53) showed that EBV does not block apoptosis per se but rather abrogates the activation of, or signalling from, the checkpoint in G(2)/M. Furthermore, analyses of BL41 and Ramos cells latently infected with P3HR1 mutant virus, which expresses only a subset of the latent viral genes, showed that LMP-1, the main antiapoptotic latent protein encoded by EBV, is not involved in the protection afforded here by viral infection. This conclusion was confirmed by analysis of clones of BL41 stably expressing LMP-1 from a transfected plasmid, which respond like the parental cell line. Although steady-state levels of Bcl-2 and related proteins varied between BL41 lines and clones, they did not change significantly during apoptosis, nor was the level of any of these anti- or proapoptotic proteins predictive of the outcome of treatment. We have demonstrated that a subset of EBV latent gene products can inactivate a cell cycle checkpoint for monitoring the fidelity and timing of cell division and therefore genomic integrity. This is likely to be important in EBV-associated growth transformation of B cells and perhaps tumorigenesis. Furthermore, this study suggests that EBV will be a unique tool for investigating the intimate relationship between cell cycle regulation and apoptosis.     

Modulation of expression of insulin and IGF-I receptor by Epstein-Barr virus and its gene products LMP and EBNA-2 in lymphocyte cell lines

The receptors for insulin and insulin-like growth factor I (IGF-I) are two closely related integral membrane glycoproteins involved in signalling of cell growth and metabolism. We have used the unique paradigm of pairs of Burkitt lymphoma cell lines (BLO2, BL30, BL41) with or without Epstein-Barr Virus (EBV) infection and cells transfected with EBV-related genes to examine effects of EBV on expression of these receptors at the gene and protein functional level. In BL30 and BL41 cells, EBV infection increased surface insulin binding and total receptor number by 2-and 18-fold, respectively. By contrast, EBV infection decreased total IGF-I receptors by 29 to 87% in all three cell lines. In general, there was a correlation between total receptor concentration and the level of insulin or IGF-I receptor mRNAs, although in one cell line insulin binding increased while receptor mRNA levels decreased slightly, suggesting posttranslational effects. BL41 cells transfected with a vector expressing the EBV latent membrane protein (LMP) exhibited a 2.6- to 3.2-fold increase in insulin receptor expression, whereas cells transfected with EBNA-2 (one of the EBV nuclear antigens) alone exhibited no effect. However, EBNA-2 appears to be required for the EBV effect on insulin receptor expression since cells infected with a mutant virus, P3JHRI, which lacks the EBNA-2 gene failed to show an increase in insulin receptor number. These data indicate that EBV infection of lymphocytes increases expression of insulin receptors while simultaneously decreasing expression of IGF-I receptors. The magnitude and sometimes even the direction of change, depends on host cell factors. A maximal increase in insulin receptors appears to require the coordinate action of several of the EBV proteins including LMP and EBNA-2. © 1993 Wiley-Liss, Inc.     

Differential regulation of Epstein-Barr virus (EBV) latent gene expression in Burkitt lymphoma cells infected with a recombinant EBV strain

Epstein-Barr virus (EBV)-negative Burkitt lymphomas (BLs) can be infected in vitro with prototype EBV strains to study how the virus may affect the phenotype of tumor cells. Studies thus far have concentrated on the use of transforming B95-8 and nontransforming P3HR1 strains. Immunological and phenotypic differences between the sublines infected with these two strains were reported. The majority of these differences, if not all, can be attributed to the lack of EBNA-2 coding sequences in the P3HR1 strain. The recent development of a selectable Akata strain has opened up new possibilities for infecting epithelial and T cells as well. We infected five EBV-negative BL lines with the recombinant Akata virus. Our results indicate that the infected cell lines BL28, Ramos, and DG75 express EBNA-1, EBNA-2, and LMP1, the viral proteins associated with type III latency, and use both YUK and QUK splices. In contrast, two EBV-negative variants of Akata and Mutu when reinfected displayed restricted type I latency and expressed only EBNA-1. All clones of infected Mutu cells used the QUK splice exclusively. The usage of Qp was observed in a majority of Akata clones. Some Akata clones, however, were found to have double promoter usage (Qp and C/Wp) but at 4 months after infection did not express EBNA-2. The results demonstrate differential regulation of EBV latency in BLs with the same recombinant viral strain and suggest that the choice of latency type may be cell dependent. The restricted latency observed for infected Akata and Mutu cells indicates that a BL may opt for type I latency in the absence of immune pressure as well.