The complexity of the Epstein-Barr virus infection in humans

The Epstein-Barr virus (EBV) was isolated 40 years ago from cultures of Burkitt lymphoma cells (BL). The tumor was encountered in Africa and exhibited characteristical geographical, clinical and pathological features. Serological studies revealed that the virus is ubiquitous in humans. The primary infection is often accompanied by the syndrome of acute infectious mononucleosis (IM). It can induce malignant proliferation of B lymphocytes in conditions of immunodeficiency. EBV can immortalize B lymphocytes in culture. These cells carry the virus as episomes and express 9 virally encoded proteins. Their immunological recognition constitutes the surveillance which is responsible for the healthy virus carrier state. The main virus reservoir is represented by a low number of resting B lymphocyte which contain the viral genome but do not express its transformation proteins. The viral genom is detectable in all African BLs, in variable proportions of nasopharyngeal carcinoma, Hodgkin’s disease, T cell lymphoma, lymphoepithelial like carcinoma, gastric carcinoma and leiomyosarcoma cases. The role of EBV in the genesis of these tumors is unknown. Acknowledgement: Supported by the the Swedish Cancer Society (Cancerfonden)     

A natural HIV p17 protein variant up‐regulates the LMP‐1 EBV oncoprotein and promotes the growth of EBV‐infected B‐lymphocytes: Implications for EBV‐driven lymphomagenesis in the HIV setting

Human immunodeficiency virus p17 matrix protein is released by infected cells and may accumulate within lymphoid tissues where it may deregulate the biological activities of different cell populations by binding to CXCR1 and CXCR2 cellular receptors. S75X, a natural p17 variant, was recently shown to enhance the malignant properties of lymphoma cells. We investigated a reference p17 protein and the S75X variant for their ability to bind to Epstein–Barr virus (EBV)‐infected primary and fully transformed B‐lymphocytes and trigger downstream effects of potential pathogenic relevance. We demonstrate that EBV infection of primary B‐lymphocytes or the ectopic expression of the latent membrane protein‐1 viral oncoprotein in EBV‐negative B‐cells up‐regulates CXCR2, but not CXCR1. Multispectral imaging flow cytometry showed that EBV‐infected primary B‐cells more efficiently bind and internalize p17 proteins as compared with activated B‐lymphocytes. The S75X variant bound more efficiently to EBV‐infected primary and fully transformed B‐lymphocytes compared with reference p17, because of a higher affinity to CXCR2, and enhanced the proliferation of these cells, an effect associated with cyclin D2 and D3 up‐regulation and increased interleukin‐6 production. Notably, the S75X variant markedly up‐regulated latent membrane protein‐1 expression at both mRNA and protein levels and enhanced the activation of Akt, ERK1/2 and STAT3 signaling, thereby contributing to EBV⁺ B‐cell growth promotion. These results indicate that EBV infection sensitizes B‐lymphocytes to CXCR2‐mediated effects of p17 proteins and provide evidence supporting a possible contribution of natural p17 variants to EBV‐driven lymphomagenesis in the human immunodeficiency virus setting.     

Role of LMP1 in immune control of EBV infection

The Epstein-Barr virus (EBV) encoded latent membrane protein (LMP1) plays a crucial role in the long-term persistence of this virus within the cells of the immune system. Not only is this protein critical for the transformation of resting B cells by EBV, it also displays pleiotropic effects on various cellular proteins expressed in the host cell. These include up-regulation of expression of B cell activation antigens, adhesion molecules and various components of the antigen processing pathway. Here we discuss how LMP1 acts like an expression 'switch' which, depending on the stage of EBV infection, manoeuvres various pathways that either modulate the immune system towards or against its survival.     

EB病毒及EB病毒感染相关淋巴瘤发病机制的研究进展

EB病毒（Epstein-Barr virus,EBV）是一个已知与人类肿瘤相关的病毒。在多种上皮、间叶和淋巴造血肿瘤中可检测到EBV的存在,与淋巴瘤关系尤为密切,特别是与Burkitt淋巴瘤（Burkitt’s lymphoma,BL）、霍奇金淋巴瘤（Hodgkin's lymphoma,HL）、弥漫性大Ｂ细胞淋巴瘤（diffuse large B-cell lymphoma,DLBCL）、移植后淋巴组织增殖性疾病（post-transplant lymphoproliferative disorders,PTLD）及NK/T细胞淋巴瘤等疾病的发生、发展、预后相关。大量的研究报道以及临床数据表明,EBV及其编码的蛋白、微小RNA在诱导淋巴细胞恶性转化的过程中发挥极其重要又复杂的作用,包括促进其增殖、抑制凋亡在内的多种信号通路中的蛋白。本文就近些年来EBV及EBV感染密切相关淋巴瘤发病机制的研究进展进行综述,旨在为进一步了解EBV感染与其导致淋巴瘤的机制提供一定的理论基础。      

Immune escape by Epstein-Barr virus associated malignancies

Persistent Epstein-Barr virus (EBV) infection remains asymptomatic in the majority of virus carriers, despite the potent growth transforming potential of this virus. The increased frequency of EBV associated B cell lymphomas in immune compromised individuals suggests that tumor-free chronic infection with this virus is in part due to immune control. Here we discuss the evidence that loss of selective components of EBV specific immunity might contribute to EBV associated malignancies, like nasopharyngeal carcinoma, Burkitt's and Hodgkin's lymphoma, in otherwise immune competent patients. Furthermore, we discuss how current vaccine approaches against EBV might be able to target these selective deficiencies.     

HIV, EBV and KSHV: viral cooperation in the pathogenesis of human malignancies

Malignancies associated with Epstein-Barr virus (EBV) and/or Kaposi's sarcoma human herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is frequently found in patients infected with HIV. Both these human gammaherpesviruses are known for their oncogenic properties, for the viral products that mimic or interfere with the functions of critical cellular proteins, and the ability to escape the immune responses. The introduction of the highly active anti-retroviral therapy (HAART) has significantly decreased the frequency of Kaposi's sarcoma (KS), non-Hodgkin's lymphoma (NHL), and primary central nervous system lymphoma (PCNSL); conversely, for some lymphomas the incidence diminished only slightly, as in Burkitt's lymphoma (BL), or had no significant variations, as Hodgkin's lymphoma (HL). These observations may indicate that HAART might have a direct impact on KSHV and EBV biology, that there is a reconstitution of the immune system in HIV-infected patients under HAART, or even that HAART perhaps has a detrimental impact in the pathogenic interactions between HIV, EBV and KSHV. The present review aim to evaluate and to discuss the data available for these hypotheses, in order to shed more light on the mechanisms for the cooperation among HIV-1, EBV and KSHV that may culminate in cell transformation and cancer development in humans.     

DNA viruses in human cancer: an integrated overview on fundamental mechanisms of viral carcinogenesis

The first experimental data suggesting that neoplasm development in animals might be influenced by infectious agents were published in the early 1900s. However, conclusive evidence that DNA viruses play a role in the pathogenesis of some human cancers only emerged in the 1950s, when Epstein-Barr virus (EBV) was discovered within Burkitt lymphoma cells. Besides EBV, other DNA viruses consistently associated with human cancers are the hepatitis B virus (HBV), human papillomavirus (HPV), and Kaposi sarcoma herpesvirus (KSHV). Although each virus has unique features, it is becoming clearer that all these oncogenic agents target multiple cellular pathways to support malignant transformation and tumor development.     

Transformation of hairy cell leukemia to EBV genome-containing aggressive B cell lymphoma

Hairy cell leukemia is a preplasmacytic B cell leukemia which is not EBV associated, although elevated titers of Epstein-Barr virus (EBV) antibodies have been seen in this leukemia and chronic lymphocytic leukemia. Hairy cells are not readily susceptible to EBV infection in vitro, even though they are EBV receptor-positive B cells. We have observed a 59-year-old patient who after 9 years of hairy cell leukemia developed a well-differentiated IgG-kappa monoclonal B cell lymphoma without further evidence of hairy cell leukemia. Pathologically, the lymphoma showed plasmacytic differentiation, and in the patient's serum, a 2 g/dl monoclonal IgG-kappa component was present. DNA extracted from the lymphomatous lymph node hybridized with DNA fragments of a reiterated sequence of EBV, IR1. The transformation, with no chemotherapy involved, from a preplasmacytic leukemia into a lymphoplasmacytic lymphoma with monoclonal gammopathy may be related to the entry of EBV into these cells. Studies at the molecular level may help understand mechanisms of malignant transformation or interconversion in lymphoproliferative disorders of the B cell type.     

Role of EBV in microRNA dysregulation in Burkitt lymphoma

Since its discovery as the first human tumor virus, Epstein-Barr virus (EBV) has been implicated in the development of a wide range of B-cell lymphoproliferative disorders, the first being Burkitt lymphoma. However, the exact mechanism by which EBV promotes oncogenesis is still matter of discussion. A role in EBV-mediated transformation has been proposed for a novel described class of small non-coding RNAs, the microRNAs (miRNAs). EBV encodes viral miRNAs, through which it may interfere with the physiological regulation exterted by cellular miRNAs. In addition, EBV-coded proteins may also disturb the well-orchestrated mechanisms of regulation of cellular function. In this review, we will focus on the role of EBV in malignant transformation of Burkitt lymphoma, with a particular insight in the interplay between the virus and cellular miRNAs.     

Bcl-2 and c-Myc co-operate in the Epstein-Barr virus-immortalized human B-cell line GM607 but do not confer tumorigenicity

Eighty-five percent of follicular lymphomas possess a characteristic t(14;18) translocation that results in the deregulated expression of the proto-oncogene BCL-2. BCL-2 overexpression alone is insufficient for full cellular transformation and at least 1 other genetic event is believed to be necessary for follicular lymphoma development. Deregulated c-Myc expression has previously been shown to cooperate with Bcl-2 to transform murine fibroblast cell lines and lead to tumor development in mice. We have developed a human model system to study early transformation in lymphoid cells using immortalized lymphoblastoid cells. We sequentially introduced BCL-2 and c-MYC, 2 proto-oncogenes known to be involved in the transformation of B cells into Epstein-Barr virus (EBV)-immortalized human B cells. We show that the c-Myc and Bcl-2 overexpression, together with EBV immortalization were insufficient to cause full cellular transformation as measured by cell proliferation rates, soft agar and tumorigenicity assays. These results show that more than 3 genetic hits (EBV infection, Bcl-2 and c-Myc overexpression) were required for the full cellular transformation of human lymphoblastoid cells. However, subtle changes in cellular proliferation and sensitivity to apoptosis were documented, at non-limiting dilutions. These changes may confer a susceptibility to the modified cells such that they are more susceptible to the acquisition of additional genetic changes and evolve towards a fully transformed state. In addition, the model system developed may be suitable for the identification of further known and novel oncogenic events involved in the full transformation of B cells.     

Epstein-Barr virus and epithelial cells: a possible role for the virus in the development of cervical carcinoma

Epstein-Barr virus (EBV) is an oncogenic herpesvirus associated with certain B cell lymphomas in humans and also with an epithelial tumour, undifferentiated nasopharyngeal carcinoma. Infection with EBV is widespread and once infected, individuals become life-long virus carriers. Epithelial cells of the pharynx have been implicated as the primary site of EBV persistence. Ectocervical epithelial cell cultures have proved useful for investigating EBV infection in vitro but only recently has EBV been shown to infect the uterine cervix in vivo. The demonstration that EBV replicates in cervical epithelium raises the possibility of venereal transmission, and suggests that the virus may be involved in cervical pathology.     

EBV infection induced transformation of benign T lymphoproliferative state in patient with chronic active EBV infection into malignant lymphoma: implication of EBV infection as additive oncogenic factor in tumorigenesis

An 11-year-old girl with chronic EBV (Epstein-Barr virus) infection, who later developed malignant lymphoma in the lung, is reported. She had an increased number of V alpha2, V beta8, CD3, CD4, and HLADR positive activated lymphocytes (20-30% of total lymphocytes) in peripheral blood. One year later, she developed lymphoma in the lung, which was V alpha2, V beta8, CD3, CD4, HLADR and IL2Rbeta positive. At that time, the population in the peripheral blood increased up to 40%, but there was no evidence of lymphoma in the bone marrow. In situ hybridization revealed lymphoma cells were EBER-1 positive but gp350/220 and LMP mRNA negative. The EBV genome was detected in the tumor, but not in the peripheral T cells. Clonal analysis of the lymphoma cells revealed monoclonal rearrangement of the TcRbeta and gamma gene, however, investigation of the terminal repeat of EBV gene did not show the monoclonal pattern. These results indicate that infection of EBV into clonally activated T cells was related with transformation from benign lymphoproliferative disease to malignant lymphoma in this patient.     

Epstein-Barr virus-related herpesvirus from a rhesus monkey (Macaca mulatta) with malignant lymphoma

A herpesvirus (RhEBV) was isolated from a lymphoblastoid cell line (LCL) that became established from a malignant lymphoma in a rhesus monkey. The predominant cell marker in the LCL was that of B lymphocytes. RhEBV-induced viral capsid (VCA) and nuclear antigens (NA) in the LCL were serologically related to similar antigens known to be induced by human Epstein-Barr virus (EBV). RhEBV was of nonhuman primate origin and was clearly differentiated from EBV in the anti-complement immunofluorescence reaction using human and non-human primate sera with antibodies to the NA induced by the respective viruses. While human sera reacted with NA induced by both EBV and RhEBV, monkey sera failed to recognize the NA induced by EBV. RhEBV-induced NA was present in nearly all the cells of a suspension prepared from the tumor tissue mass, but not in the monolayer fibroblasts derived from the tumor tissue or in the blood and lymph-node lymphocytes of clinically healthy animals. RhEBV induced in vitro transformation and establishment of LCLs from peripheral blood lymphocytes of normal rhesus and cynomolgus monkeys but not from those of 6 other non-human primate species tested. The LCLs, with predominant B-lymphocyte markers, established after treatment with RhEBV, all had evidence of the virus infection since nearly all cells in the culture expressed the virus-induced NA.     

Restricted expression of EBV encoded proteins in in vitro infected CLL cells

CLL is not associated with EBV. CLL cells separated from blood express CR2, the complement receptor that serves also as EBV receptor. Thus CLL cells can be infected in vitro with the virus, however, in contrast to normal B lymphocytes, only rare CLL clones yield transformed lines. This is due to a restricted EBV encoded protein expression in the CLL cells, they express EBNAs, the virus encoded proteins that are localized in the nucleus, but not the cell membrane associated LMP-1, that is also pivotal for the virus induced transformation of B lymphocytes. This expression pattern seems to be unique to a defined B cell maturation window that is represented by the CLL cells. We named this restricted viral expression as Type IIb. Such B lymphocytes have been encountered in lymphoid tissues of infectious mononucleosis (IM) and in post transplant lymphoproliferative disease (PTLD). Moreover, they were shown in tissues of EBV infected "humanized" mice. The EBV encoded protein expression pattern may serve as a marker for the B cell differentiation stage from which CLL clones can develop.     

Epstein-Barr virus B95.8 produced in 293 cells shows marked tropism for differentiated primary epithelial cells and reveals interindividual variation in susceptibility to viral infection

Epstein-Barr virus (EBV), a well-characterised B-lymphotropic agent is aetiologically linked to B cell lymphoproliferations, but the spectrum of diseases the virus causes also includes oral hairy leukoplakia, a benign epithelial lesion, as well as carcinomas of the nasopharynx and of the stomach. However, it is still unclear how EBV accesses and transforms primary epithelial cells. Sixteen samples consisting of primary epithelial cells from the sphenoidal sinus or from tonsils were infected with GFP-tagged recombinant B95.8 EBVs produced in the 293 cell line. The rate of infection was assessed by counting GFP-positive cells and cells expressing viral proteins. Primary epithelial cells from all samples were found to be sensitive to EBV infection but there was a marked interindividual variation among the tested samples (2-48% positive cells). This suggests heterogeneity in terms of sensitivity to EBV infection in vivo and therefore possibly to EBV-associated diseases of the epithelium. The virus showed a preferential tropism for differentiated epithelial cells (p63 negative, involucrin positive). In all cases, infected cells expressed EBV lytic proteins but also the LMP1 protein. The viral tropism for differentiated cells and the permissivity of these cells for virus replication reproduced in vitro cardinal features of oral hairy leukoplakia. We have identified a source of EBV that shows unusually strong epitheliotropism for primary epithelial cells that will allow detailed analysis of virus-cell interactions during virus infection, replication and virus-mediated transformation.     

Isolation of a normal B cell subset with a Burkitt-like phenotype and transformation in vitro with Epstein-Barr virus

Epstein-Barr virus (EBV) is causally linked with endemic Burkitt's lymphoma (BL), a tumor whose homogeneous cell surface phenotype suggests derivation from a particular subset of activated germinal centre B cells in vivo. Endemic BL also shows an unusual form of EBV infection with down-regulation of certain of the virus latent proteins which are constitutively expressed when EBV infects and transforms normal resting B cells in vitro. Here we question whether this virus:cell interaction is unique to malignant BL cells or whether it might be reproduced by in vitro infection of those particular germinal centre cells displaying the BL-like phenotype. Firstly, we show by biochemical means that a subset of normal tonsillar B cells does indeed express the globotriaosylceramide glycolipid BLA and the common acute lymphoblastic leukaemia antigen CALLA, 2 important markers of the BL phenotype. Secondly, using 2-colour immunofluorescence labelling with anti-BLA and anti-CALLA monoclonal antibodies (MAbs), 4 subsets of low buoyant density tonsillar B cells (BLA+ CALLA+, BLA+ CALLA-, BLA- CALLA+, BLA- CALLA-) have been separated by means of a FACS and tested for their susceptibility to EBV-induced growth transformation in a limiting dilution assay. The BLA+ CALLA+ (i.e., BL-like) subset contained the highest proportion of cells already actively in cycle in vivo and gave the lowest yield of transformants, perhaps reflecting the greater efficiency with which EBV transforms resting target cells. Of the cell lines established from the BLA+ CALLA+ population, a significant number retained BLA expression but CALLA was always lost. In 2 further respects, these lines resembled conventional in vitro transformants rather than lines of BL type; thus the cells expressed cellular "activation" antigens (CD23, CD39, CD30, Ki-24) characteristic of the lymphoblastoid phenotype and contained the full spectrum of EBV latent proteins.