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.     

Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein.

Infection of Epstein-Barr virus-negative human B-lymphoma cell lines with the fully transforming B95.8 Epstein-Barr virus strain was associated with complete virus latent gene expression and a change in the cell surface and growth phenotype toward that of in vitro-transformed lymphoblastoid cell lines. In contrast, the cells infected with the P3HR1 Epstein-Barr virus strain, a deletion mutant that cannot encode Epstein-Barr nuclear antigen 2 (EBNA2) or a full-length EBNA-LP, expressed EBNAs1, 3a, 3b, and 3c but were negative for the latent membrane protein (LMP) and showed no change in cellular phenotype. This suggests that EBNA2 and/or EBNA-LP may be required for subsequent expression of LMP in Epstein-Barr virus-infected B cells. Recombinant vectors capable of expressing the B95.8 EBNA2A protein were introduced by electroporation into two P3HR1-converted B-lymphoma cell lines, BL30/P3 and BL41/P3. In both cases, stable expression of EBNA2A was accompanied by activation of LMP expression from the resident P3HR1 genome; control transfectants that did not express the EBNA2A protein never showed induction of LMP. In further experiments, a recombinant vector capable of expressing the full-length B95.8 EBNA-LP was introduced into the same target lines. Strong EBNA-LP expression was consistently observed in the transfected clones but was never accompanied by induction of LMP. The EBNA2A gene transfectants expressing EBNA2A and LMP showed a dramatic change in cell surface and growth phenotype toward a pattern like that of lymphoblastoid cell lines; some but not all of these changes could be reproduced in the absence of EBNA2A by transfection of P3HR1-converted cell lines with a recombinant vector expressing LMP. These studies suggest that EBNA2 plays an important dual role in the process of B-cell activation to the lymphoblastoid phenotype; the protein can have a direct effect upon cellular gene expression and is also involved in activating the expression of a second virus-encoded effector protein, LMP.     

Persistent Epstein-Barr virus infection in a human T-cell line: unique program of latent virus expression.

The growth transforming potential of Epstein-Barr virus (EBV) for Burkitt's lymphoma and nasopharyngeal carcinoma is now extended to other neoplasia, such as Hodgkin's disease, peripheral T-cell tumor and gastric cancer. We have generated an EBV recombinant with a selectable marker at the viral thymidine kinase locus. Recombinant EBV was successfully infected into a human T-cell line, MT-2. Following incubation in the selective medium, drug resistant MT-2 cell clones were isolated and proved to be infected with recombinant EBV. EBV-infected MT-2 cell clones expressed EBNA 1 and LMP 1 and very little of EBNA 2, showing the BamHI F promoter-driven latency II form of infection, which is seen in non-B-cell tumors. This is the first report of in vitro generation of latency II type EBV infection. The present system of persistent EBV infection in T cells should be a good model for investigating the pathogenic role of EBV in non-B-cell tumors.     

The Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor.

The Epstein-Barr virus (EBV)-encoded LMP1 protein is an important component of the process of transformation by EBV. LMP1 is essential for transformation of B lymphocytes, most likely because of its profound effects on cellular gene expression. Although LMP1 is expressed in the majority of nasopharyngeal carcinoma (NPC) tumors, the effect of LMP1 on cellular gene expression and its contribution to the development of malignancy in epithelial cells is largely unknown. In this study the effects of LMP1 on the expression and tyrosine kinase activity of the epidermal growth factor receptor (EGFR) were investigated in C33A human epithelial cells. Stable or transient expression of LMP1 in C33A cells increased expression of the EGFR at both the protein and mRNA levels. In contrast, expression of the EGFR was not induced by LMP1 in EBV-infected B lymphocytes. Stimulation of LMP1-expressing C33A cells with epidermal growth factor (EGF) caused rapid tyrosine phosphorylation of the EGFR (pp170) as well as several other proteins, including pp120, pp85, pp75, and pp55, indicating that the EGFR induced by LMP1 is functional. LMP1 also induced expression of the A20 gene in C33A epithelial cells. In C33A cells, LMP1 expression increased the proliferative response to EGF, as LMP1-expressing C33A cells continued to increase in number when plated in serum-free media supplemented with EGF, while the neo control cells exhibited very low levels of viability and did not proliferate. Immunoblot analysis of protein extracts from nude mouse-passaged NPC tumors also demonstrated that the EGFR is overexpressed in primary NPC tumors as well as those passaged in nude mice. This study suggests that the alteration in the growth patterns of C33A cells expressing LMP1 is a result of increased proliferative signals due to enhanced EGFR expression, as well as protection from cell death due to LMP1-induced A20 expression. The induction of EGFR and A20 by LMP1 may be an important component of EBV infection in epithelial cells and could contribute to the development of epithelial malignancies such as NPC.     

Ethnic differences in the expression of Epstein-Barr virus latent membrane protein-1 mutations in nasopharyngeal carcinoma

The latent membrane protein-1 (LMP1) of Epstein-Barr virus (EBV) is a viral oncoprotein implicated in several EBV-associated pathologies. Many studies have characterized carboxy-terminal mutations within LMP1, errors in this area are critical since this portion contains sequences responsible for LMP1 targeting, half-life and association with host cell proteins. Although, data suggests that mutations in this area extend LMP1 half-life and increase its oncogenesis, some studies have not shown this to be true for all EBV-associated tumors. In order to evaluate 3'-end LMP1-DNA mutations in three different ethnic populations with nasopharyngeal carcinoma (NPC), we examined EBV-DNA in 34 patients of various origins (Caucasian, Chinese and Inuit). While 68% of the total group expressed EBV-antigens, only 56% of Caucasians but 86% of Inuit expressed this viral protein. Over 67% of Inuit NPC tissue contained the characteristic 30 bp deletion that was observed in only 20% of Caucasians and 33% of Chinese samples. DNA sequencing revealed that the Inuit population showed the most frequent DNA mutations and corresponding amino acid alterations in LMP1. Our results suggest that EBV-associated NPC-DNA mutations in LMP1 do not occur at equal rates in different racial groups and are more common at distinct sites in NPC tissue from Chinese and Inuit sources.     

Epstein-Barr virus latent infection membrane protein increases vimentin expression in human B-cell lines.

Latent Epstein-Barr virus (EBV) infection activates B-lymphocyte proliferation through mechanisms which are partially known. One approach to further delineate these mechanisms is to identify cellular genes whose expression is augmented in cells latently infected with EBV. Since EBV-negative Burkitt's lymphoma cells can be grown in continuous culture and EBV can establish growth-altering latent infection in these cells, some effects of EBV on B-lymphocyte gene expression can be studied by using this in vitro system. Pursuing this latter approach, we have used cDNA cloning and subtractive hybridization to identify a gene whose expression is increased after EBV infection. This gene encodes the cytoskeletal protein vimentin. Latent infection of established EBV-negative Burkitt's lymphoma cell lines with the transforming EBV strain, B95-8, resulted in dramatic increases in vimentin mRNA and protein levels, while infection with the nontransforming P3HR1 strain failed to do so. Vimentin induction was reproduced by the expression of the single EBV gene which encodes the latent infection membrane protein (LMP). An amino-terminal LMP deletion mutant did not induce vimentin. These results are of particular interest in light of the transforming potential of LMP, as demonstrated in rodent fibroblasts, and the interaction between vimentin and LMP observed in immunofluorescent colocalization and cell fractionation studies.     

Replication of latent Epstein-Barr virus genomes in Raji cells.

The replication of the 50 to 60 latent, predominantly extrachromosomal, Epstein-Barr virus genomes maintained by the Burkitt-lymphoma-derived Raji cell line was investigated by using a Meselson-Stahl density transfer approach. Samples of DNA isolated from cells cultivated for different periods in bromodeoxyuridine-supplemented medium were fractionated according to density, and the distribution of viral and cellular DNAs among the heavy-, hybrid-, and light-density species was quantitated. The results indicate that the majority of latent Epstein-Barr virus DNA plasmids each replicate once during the cell cycle.     

Restricted expression of Epstein-Barr virus latent genes in murine B cells derived from embryonic stem cells

Background

Several human malignancies are associated with Epstein-Barr virus (EBV) and more than 95% of the adult human population carries this virus lifelong. EBV efficiently infects human B cells and persists in this cellular compartment latently. EBV-infected B cells become activated and growth transformed, express a characteristic set of viral latent genes, and acquire the status of proliferating lymphoblastoid cell lines in vitro. Because EBV infects only primate cells, it has not been possible to establish a model of infection in immunocompetent rodents. Such a model would be most desirable in order to study EBV''s pathogenesis and latency in a suitable and amenable host.

Methodology/Principal Findings

We stably introduced recombinant EBV genomes into mouse embryonic stem cells and induced their differentiation to B cells in vitro to develop the desired model. In vitro differentiated murine B cells maintained the EBV genomes but expression of viral genes was restricted to the latent membrane proteins (LMPs). In contrast to human B cells, EBV''s nuclear antigens (EBNAs) were not expressed detectably and growth transformed murine B cells did not arise in vitro. Aberrant splicing and premature termination of EBNA mRNAs most likely prevented the expression of EBNA genes required for B-cell transformation.

Conclusions/Significance

Our findings indicate that fundamental differences in gene regulation between mouse and man might block the route towards a tractable murine model for EBV.     

Sequence requirements of the Epstein-Barr virus latent origin of DNA replication.

The Epstein-Barr virus (EBV) latent origin of DNA replication (oriP) is composed of two elements that contain binding sites for the sole viral gene product required for latent cycle replication, EBNA-1. One of these elements, region I, functions as an EBNA-1-dependent enhancer for RNA polymerase II-transcribed genes, may play a role in plasmid segregation, and is required for origin function in B cells latently infected with EBV. The second element, region II, contains or is very near the site of initiation of DNA replication. A genetic approach was taken to determine the contribution of the EBNA-1 binding sites in oriP to origin function. Although region I is required for the transient replication of plasmids bearing region II in EBV-infected B cells, a plasmid lacking region I but containing region II, was observed to replicate transiently in both D98/Raji and HeLa cells expressing EBNA-1. Thus, binding of EBNA-1 to region I is not absolutely required for the molecular events that lead to initiation of DNA replication at region II. Site-directed mutagenesis of the four EBNA-1-binding sites in region II, individually and in various combinations, demonstrated that only two EBNA-1-binding sites are required for region II function. The results obtained with these mutants, together with the analysis of the replicative ability of plasmids containing insertions between EBNA-1-binding sites, suggested that the spatial relationship of the two sites is critical. Mutants that contain only two EBNA-1-binding sites separated by 26 to 31 bp in region II were not maintained as plasmids over many cell generations and were greatly reduced in their ability to replicate transiently in D98/Raji cells. The EBNA-1-induced bending or untwisting of the DNA in EBNA-1-binding sites 1 and 4 in region II did not, however, demonstrate this spatial constraint. It may be concluded from these results that specific protein-protein interactions between EBNA-1 and/or between EBNA-1 and a cellular protein(s) are required for origin function.     

Interferon regulatory factor 7 regulates expression of Epstein-Barr virus latent membrane protein 1: a regulatory circuit

We have shown previously that interferon regulatory factor 7 (IRF7), a multifunctional protein intimately involved in latent Epstein-Barr virus (EBV) infection, is induced as well as activated by EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein. Since the LMP1 promoter (LMP1p) contains an interferon-stimulated response element (ISRE), we hypothesized that IRF7 might be able to regulate LMP1 expression and thus participate in a regulatory circuit between these two genes. In this study, IRF7 was shown first to activate LMP1p in transient transfection assays. Compared with EBV nuclear antigen 2 (EBNA2), the most potent viral transactivator of LMP1p, IRF7 has a lesser effect (approximately 10% that of EBNA2) on induction of LMP1p. Study with IRF7 deletion mutants showed that IRF7 functional domains have similar effects on both the beta interferon (IFN-beta) and LMP1 promoters in BJAB and 293 cells, and study with IRF7 phosphomimetic mutants showed that IRF7 phosphorylation may be involved in the activation of these two promoters. Further, the ISRE in LMP1p responds to IRF7 induction and IRF7 binds to this element. In the EBV-positive cell line P3HR1, which lacks the complete EBNA2 and EBV-encoded leader protein genes and hence expresses low-level LMP1, IRF7 alone can notably increase the endogenous LMP1 mRNA and protein levels. These results indicate that LMP1 is regulated by this host cell gene in addition to the viral factor, EBNA2, and may help to explain how LMP1 is expressed in type II latency in the absence of EBNA2. Moreover, IRF7 can regulate a viral gene in addition to a host cellular gene such as the IFN-beta gene. Together with the previous data that LMP1 can induce IRF7 expression and facilitate IRF7 phosphorylation and nuclear translocation, these results suggest a positive regulatory circuit between IRF7 and LMP1.     

EBNA1 distorts oriP, the Epstein-Barr virus latent replication origin.

The Epstein-Barr virus nuclear antigen 1 (EBNA1) protein binds and activates the latent replication origin (oriP) of the Epstein-Barr virus. We have been studying EBNA1 to determine how it activates replication at oriP. Here we demonstrate that upon binding of EBNA1 to oriP, two thymine residues become reactive to potassium permanganate (KMnO4), indicating a helical distortion at these sites. The KMnO4-reactive thymines are 64 bp apart in the region of dyad symmetry of oriP. Dimethyl sulfate protection studies indicated that EBNA1 binds on the opposite face of the helix from the reactive thymines. The nature of the helical distortion induced by EBNA1 and its possible significance to the initiation of replication are discussed.     

Defective control of latent Epstein-Barr virus infection in systemic lupus erythematosus

EBV infection is more common in patients with systemic lupus erythematosus (SLE) than in control subjects, suggesting that this virus plays an etiologic role in disease and/or that patients with lupus have impaired EBV-specific immune responses. In the current report we assessed immune responsiveness to EBV in patients with SLE and healthy controls, determining virus-specific T cell responses and EBV viral loads using whole blood recall assays, HLA-A2 tetramers, and real-time quantitative PCR. Patients with SLE had an approximately 40-fold increase in EBV viral loads compared with controls, a finding not explained by disease activity or immunosuppressive medications. The frequency of EBV-specific CD69+ CD4+ T cells producing IFN-gamma was higher in patients with SLE than in controls. By contrast, the frequency of EBV-specific CD69+ CD8+ T cells producing IFN-gamma in patients with SLE appeared lower than that in healthy controls, although this difference was not statistically significant. These findings suggest a role for CD4+ T cells in controlling, and a possible defect in CD8+ T cells in regulating, increased viral loads in lupus. These ideas were supported by correlations between viral loads and EBV-specific T cell responses in lupus patients. EBV viral loads were inversely correlated with the frequency of EBV-specific CD69+ CD4+ T cells producing IFN-gamma and were positively correlated with the frequencies of CD69+ CD8+ T cells producing IFN-gamma and with EBV-specific, HLA-A2 tetramer-positive CD8+ T cells. These results demonstrate that patients with SLE have defective control of latent EBV infection that probably stems from altered T cell responses against EBV.     

Role of latent membrane protein 2 isoforms in Epstein-Barr virus latency

The oncogenic Epstein-Barr virus (EBV) infects the majority of the human population without doing harm and establishes a latent infection in the memory B-cell compartment. To accomplish this, EBV hijacks B-cell differentiation pathways and uses its own viral genes to interfere with B-cell signalling to achieve life-long persistence. EBV latent membrane protein 2A (LMP2A) provides a surrogate B-cell receptor signal essential for cell survival and is believed to have a crucial role in the maintenance of latency by blocking B-cell activation which would otherwise lead to lytic EBV infection. These two functions demand tight control of LMP2A activity and expression levels. Based on recent insights in the function of LMP2B, an isoform of LMP2A, we propose a model for how LMP2B modulates the activity of LMP2A contributing to maintenance of EBV latency.     

Orientation and patching of the latent infection membrane protein encoded by Epstein-Barr virus.

Epstein-Barr virus is known to encode three nuclear proteins and one membrane protein (LMP) in latently infected growth-transformed cells. Studies of the plasma membrane localization and orientation of LMP by protease digestion of live cells and by immunofluorescence indicated the following. (i) At least 30% of LMP is in the plasma membrane, as opposed to other cytoplasmic membranes. (ii) A small LMP domain which corresponds to a previously proposed outer reverse turn between the first two transmembrane domains is exposed on the outer cell surface (and two other proposed outer-reverse-turn domains may be exposed), whereas all or almost all of the rest of the protein is not exposed on the outer cell surface. (iii) LMP is present in patches in the cell plasma membrane.