Epstein-Barr virus promotes epithelial cell growth in the absence of EBNA2 and LMP1 expression

We attempted to infect primary gastric epithelia (PGE) with recombinant Epstein-Barr virus (EBV) carrying a selectable marker that made it possible to select EBV-infected cells. Cells dually positive for EBV-determined nuclear antigen (EBNA) and cytokeratin were detected in 3 of 21 primary cultures after 3 days of EBV inoculation. From one culture, EBV-infected cell clones were repeatedly obtained at a frequency of 3 to 5 cell clones per 10(6) cells. EBV-infected clones had enhanced population doubling and grew to attain a highly increased saturation density, together with acquisition of marked anchorage independence. The infected clones retained the ultrastructural morphology characteristic of gastric mucosal epithelium and have been growing stably for more than 18 months (corresponding to at least 300 generations) so far, in clear contrast to the parental PGE cells, which ceased growth after 60 generations. The p53 gene of the parental PGE cells was found to be overexpressed, perhaps thereby conferring the basal potential for long-term survival in vitro. Moreover, EBV infection accelerated, to a significant extent, the growth rate and agar clonability of NU-GC-3 cells, an established EBV-negative but EBV-susceptible human gastric carcinoma cell line. Both EBV-converted PGE and NU-GC-3 clones, like EBV-positive gastric carcinoma biopsy specimens, expressed a restricted set of EBV latent infection genes characterized by the absence of EBNA2 and latent membrane protein 1 (LMP1) expression. These results indicate that EBV infection causes a transformed phenotype on PGE in the setting of possible unregulated cell cycling and renders even established gastric carcinoma cells more malignant via a limited spectrum of viral latent-gene expression. This study may reflect an in vivo scenario illustrating multiphasic involvement of EBV in carcinogenesis of gastric or other epithelial cancers.     

Infection of leukaemic B lymphocytes by Epstein Barr virus

Epstein-Barr virus (EBV) infection of B lymphocytes in vitro gives rise to immortalized lymphoblastoid cell lines. Previous reports have shown that chronic lymphocytic leukaemia (CLL) cells, although infectable by EBV, are resistant to immortalization (1-4), although a small number of CLL cell lines have been reported (5-7). In the present study we have analysed early events occurring after EBV infection in 16 CLL samples. Out of 16 samples, 15 could be infected by the virus and expressed the full EB viral nuclear antigen (EBNA) complex but only one out of 16 expressed the latent membrane protein (LMP). The five CLLs in which we could investigate the presence of viral episomes showed circularized EBV by 16 hours after infection. The sequence of EBNA expression and genome circularization mirrored that seen in normal B cells, although genome amplification was not detected. The only CLL sample which expressed LMP after EBV infection was induced to proliferate for 2-3 weeks, but no cell line was established. Immortalized cell lines were obtained from three out of 16 samples tested, but all were polyclonal for light chain expression and had arisen from the CD5-negative, normal B-cell population. Thus the inability of EBV to induce proliferation of most CLL cells correlated with the absence of LMP expression which is invariably expressed during immortalization of normal B cells. This novel type of restricted gene expression could be compatible with evasion of host immune responses and consequent long-term survival of the cell in vivo.     

Distribution of monoclonal antibodies in intestinal and urogenital secretions of mice bearing hybridoma ''backpack'' tumours

Primary Epstein-Barr virus (EBV) infection may manifest itself as a benign lymphoproliferative disorder, infections mononucleosis (IM). EBV infection has been characterized in lymphoreticular tissues from nine patients with IM using the abundantly expressed EBV-encoded nuclear RNAs (EBERs) as a marker of latent infection. Expression of the virus-encoded nuclear antigen (EBNA) 2 and of the latent membrane protein (LMP) 1 was seen in variable proportions of cells in all cases. Double labelling revealed heterogeneous expression patterns of these proteins. Thus, in addition to cells revealing phenotypes consistent with latencies I (EBNA2-/LMP1-) and III (EBNA2+/LMP1+), cells displaying a latency II pattern (EBNA2-/LMP1+) were observed. Cells expressing EBNA2 but not LMP1 were also detected; whilst this may represent a transitory phenomenon, the exact significance of this observation is at present uncertain. EBER-specific in situ hybridization in conjunction with immunohistochemistry revealed expression of the EBERs mainly in B-lymphocytes, many of which showed features of plasma cell differentiation. By contrast, convincing evidence of latent EBV infection was not found in T-cells, epithelial or endothelial cells. Double-labelling immunohistochemistry revealed expression of the replication-associated BZLF1 protein in small lymphoid cells, often showing plasmacytoid differentiation. There was no unambiguous expression of this protein in other cell types. These results suggest that B-cells are the primary target of EBV infection and that plasma cells may be a source of infectious virus found in the saliva of IM patients.     

Epstein-Barr virus (EBV) can immortalize B-cll cells activated by cytokines

B-type of chronic lymphocytic leukemia (B-CLL) cells are inert to the potent transforming action of Epstein-Barr virus (EBV). The mitogenic action of Staphylococcus aureus Cowan I (SAC), MP6-thioredoxin, and interleukin 2 (IL-2), agents previously shown to induce proliferation in normal as well as in B-CLL cells, lifted this block, and EBV-positive cell lines could be established. It was not possible to establish cell lines of leukemic origin from cultures that were incubated with EBV alone or cytokine mix alone. CLL-cells infected with EBV only, expressed the viral nuclear antigen complex (EBNA), but not the viral latent membrane protein (LMP). They were not activated as measured by cell size and 3H-thymidine incorporation. In contrast, cells incubated with EBV and cytokine mix expressed both EBNA and LMP in parallel with enlargement and increased 3H-thymidine incorporation. These results emphasize that LMP expression is a prerequisite for growth transformation and immortalization and that cytokine activation signals are required for its expression in B-CLLs. Cells incubated with SAC/MP6-thioredoxin/IL-2 did not express any of the viral antigens, but were activated with regard to the mentioned parameters. Nine cell lines were established from six patients. From each of the three patients, we obtained 'twin'-pair lines: one corresponding to the malignant cell and the other to a normal B-lymphoblastoid cell. Thus, malignant and normal B-cell counterparts, from the very same donor, are at hand for comparative studies. The cell lines have been carried out for more than 12 months in culture. We conclude that B-CLL that are refractory to EBV-transformation can be rendered susceptible through in vitro cytokine activation.     

Regional differences in the arterial response to vasopressin: role of endothelial nitric oxide

Two Epstein-Barr virus (EBV) gene products, latent infection membrane protein 1 (LMP1), expressed mainly in latent infection, and BHRF1, expressed in lytic infection, have the ability to promote cell survival. LMP1 protects human B cells from apoptosis by upregulating expression of Bcl-2 and A20. We have demonstrated that LMP1 transfectants of Jurkat T cells are resistant to apoptosis induced by serum depletion without affecting the Bcl-2/Bax system. Overexpression of LMP1 in epithelial cells inhibits apoptosis induced by TNF-alpha, but not by anti-Fas antibodies. These results indicate that the anti-apoptotic mechanism of LMP1 differs among different cell types. BHRF1 can prevent apoptosis induced by TNF-alpha and anti-Fas antibodies in epithelial cells. The implication of the anti-apoptotic function of LMP1 and BHRF1 is reviewed in relation to EBV infection.     

Analysis of major histocompatibility complex class I, TAP expression, and LMP2 epitope sequence in Epstein-Barr virus-positive Hodgkin's disease

The Epstein-Barr virus (EBV)-encoded latent membrane proteins, LMP1 and LMP2, are consistently expressed by the malignant Hodgkin/Reed-Sternberg (HRS) cells of EBV-associated Hodgkin's disease (HD). Cytotoxic T lymphocyte (CTL) responses to both of these proteins have been shown in the blood of EBV-seropositive individuals, yet in HD the apparent failure of the CTL response to eliminate HRS cells expressing LMP1 and LMP2 in vivo has given rise to the suggestion that HD may be characterized by the presence of defects in antigen processing/presentation or in CTL function. This study has used immunohistochemistry to show high-level expression of major histocompatibility complex (MHC) class I molecules by the HRS cells of EBV-associated HD and either low level or absence of expression of MHC class I molecules on HRS cells of EBV-negative tumors. In addition, HRS cells expressed high levels of transporter-associated proteins (TAP-1, -2), irrespective of the presence of latent EBV infection. These results suggest that global downregulation of MHC class I molecules does not account for the apparent ability of EBV-infected HRS cells to evade CTL responses, but may be important in the understanding of EBV-negative disease. We have also sequenced an epitope in LMP2A (CLGGLLTMV) that is restricted through HLA A2.1, a relatively common allele in Caucasian populations, and showed that this epitope is wild type in a small group of EBV-associated HLA A2.1-positive HD tumors. This result may be relevant to proposed immunotherapeutic approaches for EBV-positive HD patients that target CTL epitopes.     

Epstein-Barr virus nuclear antigen 2 transactivates the long terminal repeat of human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1)-infected subjects show a high incidence of Epstein-Barr virus (EBV) infection. This suggests that EBV may function as a cofactor that affects HIV-1 activation and may play a major role in the progression of AIDS. To test this hypothesis, we generated two EBV-negative human B-cell lines that stably express the EBNA2 gene of EBV. These EBNA2-positive cell lines were transiently transfected with plasmids that carry either the wild type or deletion mutants of the HIV-1 long terminal repeat (LTR) fused to the chloramphenicol acetyltransferase (CAT) gene. There was a consistently higher HIV-1 LTR activation in EBNA2-expressing cells than in control cells, which suggested that EBNA2 proteins could activate the HIV-1 promoter, possibly by inducing nuclear factors binding to HIV-1 cis-regulatory sequences. To test this possibility, we used CAT-based plasmids carrying deletions of the NF-kappa B (pNFA-CAT), Sp1 (pSpA-CAT), or TAR (pTAR-CAT) region of the HIV-1 LTR and retardation assays in which nuclear proteins from EBNA2-expressing cells were challenged with oligonucleotides encompassing the NF-kappa B or Sp1 region of the HIV-1 LTR. We found that both the NF-kappa B and the Sp1 sites of the HIV-1 LTR are necessary for EBNA2 transactivation and that increased expression resulted from the induction of NF-kappa B-like factors. Moreover, experiments with the TAR-deleted pTAR-CAT and with the tat-expressing pAR-TAT plasmids indicated that endogenous Tat-like proteins could participate in EBNA2-mediated activation of the HIV-1 LTR and that EBNA2 proteins can synergize with the viral tat transactivator. Transfection experiments with plasmids expressing the EBNA1, EBNA3, and EBNALP genes did not cause a significant HIV-1 LTR activation. Thus, it appears that among the latent EBV genes tested, EBNA2 was the only EBV gene active on the HIV-1 LTR. The transactivation function of EBNA2 was also observed in the HeLa epithelial cell line, which suggests that EBV and HIV-1 infection of non-B cells may result in HIV-1 promoter activation. Therefore, a specific gene product of EBV, EBNA2, can transactivate HIV-1 and possibly contribute to the clinical progression of AIDS.     

CD21-Dependent infection of an epithelial cell line, 293, by Epstein-Barr virus

Epstein-Barr virus (EBV) is invariably present in undifferentiated nasopharyngeal carcinomas, is found sporadically in other carcinomas, and replicates in the differentiated layer of the tongue epithelium in lesions of oral hairy leukoplakia. However, it is not clear how frequently or by what mechanism EBV infects epithelial cells normally. Here, we report that a human epithelial cell line, 293, can be stably infected by EBV that has been genetically marked with a selectable gene. We show that 293 cells express a relatively low level of CD21, that binding of fluorescein-labeled EBV to 293 cells can be detected, and that both the binding of virus to cells and infection can be blocked with antibodies specific for CD21. Two proteins known to form complexes with CD21 on the surface of lymphoid cells, CD35 and CD19, could not be detected at the surface of 293 cells. All infected clones of 293 cells exhibited tight latency with a pattern of gene expression similar to that of type II latency, but productive EBV replication and release of infectious virus could be induced inefficiently by forced expression of the lytic transactivators, R and Z. Low levels of mRNA specific for the transforming membrane protein of EBV, LMP-1, as well as for LMP-2, were detected; however, LMP-1 protein was either undetectable or near the limit of detection at less than 5% of the level typical of EBV-transformed B cells. A slight increase in expression of the receptor for epidermal growth factor, which can be induced in epithelial cells by LMP-1, was detected at the cell surface with two EBV-infected 293 cell clones. These results show that low levels of surface CD21 can support infection of an epithelial cell line by EBV. The results also raise the possibility that in a normal infection of epithelial cells by EBV, the LMP-1 protein is not expressed at levels that are high enough to be oncogenic and that there might be differences in the cells of EBV-associated epithelial cancers that have arisen to allow for elevated expression of LMP-1.     

Epstein-Barr virus-associated dural leiomyosarcoma in a man infected with human immunodeficiency virus. Case report

A 35-year-old man infected with human immunodeficiency virus presented with cervical myelopathy of 2 months duration. Clinical and radiographic evaluation revealed a discrete, subdural mass at C-6. At surgery, the mass proved to have a dural attachment and thus clinically, radiographically, and grossly, it resembled meningioma. Histopathological analysis revealed a leiomyosarcoma that stained diffusely for muscle-specific actin. Electron microscopy revealed basal lamina surrounding the tumor cells and intracytoplasmic bundles of myofilaments. Epstein-Barr virus (EBV) was demonstrated within tumor cell nuclei by in situ hybridization for EBER1 messenger RNA and immunohistochemical staining for EBNA2 protein. Epstein-Barr virus latent membrane protein (LMP1) was not detected. This is the first documentation of an EBV-associated smooth-muscle tumor of the dura, and the first demonstration that tumors in this location contain EBV in an unusual form of latency not seen in lymphoid cell lines. With increasing numbers of individuals being afflicted with long-term immunosuppression, EBV-associated dural leiomyoma and leiomyosarcoma may be encountered more frequently in the future.     

Epstein-Barr virus contributes to the malignant phenotype and to apoptosis resistance in Burkitt's lymphoma cell line Akata

In the present study, we established an in vitro system representing the Burkitt's lymphoma (BL)-type Epstein-Barr virus (EBV) infection which is characterized by expression of EBV-determined nuclear antigen 1 (EBNA-1) and absence of EBNA-2 and latent membrane protein 1 (LMP1) expression. EBV-negative cell clones isolated from the EBV-positive BL line Akata were infected with an EBV recombinant carrying a selectable marker, and the following selection culture easily yielded EBV-infected clones. EBV-reinfected clones showed BL-type EBV expression and restored the capacity for growth on soft agar and tumorigenicity in SCID mice that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. Moreover, it was found that EBV-positive cells were more resistant to apoptosis than were EBV-negative cells. EBV-infected cells expressed the bcl-2 protein, through which cells might become resistant to apoptosis, at a higher level than did uninfected cells. This is the first report that BL-type EBV infection confers apoptosis resistance even in the absence of expression of LMP1 and BHRF1, both of which are known to have an antiapoptotic function. Surprisingly, transfection of the EBNA-1 gene into EBV-negative Akata clones could not restore malignant phenotypes and apoptosis resistance, thus suggesting that EBNA-1 alone was not sufficient for conferring them. Our results suggest that the persistence of EBV in BL cells is required for the cells to be more malignant and apoptosis resistant, which underlines the oncogenic role of EBV in BL genesis.     

Immunohistochemical detection of the Epstein-Barr virus-encoded latent membrane protein 2A in Hodgkin's disease and infectious mononucleosis

We describe two new monoclonal antibodies specific for the Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) that are suitable for the immunohistochemical analysis of routinely processed paraffin sections. These antibodies were applied to the immunohistochemical detection of LMP2A in Hodgkin's disease (HD). LMP2A-specific membrane staining was seen in the Hodgkin and Reed-Sternberg (HRS) cells of 22 of 42 (52%) EBV-positive HD cases, but not in 39 EBV-negative HD cases. In lymphoid tissues from patients with acute infectious mononucleosis (IM), interfollicular immunoblasts were shown to express LMP2A. This is the first demonstration of LMP2A protein expression at the single-cell level in EBV-associated lymphoproliferations in vivo. The detection of LMP2A protein expression in HD and IM is of importance in view of the proposed role of this protein for maintaining latent EBV infection and its possible contribution for EBV-associated transformation. Because LMP2A provides target epitopes for EBV-specific cytotoxic T cells, the expression of this protein in HRS cells has implications for the immunotherapeutic approaches to the treatment of HD.     

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.     

v-representability for noninteracting systems

The Epstein-Barr virus (EBV) induces unlimited growth of B lymphocytes in vitro, a phenomenon known as immortalization. The elucidation of the mechanisms by which EBV de-regulates B-cell proliferation in vitro will permit an understanding of how the virus contributes in vivo to the genesis of Burkitt's lymphoma (BL) and of lymphoproliferations in immunosuppressed patients. At present, no single EBV immortalizing gene has been identified, and the hypothesis has been made that many viral genes cooperate in establishing an autocrine loop of secretion leading to immortalization. Constitutive expression of B-cell surface molecules such as CD21 and CD23, specifically implicated in the control of B-cell proliferation, is indeed induced at the surface of immortalized B lymphocytes. The expression of the viral nuclear antigen 2 (EBNA2) has been shown to be in part responsible for CD21 and CD23 up-regulation, and EBNA2 is suspected to be a transactivator of cellular genes, although this point remains to be demonstrated. The role of EBNA2 gene, independently of other viral genes, has been investigated by transfection into B-lymphoma lines, but conflicting results have been reported. To further investigate its role in the regulation of CD21 and CD23 molecules, we have compared the effects of EBNA2 expression in 2 sets of B-lymphoma lines infected with P3HR1 EBV strain, and/or transfected with EBNA2 gene. We report here that: (i) EBNA2 expression is not a sufficient condition to induce CD21 and CD23 upregulation, EBNA2's effects are highly dependent on the cellular context, and moreover can be modified by infection with P3HR1 virus; (ii) EBNA2 induces activation of CD23 expression in a very particular way, namely, an increased quantity of CD23 steady-state RNA coding for the form A of the protein, which is not detectable at the cell surface but directly secreted.