The Epstein-Barr virus receptor on two nasopharyngeal carcinoma model cell lines

It was reported that the OKB7 monoclonal antibody to C3d receptor could directly inhibit Epstein-Barr virus (EBV) attachment to and infection of B-lymphocytes. So we tested whether the OKB7 could inhibit superinfection of two epithelial NPC model cell lines (D98/HR-1 and NPC-KT) with EBV. Pretreatment of B-lymphocytes with the OKB7 significantly inhibits EBV infection. However, pretreatment with the OKB7 had no effect on superinfection of D98/HR-1 and NPC-KT cells. These data suggest that an EBV receptor, unrelated to C3d receptor, exists.     

Induction of the Epstein-Barr virus (EBV) cycle in latently infected cells by n-butyrate.

n-Butyrate was found to increase the number of virus producer cells to a dramatic extent in the Epstein-Barr virus (EBV)-carrying P3HR-1 and B95-8 lines. Induction was also seen in the nonproducer Raji and the low producer Daudi lines, but at a mucch lower level. The virus containing supernatant of the butyrate treated P3HR-1 cells induced preferentially EBNA in EBV-negative Ramos target cells, whereas the spontaneously produced virus induced predominantly EA in Raji indicator cells. This suggests a possible difference in the biological properties of the butyrate induced vs the prototype virus. In addition to providing a convenient method to obtain a high yield of viral-DNA and virus antigen-producing cells in the severely restricted EBV system, the findings raise interesting questions on the mechanism of EBV induction, and its possible relationship to the known differentiation inducing ability of n-butyrate.     

Heterogeneity of Epstein-Barr virus. II. Induction of early antigens (EA) by complementation.

Cells of Epstein-Barr virus (EBV)-negative human B lymphoma lines BJA and Ramos were converted into EBV genome carriers by virus isolates from P3HR-1 and B 95-8 cells (Fresen and zur Hausen, 1976). Cloning of P3HR-1 virus-converted BJA cells resulted in clones with two different Epstein-Barr nuclear antigen (EBNA) patterns: a faint granular EBNA staining and clones with a brilliant EBNA expression (Fresen et al., 1977). The latter always segregated EBNA-negative cells from which one EBNA-negative subclone (B1-28) was isolated. Induction of early antigens (EA) was studied by infecting parental lines (BJA and Ramos), converted lines (BJA-HR1K, BJA-B 95-8, Ramos-HR1K, Ramos-B 95-8), the BJA-HR1K clones A5 (faint granular EBNA expression) and B1-19 (brilliant EBNA expression), the EBNA-negative subclone B1-28, and Raji cells with EBV from P3HR-1 and B 95-8 cells, respectively. The following results were obtained: (1) EA induction by P3HR-1 virus is enhanced on the average 14-fold in EBV genome-harboring cells when compared to genome-negative lines. (2) B 95-8 virus induces EA only in P3HR-1 virus-converted cells and to a small extent also in Raji cells. A significant EA induction occurs in the A5 clone of BJA-HR1K, whereas the brilliantly EBNA-expressing B1-19 clone is not induced. B 95-8 virus-converted cells cannot be induced by B 95-8 virus. (3) EA induction following infection of EBV genome-carrying cells is directly proportional to the dilution of the infecting virus. In EBV genome-free cells, EA induction is reduced by the square of the dilution factor. These results imply that resident genomes complement superinfecting genomes in EA induction by EBV and that two different populations of genomes (present in P3HR-1 virus isolates) are required for EA induction following infection of B lymphoblasts.     

Infection of the human T-cell-derived leukemia line Molt-4 by Epstein-Barr virus (EBV): induction of EBV-determined antigens and virus reproduction

We have reported previously that human T-cell-derived Molt-4 cells become susceptible to Epstein-Barr virus (EBV) infection after implantation of functional EBV receptors into the plasma membranes of Molt-4 cells (Volsky et al., 1980). In the present work, we expand this finding by analyzing the following: (i) Virus adsorption vs viral penetration—using [³H]thymidine-labeled EBV, we demonstrate that the virus could adsorb to both the untreated and the receptor-implanted Molt-4 cells. However, only the altered cells became susceptible to EBV penetration followed by the viral antigen synthesis; (ii) EBV substrain specificity of infection—EBV P3HR-1 virus induced the nuclear (EBNA), early (EA), and virus capsid (VCA) antigens, whereas EBV B-95-8 virus induced only EBNA; (iii) virus reproduction—in situ hybridization was used to demonstrate the EBV-DNA synthesis in the P3HR-1 virus-infected cells. In addition, immature herpes-like particles were observed in electron micrographs of infected cells. It is concluded that EBV infection of human T-cell-derived Molt-4 cells may lead to a full viral-lytic cycle in a portion of infected cells. The results suggest, however, that the primary EBV productive infection may not necessarily involve any immunofluorescence-detectable EBNA synthesis.     

Infection of human epithelial cells by Epstein-Barr virus (EBV). II. Biochemical characterization of EBV-determined proteins synthesized in epithelial cells

Primary cultures of epithelial cells were grown from tonsils of patients with diseases not related to EBV. The cells were implanted with EBV receptors and exposed to EBV of the transforming (B95-8, AG-876) and nontransforming (P3HR-1) strains. The EBV-infected and control cells were pulsed with [35S]methionine at 18-24 h after infection, and cell extracts were prepared for immunoprecipitation with anti-EBV sera and analysis by gel electrophoresis and autoradiography. About 20 EBV-determined proteins ranging from 22 to 185 kDa were detected in P3HR-1 virus-infected epithelial cells. Only a few polypeptides were detected in extracts of cells infected with AG-876 virus while no EBV-specific proteins were immunoprecipitated from extracts of B95-8 virus-infected cells. These results demonstrate that the system of EBV receptor-implanted normal human epithelial cells can be used for direct biochemical analysis of EBV infection in the epithelial tissue.     

Differences in Epstein-Barr virus (EBV) receptors expression on various human lymphoid targets and their significance to EBV-cell interaction

This study was aimed at quantitating, by means of fluorescence-activated cell sorter (FACS), EBV binding to different types of target cells, and at learning about a possible relation between EBV receptor density and the fate of cell-surface bound virus. We used fluoresceinated virus preparations of two strains of EBV (B95-8: lymphocyte transforming strain; P3HR-1: non-transforming strain) to analyze quantitatively the expression and density of EBV receptors on different human lymphoid cell lines and on B lymphocytes from both EBV-seropositive and -seronegative donors. FACS analysis was also used as a tool to approximate the cell surface area of the different lymphoid cells examined. Our results indicate that: (a) after accounting for the difference in cell surface dimensios, the fluorescence intensity of EBV-bound Raji (a B line) cells was three to four times higher per unit area than that of EBV-bound fresh B lymphocytes from an EBV-seropositive donor; (b) Molt-4 (a T line) cells bound about 21-fold less P3HR-1 EBV and 6-fold less B95-8 EBV than Raji cells per unit area; (c) B lymphocytes from EBV-seronegative adult donors bound only about one third as much virus as B cells from seropositive individuals; (d) two B lymphocyte sub-populations can be identified in the peripheral blood in regard to their ability to bind EBV, regardless of the EBV antibody status of the donor; (e) the EBV receptor on Molt-4 cells appears structurally different from the one found on Raji cells since EBV binding to Molt-4 cells was not blocked by a monoclonal antibody (OKB7) specific to the complement receptor (CR2). Further, in contrast to Raji cells, Molt-4 expressed a differential binding activity for each of the two EBV strains used. Taken together, the important differences observed in regard to EBV attachment to various targets also appear to relate to the fate of cell-surface bound virus: i.e., virus penetration might be determined, at least in part, by the density of EBV receptors on the target cell surface; thus the receptor density may play a major role in viral infection.     

Analysis of epstein-Barr virus (EBV) of P3HR-1: isolation of EBV with EBNA induction ability in human cord lymphocytes and without EA induction ability in Raji cells

A subline of P3HR-1 cells was isolated through a prolonged (over 1 year) propagation of the cells at a non-EBV-productive condition followed by cell cloning procedures. Cloned cells thus obtained, designated DHR1, produced EBV when brought back to the EBV-productive condition. Restriction enzyme analysis of the viral DNA revealed that DHR1 EBV is composed of an apparently homogeneous EBV population, and it displays a similar but not identical genome organization compared with HR-1 EBV. The characteristic biological properties of DHR1 EBV included the ability to induce EBV-associated nuclear antigen (EBNA) in human cord lymphocytes and the inability to induce EBV-associated early antigen (EA) in Raji cells. These are in striking contrast to the behavior of the parental HR-1 EBV. Thus, P3HR-1 cultures after a period of nonproductivity reinitiated the production of virus with an apparently homogeneous and unique population of EBV distinguishable from the original HR-1 virus.     

Induction and biological characterization of the Epstein-Barr virus (EBV) carried by the Jijoye lymphoma line

In contrast to all other EBV isolates tested, the P3HR-1 line, a clonal derivative of the Burkitt lymphoma line Jijoye, releases nontransforming, cytopathic virus, which induces an abortive cycle in superinfected, EBV-receptor-positive lymphoid cell lines. We have examined the question whether P3HR-1 virus is a unique mutant or if the original, Jijoye-associated virus had already similar biological characteristics,. Jijoye cells were induced with sodium butyrate and with anti-IgM, respectively. Transforming virus was recovered, capable of inducing EBNA in cord blood cells. Both human cord blood and marmoset lymphocytes were immortalized by Jijoye virus. Both derived lines showed a typical lymphoblastoid (LCL) phenotype, including agarose clonability. It is concluded that P3HR-1 virus is a mutant of the original transforming Jijoye virus. Moreover, the results suggest that the phenotypic properties of the transformed lines are determined at the cellular, not the viral genome level.     

Infection of normal human thymic epithelial cells by Epstein-Barr virus (EBV) following implantation of EBV receptors

Normal human thymic epithelial cells cannot be infected and transformed by Epstein-Barr virus (EBV). Measurements using fluorescein-labelled EBV and cytofluorograph revealed that the cells do not have receptors for the virus. Following transplantation of functional EBV receptors onto epithelial cell membranes, however, the cells were infected with EBV and expressed EBV-determined nuclear antigen (EBNA). No EBV associated antigens characteristic of the lytic cycle of the virus were detected. This method of in vitro infection by transforming viruses such as EBV may provide ways of deriving functioning thymic epithelial cell lines.     

Pattern of Epstein-Barr virus (EBV)-specific proteins synthesized during primary lytic infection of mouse lymphocytes by EBV

Normal mouse lymphocytes were implanted with EBV receptors and exposed to the virus of P3HR-1 strain. 5% of the cells expressed early (EA) and viral capsid (VCA) antigens as assayed by immunofluorescence 24 h after the infection. Only 0.1% of cells expressed nuclear-like antigen (EBNA) 48 h post-infection. When labelled metabolically with [35S]methionine, extracted, immunoprecipitated with EBV-positive sera, and analyzed by SDS-gel electrophoresis and autoradiography, about 20 EBV-determined proteins ranging from 19 to 165 kd were detected. Their pattern and relative quantitative expression differed from those in P3HR-1 virus superinfected Raji cells. Polypeptides of approximate molecular size 78, 72, 65, 48 and 26.5 kd were predominant in EBV-infected mouse lymphocytes. In contrast, 130, 98, 59, 50.5 and 36 kd proteins were predominant in the induced Raji cells. Our results demonstrate that rodent lymphocytes can be used for the direct biochemical analysis of EBV-translational products during primary lytic infection in normal cells.     

EB病毒感染对鼻咽癌细胞生长和凋亡的影响

目的:探讨EB病毒(EBV)感染对鼻咽癌细胞系生长和细胞凋亡的影响。方法:用EBV直接感染人鼻咽癌细胞系CNE1;用免疫组化(LSAB)法检测EBV-LMP1和bcl-2蛋白的表达;用MTT法测定鼻咽癌细胞系的生长能力;用流式细胞术和TUNEL法检测癌细胞凋亡。结果:感染EBV的鼻咽癌细胞系(E-CNE1)的EBV-LMP1表达阳性,生长能力较未感染EBV的CNE1明显增强(P＜0.01),2种鼻咽癌细胞系均无凋亡发生,而均仅有2%~3%的细胞表达bcl-2蛋白。结论:EBV感染和LMP1表达可促进鼻咽癌细胞生长,但对鼻咽癌细胞的bcl-2表达和细胞凋亡无影响。     

Human B cell line deficient in the expression of B cell-specific glycoproteins (GP 27,35).

A human B lymphoid cell line, P3HR-1, expresses only low levels of the 27 000 and 35 000 mol.wt. B cell-specific glycoproteins (GP 27,35). Indirect antibody-binding and quantitative absorption tests with a xenoantiserum against the antigens showed that P3HR-1 cells have on their surface about 1% of the amount found on other human B lymphoblastoid cell lines. The deficit of the glycoproteins on the surface of P3HR-1 cells could be accounted for by a reduced rate of synthesis in these cells. A simple relationship between the reduced expression of GP 27,35 on P3HR-1 cells and their inability to bind Epstein-Barr virus (EBV) or express complement receptors was excluded because other B lymphoid cells which expressed neither virus-binding sites nor complement receptor had normal amounts of GP 27,35 on their surface. However, antibodies against GP 27,35 could block the absorption of EBV by EBV receptor-positive B cells.     

B cell activation by the nontransforming P3HR-1 substrain of the Epstein-Barr virus (EBV)

The P3HR-1 substrain of Epstein-Barr virus does not transform B cells. This defect is known to be determined by the loss of the coding sequence for the nuclear antigen EBNA-2. The virus can attach to and enter resting B cells. The initial events after EBV infection are reminiscent of those induced by polyclonal B cell activators. Similar to the effect of these, P3HR-1 virus lowers membrane IgD expression on B cells and abrogates the transient elevation of activation markers BB-1 and LB-1 induced by the culture conditions. An important event of B cell activation is the acquisition of competence to respond to specific growth factors produced by T cells. This was induced by the P3HR-1 virus. The infected B cells had elevated [3H]thymidine incorporation when exposed to the supernatant of PHA-treated T cells. The EBV receptor is identical with the complement receptor CR2. Ligand binding to CR2 has been shown both with mouse and human B cells to deliver certain activation signals. Therefore, it is possible that the early step of activation by EBV is initiated through the binding to the receptor and is thus a cell surface event.     

CD21-independent Infection of Epstein-Barr Virus in Human Signet Ring Gastric Carcinoma Cell Line

Epstein Barr virus ( EBV), a ubiquitous human her pesvirus, is the etiologic agent of infectious mononucleo sis, and is closely associated with several human malig nancies including Burkitt′s lymphoma (BL), undifferenti ated nasopharyngeal carcinoma (NPC) and opportunisticlymphoma in immunocompromised hosts. In recent years,there have been increasing evidences of association of EBVwith additional malignancies, such as gastric carcinoma.EBV has been found in tumor cells of m…     

Studies on an epithelial/Burkitt hybrid cell line prepared from a lymphoblastoid cell line with one EBV genome equivalent per cell.

An epithelial hybrid cell line was prepared by fusing D98 cells to an HR-1 cell line previously shown to contain only one EBV genome equivalent per cell. At an early passage level, p 11, the EBV genome could not be induced to synthesize EBV antigens after treatment with iododeoxyuridine (IUdR). When the cells were retested for this property at passage 34, full induction of the EBV genome was observed. The ability of induction of EBV antigens and virus DNA depended on a spontaneous increase in the level of EBV DNA in the cells as they were passaged.     

Upregulation of LMP1 Expression by Histone Deacetylase Inhibitors in an EBV Carrying NPC Cell Line

OBJECTIVES: In about 60% of Epstein-Barr virus (EBV) carrying nasopharyngeal carcinomas (NPC) LMP1 expressing cells can be detected. The frequency of LMP1 positive cells and the expression level varies from cell to cell in the different tumors. Cell lines derived from EBV positive NPCs loose the virus during in vitro culture. The in vitro infected NPC cell line TWO3-EBV used in our study carries the neomycin-resistance gene containing EBV and expresses low level of LMP1. With this cell line it was thus possible to study the regulation of LMP1 expression by modification of chromatin acetylation state. STUDY DESIGN: The TWO-EBV cell line was treated with n -butyrate (NB) or trichostatin A (TSA). RESULTS: Shown by immunoblotting, the LMP1 level was elevated in the treated samples. Already 2 h after TSA exposure LMP1 expression was higher and it increased up to 24 h. Immunofluorescence staining showed that nearly all cells were LMP1 positive. Neither EBNA2 nor BZLF1 were induced. Tested first 2 h after the treatment, acetylated histone H3 and H4 were already detectable, and their level increased up to 8 h. Chromatin immunoprecipitation (ChIP) verified that the LMP1-promoter (LMP1p) (ED-L1) was acetylated after TSA treatment. CONCLUSION: EBV carrying epithelial cells do not express EBNA-2. We showed that LMP1 expression was upregulated by histone deacetylase inhibitors in an in vitro infected, EBV carrier NPC cell line.     

Epstein-Barr virus (EBV)--lymphoid cell interactions. II. The influence of the EBV replication cycle on natural killing and antibody-dependent cellular cytotoxicity against EBV-infected cells.

We investigated the influence of the Epstein-Barr virus (EBV) replication cycle on natural killing (NK) activity and antibody-dependent cellular cytotoxicity (ADCC) against EBV-infected cells. Peripheral blood lymphocytes from healthy EBV-seropositive and -seronegative donors were separated on Ficoll-Hypaque gradients and used as effector cells in the standard 51Cr release assay to measure NK and ADCC. EBV-genome positive RAJI and DAUDI cells superinfected with either the non-transforming P3HR-1 EBV or the transforming B95-8 EBV were used as targets. The results obtained show that most normal individuals have ADCC and NK activity against P3HR-1 EBV-infected RAJI cells. Both the cytotoxic activities increased with the proportional increase in effector/target (E/T) ratios, assay incubation time, dose of the infecting virus and the time of pre-infection with EBV. Moreover, the data obtained indicate that different immune mechanisms are effective at different stages of the virus replication cycle. During the early stages of virus replication, EBV-superinfected cells are more susceptible to ADCC than to NK, whereas in later stages the susceptibility to NK is increased significantly and appears to play a more dominant role. The nature of the target cells or the strain of EBV used to superinfect these targets did not influence their susceptibility to ADCC and NK activity; however some quantitative differences were found. Using metabolic inhibitors such as cytosine arabinoside, phosphonoacetic acid, actinomycin D, cycloheximide and puromycin, it was found that new DNA synthesis is not essential but some RNA and protein synthesis is necessary, late in the viral cycle, for the superinfected cells to become susceptible to NK and ADCC.     

A membrane-located glycosphingolipid of monocyte/granulocyte lineage cells induces growth arrest and triggers the lytic viral cycle in Epstein-Barr virus genome-positive Burkitt lymphoma lines

Gangliosides are known to influence cell growth and differentiation. The neolacto series ganglioside IV³NeuAc-nLc₄ (2→3-sialosylparagloboside) is present in members of the monocyte/granulocyte lineage, but is not found in cells that belong to the lymphocyte lineage. In this study we demonstrated that IV³NeuAc-nLc₄ inhibits the proliferation of Epstein-Barr virus (EBV) genome-positive Burkitt lymphoma cells of the lines Raji and P3HR-1K. IV³NeuAc-nLc₄-induced growth inhibition is associated with an increase in G₀/G₁ phase cells and a reduced expression of CD21 and HLA-DR antigens on Raji cells. These data suggest that IV³NeuAc-nLc₄ may affect differentiation of lymphoma cells. Additionally, the increased expression of viral mRNA species which are characteristic for the lytic viral cycle in the non-producer line Raji and the enhanced release of virions from the producer line P3HR-1K demonstrate that IV³NeuAc-nLc₄ activates the replication of EBV. Growth inhibition and termination of the viral latency suggest that IV³NeuAc-nLc₄ present in monocyte/granulocyte lineage cells may be an effector of the natural defense against EBV persistency and transformation. Received: 20 December 1998     

EB病毒感染对鼻咽癌细胞生长和凋亡的影响

目的:探讨EB病毒(EBV)感染对鼻咽癌细胞系生长和细胞凋亡的影响。方法:用EBV直接感染人鼻咽癌细胞系CNE1;用免疫组化(LSAB)法检测EBV-LMP1和bcl-2蛋白的表达;用MTT法测定鼻咽癌细胞系的生长能力;用流式细胞术和TUNEL法检测癌细胞凋亡。结果:感染EBV的鼻咽癌细胞系(E-CNE1)的EBV-LMP1表达阳性,生长能力较未感染EBV的CNE1明显增强(P<0.01),2种鼻咽癌细胞系均无凋亡发生,而均仅有2%~3%的细胞表达bcl-2蛋白。结论:EBV感染和LMP1表达可促进鼻咽癌细胞生长,但对鼻咽癌细胞的bcl-2表达和细胞凋亡无影响。