Stress and the transformation of lymphocytes by Epstein-Barr virus

Although various stressors appear to influence herpesvirus infections, the underlying mechanisms have not been studied. A prospective design was used to examine the effects of examination stress and loneliness on the transformation of B lymphocytes in mixed cultures of T and B lymphocytes by Epstein-Barr virus (EBV). Three blood samples were drawn from 42 EBV-seropositive medical students, with the baseline sample taken 1 month before their final examinations, the stress sample drawn on the first day of final examinations, and the third sample taken the first week after their return from summer vacation. A median split on the UCLA Loneliness Scale divided the subjects into high- and low-scoring loneliness groups. There were significant effects for change over trials, with the lowest transformation levels (i.e., more virus required to transform cells) found in the stress sample. There was also a significant main effect for loneliness, in which high loneliness was associated with lower transformation levels. Possible immunological pathways for the observed changes are discussed.     

Efficiency of transformation of lymphocytes by Epstein-Barr virus.

We explored host cell and viral factors which influence the susceptibility of lymphocytes to immortalization by the Epstein-Barr virus (EBV). We found that immortalization by the B95-8 strain of EBV follows “one-hit” kinetics. With a limiting dilution technique, approximately 1 in 20 virus particles is transforming when assayed on human umbilical cord leukocytes (HUCL). Mixed mononuclear leukocytes from adult human and marmoset blood are 100- and 1000-fold, respectively, less sensitive to immortalization than HUCL. Pretreatment of cord blood leukocytes with phytohemagglutinin results in a 50% increase in their transformability. Lipopolysaccharide (LPS) from Escherichia coli increases the number of transformation events observed by 300–500%. LPS appears to enhance transformation by mechanisms other than stimulation of cellular DNA synthesis. A transformed center assay employing either autochthonous lymphocytes or human placental cells as feeders was used to determine the number of umbilical cord cells which were immortalized. At multiplicities of infection of 30–100 particles per cell, multiplicities which saturate nearly all the susceptible cells, approximately 1 in 200 to 1 in 500 mixed mononuclear cells transformed. When the mixed population of cells was depleted of T lymphocytes, there was a fourfold increase in the number of transformed cells. When correction is made for the plating efficiency of transformed cord blood leukocytes, it is calculated that at least 10% of virus-exposed cells establish permanent lines. This number is probably still an underestimate since we have not yet used a population which is composed entirely of susceptible cells.     

Identification of the target cells in human B lymphocytes for transformation by Epstein-Barr virus.

Lymphocytes from human umbilical cord blood were purified and then separated into rosette-forming cells (T cells) and non-rosette-forming cells (non-T cells). Non-rosette-forming cells were further divided by the technique of rosette formation, after neuraminidase treatment, into surface immunoglobulin (SIg)-carrying cells (B cells) and cells lacking SIg but carrying Fc receptors (null cells). The three cell subsets, T, B, and null cells, were examined for susceptibility to transformation by the B95-8 strain of Epstein-Barr virus (EBV) using the criteria of colony formation by transformed cells and/or transformation efficiency as judged by the days required for the first appearance of transformation. Not only the T cells but also the null cells were unsusceptible to transformation by EBV. In contrast, B cells were highly susceptible. In a study of the quantitative relationship between the target cells for viral transformation and those B cells which possessed SIg after an acid pH treatment, 10 lymphocyte preparations from three cord blood samples and seven adult peripheral blood samples were tested individually. The fraction size of transformable cells was determined by the growth-curve procedure for transformed cells while the procedure of direct membrane immunofluorescence with anti-IgM (μ-specific) serum and polyvalent anti-Ig serum was used to determine the fractions of SIg and SIg(M) cells. The actual fraction of EBV target cells was nearly equal to that of SIg(M) B cells but not to that of the total SIg B cells. Thirty-four lymphocyte preparations from eight cord and 26 adult peripheral blood samples were examined for the percentage of SIg B cells and for susceptibility to EBV transformation as assayed by the colony-formation procedure. EBV susceptibility and the fraction of SIg(M) B cells, but not of total SIg B cells, correlated nicely, suggesting that SIg(M)-bearing cells were probably the major target among the B lymphocytes for transformation by EBV.     

Plasmodium falciparum products enhance human lymphocyte transformation by Epstein-Barr virus

Supernatants obtained from the in vitro culture of Plasmodium falciparum infected erythrocytes induced prolonged lymphocyte survival in culture for more than 8 weeks in six cultures and permanent cell lines were established in four of these. The cells in the latter showed lymphoblastoid features similar to those seen in parallel cultures to which transforming Epstein-Barr (EB) virus instead of P. falciparum derived substances had been added. Cells from the same donors stimulated with other mitogens (pokeweed mitogen, Salmonella paratyphi culture supernatants) ceased to proliferate and died after 3-4 weeks. A 195 Kd polypeptide obtained from P. falciparum parasites also exhibited the potential to transform normal lymphocytes. Characterization of the cell lines indicated a B lymphocyte origin and the presence of EB virus in these lines suggests the possibility that P. falciparum products may activate latent EB virus genomes. These observations appear relevant to both the choice of P. falciparum derived antigens as vaccines, and to the interaction of EB virus and malaria in the aetiology of African Burkitt's lymphoma (BL).     

The production of anti-HBc IgM antibodies by human lymphocytes transformed by the Epstein-Barr virus

In transformation by Epstein-Barr virus of lymphocytes derived from patients with acute hepatitis B, continuous cell cultures were obtained which produced anti-HBc IgM antibodies. These cell lines underwent from 50 to 150 passages. The level of the specific immunoglobulin production was shown to have a trend to decline; however, after cloning the antibody production became more stable. The antibodies produced by the cloned lymphocyte culture could not neutralize the antigen (unlike polyclonal antibodies) which indicated a high efficacy of cloning. When the solid phase was sensitized with produced immunoglobulins, specific activity of binding of HBc antigen detectable by anti-HBc conjugate with horseradish peroxidase was demonstrated. The study of sedimentation properties of antibodies produced by transformed lymphocytes showed their sedimentation constant to be 19S.     

Reversible inhibition by phosphonoacetic acid of human B lymphocyte transformation by Epstein-Barr virus.

B lymphocytes purified by immunoabsorbent chromatography from the peripheral blood lymphocytes of adults provide highly effective targets for infection and transformation by Epstein-Barr virus (EBV). Using this system, the kinetics of DNA synthesis induction due to EBV infection have been characterized. The kinetics show two phases: an early phase, lasting 3 to 4 days, the rate and absolute level of which are dependent upon multiplicity of infection; and a later phase, representing normal exponential growth, the level but not rate of which is dependent upon multiplicity of infection. The induction of DNA synthesis begins at a time (24 hr) which agrees well with the published times for the appearance of the Epstein-Barr virus nuclear antigen (EBNA). Absorption and penetration of the cells by EBV appear to require 1 to 2 hr. The induction of DNA synthesis proceeds normally during the first phase in the presence of phosphonoacetic acid (PAA; 200 μg/ml). Thereafter, DNA synthesis remains at a plateau. Blast-transformed EBNA-positive cells may be isolated from these cultures. Removal of PAA at any time up to 28 days postinfection results in resumed proliferation and outgrowth. These cells express four phenotypic properties of transformation by EBV: (1) induction of DNA synthesis, (2) EBNA expression, (3) blast transformation, and (4) immortalization (survival for at least 28 days). However, they cannot grow out. It is therefore proposed that the phenomenon of EBV infection in the presence of PAA may be termed “abortive transformation.”     

Epstein-Barr virus: transformation of lymphocytes separated by size or exposed to bromodeoxyuridine and light.

Experiments designed to assess the physiological state of cells susceptible to transformation by Epstein-Barr virus (EBV) indicated that transformation does not require cellular DNA synthesis at the time of virus exposure and that a resting lymphocyte can be the target cell. The following results support this conclusion: Lymphocyte preparations from different human umbilical cords vary in extent of spontaneous DNA synthesis, but EBV-induced transformation is independent of this variation. Increases in spontaneous DNA synthesis which occur after several days in culture are not accompanied by increased cell sensitivity to transformation. Transformation occurs in highest frequency in partially purified cell subpopulations with low levels of DNA synthesis. Conversely, lymphocyte subpopulations with high rates of spontaneous DNA synthesis are relatively refractory to transformation. The fraction of cells transformed by EBV (which we estimate to be about 10% after correction for plating efficiency) exceeds the fraction in DNA synthesis at the time of virus exposure (less than 1%). Treatment of cells with bromodeoxyuridine (BrdU) followed by light before virus exposure does not impair the ability of EBV to stimulate DNA synthesis in human leukocytes or to transform marmoset leukocytes. However BrdU-light treatment is inhibitory to transformation if treatment is delivered about 24 hr after virus exposure or thereafter.     

Novel nuclear antigens recognized by human sera in lymphocytes latently infected by Epstein-Barr virus

We have used three latently infected cell lines, X50-7, JC-5, and Raji, to identify two new nuclear antigen complexes by Western immunoblotting with human anti-EBNA (Epstein-Barr Virus Nuclear Antigen) sera. One antigen complex, termed EBNA III, is composed of a group of high molecular weight proteins between 130 and 160 kDa and the other antigen complex, termed EBNA IV, is a size-related group of polypeptides between 28 and 62 kDa. Both the EBNA III and EBNA IV groups of proteins display variation in size among the different strains of EBV. Cell fractionation of X50-7, JC-5, Raji, and C16, a cell clone of P3HR1, showed that both new antigen complexes were completely recovered from the nuclei of latently infected lymphocytes as were previously described EBNA I and II. Because these new antigens are only detected by anti-EBNA sera in EBV infected cells, it seems likely that they may be encoded by the viral genome and play some role in the immortalization of lymphocytes by the virus.     

Early events in the infection of human B lymphocytes by Epstein-Barr virus: the internalization process

The early events in the infection of normal B lymphocytes and B lymphoblastoid cells by Epstein-Barr virus (EBV) were examined by electron and immunoelectron microscopy and by infectivity and inhibition studies. Purified EBV remained on the cell surface at 4 degrees and appeared as 250-nm ovoid particles in contact with the cell membrane through 50-nm envelope projections. Internalization of EBV in normal B lymphocytes into large (300-500 nm) uncoated vacuoles was initiated within 2 to 5 min at 37 degrees. At this stage approximately 1/3 of cell-associated virus was located in cellular invaginations while another 1/3 was in cell vacuoles. Direct fusion of EBV with the outer cell membrane was not observed. Instead, viral deenvelopment and nucleocapsid transit into the cytoplasm occurred from the large endocytic vesicles within 15 to 30 min at 37 degrees and did not involve lysosomal enzymes. During this time, the viral envelope became amorphous and its separation from the nucleocapsid was evident. After 60 to 90 min at 37 degrees, viral nucleocapsids were visualized in close proximity to the cell nucleus. Weak bases such as chloroquine, methylamine, and ammonium chloride retarded viral deenvelopment and fusion inside the endocytic vacuoles, resulting in abrogation of viral infectivity and accumulation of intact virions within cell vacuoles. These studies indicate that EBV enters normal B lymphocytes by a different endocytic pathway than the clathrin-receptosome-lysosome pathway utilized by many other ligands, including a number of viruses, to enter cells. In contrast to the pathway of entry into normal B lymphocytes, EBV entered B lymphoblastoid cells by direct fusion with the outer cell membrane within 2 to 5 min at 37 degrees.     

Early events in Epstein-Barr virus infection of human B lymphocytes.

The sequence of Epstein-Barr virus (EBV) and B lymphocyte changes in the 3 days following acute infection was analyzed. By 16 hr the average infected lymphocyte had 1 EBV episome. Nuclear protein-2 (EBNA-2) and EBNA-leader protein (-LP) were detected by 12 hr, and by 32 hr were at the levels of stable EBV infection in lymphoblastoid cell lines (LCLs). At 12 hr, all EBNA-LP and EBNA-2 RNAs were initiated from the Pw promoter. By 36 hr a significant EBNA-LP and EBNA-2 RNA fraction initiated from the upstream Pc promoter. Throughout acute infection, a similar fraction of potentially bicistronic EBNA-LP mRNAs had first exon splices which would result in EBNA-LP translation. By 36 hr c-myc RNA was transiently induced, and CD21 and CD23 RNAs were beginning to increase. This coincided with low-level EBNA-1, EBNA-3A, B, and C, and latent membrane protein-1 (LMP-1) expression. By 46 hr, EBNA-1, the EBNA-3s, and LMP-1 were near the levels ordinarily found in LCLs and a substantial fraction of lymphocytes were in S phase. These results are compatible with a key role for EBNA-2 (or EBNA-LP) in regulating virus and cell gene expression. High-level expression of the EBV-encoded small RNAs, EBERs, was delayed beyond 36 hr and may, therefore, be activated by other virus or cell genes. A 65-kDa virion protein persisted in acutely infected cells. This protein could be a mediator of virus or cell gene expression.     

Human antibodies to cedar pollen secreted by human peripheral lymphocytes transformed by Epstein-Barr virus]

To establish immortalized human B-cells secreting antibodies to cedar pollen allergens, peripheral blood lymphocytes from 13 donors were transformed with Epstein-Barr virus. Of 5000 micro culture wells with transformed cell growth, supernatants from 88 wells were found to contain antibodies to pollen allergens. Fourteen supernatants reacted with a cedar allergen Cry j1 and 10 reacted with Cry j2. IgM class antibodies were predominant.     

Immunization of human lymphocytes with Epstein-Barr virus capsid antigens in vitro

Research Center for Development and Introduction of Modern Methods of Molecular Diagnosis, Ministry of Health of the USSR, Moscow. (Presented by Academician of the academy of Medical Sciences of the USSR A. I. Borob'ev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 111, No. 1, pp. 62–64, January, 1991.     

Isoprinosine enhances the activation of sensitized lymphocytes by Epstein-Barr virus antigens

Isoprinosine, a synthetic purine derivative and a potentially useful immunostimulating agent, was tested for its ability to enhance lymphocyte responses to Epstein-Barr virus (EBV) antigens and to autologous EBV-transformed lymphocytes. Isoprinosine significantly enhanced the response of sensitized lymphocytes (i.e. from EBV-seropositive individuals) to EBV antigens, while it has no effect on the lack of response of unsensitized lymphocytes (i.e. from EBV-seronegative individuals) to these antigens. In addition, isoprinosine enhanced lymphocytes response to autologous EBV-transformed cells, and potentiated the generation of cytotoxic lymphocytes. From these observations, and since immunosuppression is commonly observed in EBV-associated malignancies and other EBV-induced disorders, it may be important to point out that the use of isoprinosine as an immunostimulating agent in patients with these diseases deserves serious consideration.     

Transfection of human lymphocytes with cloned Epstein-Barr virus (EBV) DNA

Human primary cord-blood lymphocytes were transfected, using the DEAE-dextran technique, with a set of seven largely overlapping clones jointly covering the whole M-ABA Epstein-Barr virus (EBV) genome. Three fragments, cosmids cMB-14 and cM301-99 and plasmid pM966-20, were able to stimulate transient cellular DNA synthesis, blastic transformation, and clumps formation, as well as to prolong the life span from a maximum of 2 weeks in control cultures to up to 6 weeks. The fragments stimulating DNA synthesis also expressed this property in mutual combinations or when combined with cosmid cMSal-A or cM302-21. Their use with any other fragments in cotransfection did not result in further DNA synthesis stimulation. Cosmids cM302-23 and cMSal-B suppressed this effect. Cosmid cM301-99 but not cM302-23 induced transient EBNA-1 formation in about 1% of lymphocytes. Lymphocytes transfected with single fragments or their combinations failed to grow into immortalized cell lines. The results suggest that transient expression of viral functions at levels achievable by transfection is not sufficient for cell immortalization.     

EB病毒对人胚鼻咽上皮细胞的转化

为观察EBV和／或促癌物四癸酸佛波醇二酯（TPA）对其的转化作用，以人胚鼻咽上皮作体外原代组织培养，采用自B95－8细胞分离的EB病毒直接感染或结合TPA处理体外培养的人胚鼻咽上皮细胞，着重观察感染细胞在半固体培养基中的集落形成率；并采用PCR扩增法探讨EB病毒是否直接进入鼻咽上皮细胞。结果显示：单独EB病毒或灭活（56℃，30分钟）EB病毒加TPA感染时，病毒不能进入细胞导致表型改变；活性EB病毒结合TPA同时处理或先用EB病毒后用TPA处理时，EB病毒能直接进入细胞并导致细胞集落形成率明显增高（P＜0．05）。从而表明EB病毒体外能部分转化人胚鼻咽上皮细胞，其转化作用依赖于TPA的存在和病毒基因组的完整。     

Inhibition of Plasmodium falciparum growth by IgG antibody produced by human lymphocytes transformed with Epstein-Barr virus.

Supernatants from Epstein-Barr virus (EBV)--stimulated B lymphocytes obtained from two adult Gambians who were partially immune to malaria markedly inhibited the growth of Plasmodium falciparum in vitro (55-95% inhibition). When 22 separate colonies were derived by micromanipulation from one of these primary cultures and their supernatants assayed, the degree of inhibition correlated with levels of IgG fluorescent antibody and total IgG. The inhibitory anti-P. falciparum IgG immunoprecipitated an antigen of mol. wt 195,000, identified as the major schizont surface glycoprotein by dual biosynthetic labelling with 3H-glucosamine or 35S-methionine. Other studies on the analogous schizont surface protein of rodent malarias have shown that this antigen stimulates protective immunity. Production of this inhibitory antibody by adult Gambians may therefore contribute to their immunity to malaria. Human antibodies produced by EBV-stimulated B lymphocytes may be used to identify other important P. falciparum antigens and have potential applications for immunotherapy.     

Induction of nuclear antigen (EBNA) synthesis without transformation in human cord lymphocytes by P3HR-1 strain of Epstein-Barr virus.

Various preparations of P3HR-1 strain of Epstein-Barr virus (EBV) were examined for transforming activity of human cord lymphocytes by a clonal transformation procedure. The B95-8 strain of EBV was used as a positive control. No transformation was evident in all the tests with P3HR-1 virus. However, formation of EBV-associated nuclear antigen (EBNA) without detectable induction of DNA synthesis was demonstrated.     

Inhibition of EB virus transformation of non-adherent human lymphocytes by co-cultivation with adult fibroblasts

Transformation of a special population of non-adherent human lymphocytes by EB virus (EBV) is reversibly inhibited by co-cultivation on adult human fibroblasts. Neither fluid from adult fibroblast cultures nor extracts of fibroblasts inhibited such transformation, and the growth of already transformed lymphocytes was not inhibited on adult fibroblasts. The EBV-associated nuclear antigen (EBNA) was detectable in inhibited cultures, with a maximum level of about 15% at 14 days after which it decreased gradually. Reversal of inhibition at 28 days, by either addition of phytohaemagglutinin or by removal of the lymphocytes from the adult fibroblasts, resulted in a prompt increase in the percentage of EBNA-positive cells and typical lymphoblastoid outgrowth. Transformation of EBV-infected non-adherent lymphocytes could be inhibited by the addition of adult fibroblasts up to 2–4 days after infection. The results indicate that, in the EBV-infection of non-adherent lymphocytes on adult fibroblasts, a block resulting in inhibition of transformation occurs between the production of EBNA and the onset of autonomous proliferation of the infected lymphocytes.Supported by a grant from the National Health and Medical Research Council, Canberra.     

Transformation of lymphocytes by Epstein-Barr virus requires only one-fourth of the viral genome

Inactivation studies were performed to measure the target sizes of three Epstein-Barr virus (EBV) -associated functions: (i) transformation of lymphocytes; (ii) stimulation of cell DNA synthesis in lymphocytes; and (iii) induction of the EBV nuclear antigen (EBNA) expression in lymphocytes. The target size of the plaque-forming function of herpes simplex virus type 1 (HSV-1) was studied in parallel and was used as a standard for comparison with the measured target sizes. When ionizing irradiation was used as an inactivating agent, the target size of EBV transforming function was found to be as large as that determined for the plaque-forming function of HSV-1. A similar size was measured for the EBV function required to stimulate cell DNA synthesis. In contrast, the target size of the viral function needed for induction of EBNA expression was found to be only 35% of that observed for HSV-1 plaque-forming function. Inactivation studies were also performed using the chemical mutagen, N-acetoxyacetylaminofluorene. The target size measured using this agent for the transforming function of EBV was about 25% of that determined for plaque-forming function of HSV-1. This result indicates that only a portion of the viral DNA encodes information necessary for the initiation and maintenance of transformation by EBV.