Abortive expression of the Epstein-Barr virus (EBV) cycle in a variety of EBV DNA-containing cell lines, as reflected by nucleic acid hybridization in situ.

A variety of Epstein-Barr virus (EBV) DNA-containing cell lines have been tested for the expression of the EBV-associated antigens EBNA (nuclear antigen), EA (early antigen), and VCA (viral capsid antigen), and for the presence of cells containing disproportionate amounts of EBV DNA. The antigen tests utilized immunofluorescence and 125I-labelled antibodies combined with autoradiography. EBV-DNA was detected by in situ hybridization with 3H-labelled EBV RNA complementary to P3HR-1 EBV DNA (P-EBVcRNA). The P-EBVcRNA has been shown to represent the majority of the P3HR-1 EBV DNA sequences. It was concluded that EBV DNA-containing cell lines can be divided into those that express only EBNA, those that express EBNA and EA and those that express EBNA, EA and VCA and also contain cells that undergo disproportionate EBV DNA synthesis. Consequently, in some cell lines there is an abortive expression of the EBV cycle in that some cells spontaneously express EA but fail to continue further to viral DNA synthesis. A similar pattern can be found after experimental induction of the EBV cycle, suggesting that related mechanisms govern the spontaneous expression of the EBV cycle and the extent of its inducibility.     

Somatic cell hybrids between human lymphoma cell lines. V. IdUrd inducibility and P3HR-1 superinfectability of Daudi/HeLa (DAD) and Daudi/P3HR-1 (DIP-1) cell lines.

We have studied two types of somatic cell hybrid with regard to expression of the Epstein-Barr virus (EBV) cycle and its regulation. The first, DIP-1, a hybrid formed between two human lymphoma EBV producers (Daudi and P3HR-1), contained EBV DNA, expressed the virus-determined nuclear antigen (EBNA), andwas a producer of the EBV-associated antigens EA (early antigen) and VCA (viral capsid antigen). The second, DAD, a hybrid series of clones formed between Daudi and a HeLa cell derivative (D98), differed with regard to the expression of EBNA, EA, VCA and the content of EBV DNA. EA was regularly induced in the EBV DNA-containing hybrids following treatment with iododeoxyuridine (IdUrd). This induction was greater in lines spontaneously expressing EA. In two hybrids, DIP-1 and DAD10, VCA and virus DNA synthesis were also induced in the presence of IdUrd, the latter being detected by in situ hybridization with P3HR-1 EBV complementary RNA. Finally, while DIP-1 was superinfectable by the P3HR-1 EBV strain, the DAD series of hybrids were refractory to P3HR-1 superinfection and lacked EBV receptors.     

Effect of retinoic acid (RA) on the Epstein-Barr virus (EBV)-inducing effect of sodium butyrate

Sodium butyrate is a powerful inducer of the Epstein-Barr virus (EBV) cycle in the P3HR-1 cell line. Retinoic acid (RA) was found to reduce the butyrate induction of early antigen (EA) and viral capsid antigen (VCA) by 26-41%. This is similar to the previously reported effect of RA on IUDR and TPA induction, with certain quantitative differences between the systems.     

Ultraviolet inactivation of Epstein-Barr virus: effect on synthesis of virus-associated antigens.

The relative sensitivity to ultraviolet (UV) light of genome functions of the P3HR-1 strain of Epstein-Barr virus (EBV) was studied. The formation of viral capsid antigen (VCA) appeared to be more sensitive than that of early antigen (EA), while the synthesis of membrane antigen (MA) was most resistant, as seen on examination in the presence of cytosine arabinoside (Ara-C). However, the appearance of both VCA and EA, but not that of MA, was delayed with UV-irradiated virus, in either the presence or absence of Ara-C. The synthesis of EA and VCA induced by UV-irradiated virus was suppressed in the presence of Ara-C, while that of MA was not.     

Segregation of the EBV-determined nuclear antigen (EBNA) in somatic cell hybrids derived from the fusion of a mouse fibroblast and a human Burkitt lymphoma line

Four independently derived hybrids between the mouse fibroblast line A9 and the human, Burkitt-lymphoma-derived lymphoblastoid cell line Daudi were studied for the presence of the Epstein-Barr virus (EBV) genome, the EBV-determined nuclear antigen (EBNA), other EBV-associated antigens, human surface immunoglobulin and the presence of human chromosomes. The four lines differed in the number of their EBV genomes. There was a parallelism between this number, as detected by c/RNA/DNA hybridization, and the frequency of EBNA-positive nuclei. None of the other EBV-antigens, EA, VCA or MA, was expressed at any time, either in the untreated hybrid cells or after IUDR-treatment. The hybrids did not carry detectable surface-associated immunoglobulin or EBV-receptors. The presence of the EBV genome was coincident with the maintenance of human chromosomes, but the hybrids that have lost detectable viral genomes and EBNA still contained a considerable number of human chromosomes, suggesting that the viral genome may be associated with a few chromosomes only.     

Effect of local radiotherapy on the antibody levels against EBV-induced early and capsid antigens (EA and VCA) in patients with certain malignant tumours

Antibodies to tumour-related and EBV-associated intracellular antigens were studied in 14 cases of Burkitt's lymphoma, 50 of nasopharyngeal carcinoma, 10 of maxillary carcinoma and 95 cases of other tumours, all of whom received radiotherapy in Nairobi. For up to 1 month after radiotherapy the cases of Burkitt's lymphoma recorded an increase in antibodies to early antigens (EA) amounting to, on average, about one titre step (p<0.001). During the same interval there was a small but significant increase in antibodies to viral capsid antigen (VCA) (mean 0.6 titre step; p<0.001). The patients with nasopharyngeal and maxillary carcinoma, too, showed a small but statistically significant increase in antibodies to VCA 2-6 months after radiotherapy (p<0.01 and 0.001, respectively), but no increase in antibodies to EA. On the contrary, in nasopharyngeal carcinoma patients there was a significant decrease in antibodies to EA 2-3 months after radiotherapy. In the group of other tumours there was no evidence of a change in antibodies to the intracellular antigens studied during any of the relevant intervals.     

Growth and antigenic properties of a biopsy-derived Burkitt's lymphoma in thymus-less (nude) mice

A Burkitt's lymphoma was transferred to the congenitally athymic mouse mutant nude with biopsy material from a 7-year-old Kenyan girl. The tumor grows locally at the site of inoculation with no distant metastases. The established tumor has been maintained for six passages so far with preservation of histological and cytological appearance. The mouse-passaged tumor has a normal human diploid female chromosome complement. Isozyme studies have shown tumor to be of the same glucose-6-phosphatedehydrogenase (G-6-PD) and phosphoglucomutase₁ phenotype (B and 2-1 respectively) as tumor biopsy from the patient. The mouse-passaged line maintained surface IgM, similarly to the original tumor and the derived tissue culture line, but lost the IgG coating characteristic for the original tumor but absent from two subsequent biopsies and from the derived tissue culture line. This is in line with previous observations indicating that surface-associated IgG on Burkitt biopsies is due to coating from the outside, whereas surface-associated IgM is a cell marker. Whereas the biopsy cells of the patient were positive for EBV-associated membrane antigen (MA), but not for early antigen (EA) and viral capsid antigen (VCA), the mouse-passaged line was positive for all three. This suggests that the restrictive influence of the human host on the production of EA and VCA in the Burkitt tumor is raised in the mouse host. The serum of the tumor-bearing nude mice contained anti-human antibodies, but no detectable EBV (anti-MA, EA and VCA)-antibodies.     

Combined effect of the extracts from Croton tiglium,Euphorbia lathyris or Euphorbia tirucalli and n-butyrate on Epstein-Barr virus expression in human lymphoblastoid P3HR-1 and Raji cells

The combined usage of n-butyrate and 12-O-tetradecanoylphorbol-13-acetate (TPA) or the oily extracts from Croton tiglium, Euphorbia lathyris or Euphorbia tirucalli exerted a marked effect on induction of Epstein-Barr virus (EBV)-associated early (EA) and viral capsid (VCA) antigens in EBV genome-carrying human lymphoblastoid cell lines. In producer P3HR-1 cells, the enhancing effect of the 2 components was additive both for EA and VCA, while in non-producer Raji cells, a synergistic increase of EA was observed. The possible implication of these findings relating to the cause of EBV-associated diseases is discussed.     

Early events in transformation of human cord leukobytes by Epstain-Barr virus: induction of DNA synthesis, mitosis and the virus-associated nuclear antigen synthesis

The present investigations were undertaken to establish the early events in EBV-induced transformation of human cord lymphocytes with particular reference to the induction of DNA synthesis, mitosis and viral synthesis. When cord leukocytes were exposed to a 3 h pulse of²H-TdR at 3 h intervals following EBV infection and examined by autoradiography, blastoid cells with labelled nuclei appeared 12-24 h after virus exposure, reaching a maximum of 7.5% in 15 h. When the infected leukocytes were reincubated after each pulse for an additional 20-40 h with colchicine added 10 h prior to termination of incubation, 18% of the positive cells were in mitosis. The cells were examined by immunofluorescence for the synthesis of EBV-associated antigens. Early antigen (EA)—and viral capsid antigen (VCA)—were not detected, but EBV-associated nuclear antigen (EBNA) synthesis became evident as early as 2 days after infection. The frequency of EBNA-positive cells, mostly blastoid cells, increased with continued incubation, and at 10 days the majority of the cells were positive. These findings strongly suggest that the human cord leukocytes are non-permissive for replication of EBV, and that the virus can induce in them early DNA synthesis followed by high-frequency mitosis and cell proliferation, in close association of the viral genome with the host-cell genome as shown by the synthesis of EBV-specific “footprint”.     

Comparative study of anti EBV antibodies in non-Hodgkin's lymphomams and angio-immunoblastic lymphadenopathies (author's transl)]

Antibody titers to Epstein-Barr virus (EBV)--related antigens (viral capsid antigen : VCA; early antigen : EA; and EBV associated nuclear antigen : EBNA) were determined in the sera of 86 patients and 150 matched control subjects. The patients belonged to four histological groups : diffuse and nodular non-hodgkin's lymphomas angio-immunoblastic lymphadenopathies and apparented syndroms. The incidence of antibodies to other herpes-viruses (cytomégalovirus, herpes simplex virus, and varicella zoster virus) was compared. There was a significantly higher incidence of anti VCA and anti EA titers in some patients, not associated with an increase in titres of antibodies to other herpes viruses.     

Antibody reactions to herpesvirus saimiri (HVS)-induced early and late antigens (EA and LA) in HVS-infected squirrel, marmoset and owl monkeys

Vero cells were infected with Herpesvirus saimiri. Early (EA) and late (LA) viral antigens were distinguished by inhibiting viral DNA synthesis with cytosine arabinoside (Ara C), or allowing it to proceed uninhibited until CPE was visible. The difference in the antigenic specificity of EA and LA was confirmed by direct fluorescence tests, in combination with blocking experiments. Sera that were anti-EA positive according to the indirect test were capable of blocking the EA staining reaction with direct EA+- conjugates, whereas anti-LA+EA— sera were unable to do so. Healthy adult squirrel monkeys had antibodies to late antigens (LA) but not to early antigens (EA), as judged by immunofluorescence tests. Isolation-reared, antibody-free squirrel monkeys responded with anti-EA and anti-LA antibodies after HVS inoculation. Uninoculated, cage-mate squirrel monkeys became infected by horizontal transmission and responded with LA and EA antibodies approximately 2 months after being housed together with the infected animals. Simultaneously isolated controls remained antibody-free. HVS-inoculated, tumor-bearing marmoset and owl monkeys developed antibodies to both LA and EA in the majority of the cases studied, although in some owl monkeys there was only LA and no EA antibody development. Two different patterns of EA staining could be distinguished, trabecular and punctate. There was a conspicuous difference between the species with regard to the timing of antibody development. Squirrel monkeys, the natural host species of the virus, but resistant to its oncogenic action, responded with antibody development within 14–17 days after inoculation. Marmoset and owl monkeys did not respond until 28–80 days. Conceivably, the natural host species has been selected for a high degree of genetic responsiveness against virus-determined antigens. This may be partly or entirely responsible for its resistance to the oncogenic effect of this virus.     

Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines

Anti-complement immunofluorescence (ACIF) was used to study the complementfixing antigens of human lymphoblastoid cell lines. These cell lines carry the Epstein-Barr virus (EBV) genome although only producer cultures synthetize EBV-specific antigens (virus capsid antigen, VCA and early antigen, EA) detectable by direct and indirect immunofluorescence, usually in less than 5% of the cells. The ACIF test revealed an antigen localized in the nucleus of the lymphoblastoid cells. In contrast to EA and VCA, this antigen was present in over 90% of the cells of both producer and non-producer cultures. The antigen was shown to be specific for EBV by comparing the reactions of 52 sera in the ACIF test. Sera giving the nuclear reaction contained antibodies to VCA, EA or antigens detectable by complement fixation tests on cell extracts, but sera without EBV antibodies failed to give the reaction. Weak, equivocal or discordant reactions occurred with six sera with low titres in VCA, EA or complement fixation tests. Cell lines derived by transformation of human and primate lymphocytes by EBV gave the nuclear reaction. Control cells with no known association with EBV were non-reactive. These included foetal lymphocytes transformed by phytohaemagglutinin, cell lines derived from breast cancer, glioma, normal glia, pleuritis maligna and myeloma, and two marmoset lymphoid lines carrying Herpesvirus saimiri (HVS). In preliminary experiments, the ACIF test was used as a tool to trace the EBV genome at the cellular level. Cells from two Burkitt lymphoma biopsies, one tested after biopsy and one after passaging in nude mice, contained an EBV-specific antigen. Three clones of cells derived from hybrids of mouse somatic cells and a human lymphoblastoid cell line also contained such an antigen, but the number of reactive cells varied from clone to clone. A fourth clone was non-reactive.     

Differential inhibition of epstein-barr virus induction by the amino acid analogue,L-canavanine

The effect of an amino acid analogue, L-canavanine, on the synthesis of Epstein-Barr virus (EBV) antigens was investigated in lymphoblastoid cells. The analysis revealed that after infection of BJAB and NC-37 cells with P3HR-I EBV synthesis of early antigen (EA) was not affected by canavanine in concentrations up to 8.4 mM. The synthesis of EBV-determined nuclear antigen (EBNA) and of viral capsid antigen (VCA) was significantly inhibited at concentrations higher than 2.8 mM. Spontaneous induction of EA in P3HR-I cells was not affected by canavanine. On the other hand, EA induction by the tumor promoter TPA, by iododeoxyuridine (IdUrd), by antiserum to human IgM and by n-butyric acid was clearly inhibited by this treatment. Application of 0.3 mM canavanine resulted in more than 95% inhibition of EA induction by TPA. Under these conditions cell growth and incorporation of radiolabelled amino acids into an acid-insoluble fraction was significantly impaired. Differential treatment of the cells with canavanine established that EA induction was completely suppressed when the cells were treated concomitantly with canavanine and TPA. Subsequent treatment with canavanine after prior exposure to TPA resulted in some viral antigen induction depending on the time period of TPA exposure. Pretreatment of the cells overnight with canavanine followed by washing and addition of the tumor promoter did not suppress EA induction by TPA. These data support the concept that EA induction by superinfection follows a different pathway from antigen induction by chemical inducers.     

Differential induction of Epstein-Barr virus-related antigens in heterokaryon cultures.

Different paterns of induction of Epstein-Barr virus (EBV)-related antigens were observed in heterokaryons produced by Sendai virus-mediated fusion of producer and non-producer human lymphoblastoid cells with various other cell types. EBV-related early antigens (EA) and viral capsid antigen (VCA) could obviously be induced in heterokaryons between producer cells (P3HR-1 and QIMR-WIL), normally expressing these natigens at very low frequency, and human FL or HeLa cells. Positive cells were detected as early as 3 h after fusion and there often followed a rapid increase in positive cells. In contrast, in heterokaryons between non-producer cells (Raji and NC-37) and FL or HeLa cells, only EA but not VCA was induced. EA induction was also evident in fusion of human lymphoblastoid cells with monkey cells (Vero) but with mouse cells (L-M(TK-) C11D and MCB-2) no EBV induction occurred. The EBV induction in heterokaryons was significantly enhanced by 5-iododeoxyuridine treatment.     

Immunofluorescence and anti-complement immunofluorescence absorption tests for quantitation of Epstein-Barr virus-associated antigens.

Immunofluorescence absorption methods are described which permit quantitative estimation and differentiation of Epstein-Barr virus (EBV)-associated antigens (virus capsid antigen, VCA, early antigen, EA and EBV-determined nuclear antigen, EBNA) in cell extracts. EBNA was present in all cell lines (producer and non-producer) which carried the EBV-genome, while VCA and EA were present in producer lines only. All the antigens were absent from a lymphoid cell line (MOLT-4) which lacked the EBV-genome, as well as from leukemia cells from peripheral blood. The techniques demonstrated antigenic identity of the various antigens when prepared from different cell lines.     

Epstein-Barr virus antibodies in tonsillar carcinoma patients;.

Sera from 18 tonsillar carcinoma patients and from 18 matched control subjects were examined for the presence of antibodies to viral capsid antigen (VCA), early antigen (EA) and nuclear antigen (EBNA) of EB virus. Antibodies to all three antigens were found more frequently and in significantly higher titres in the tonsillar carcinoma patients than in control subjects.     

Sensitivity of Epstein-Barr virus (EBV) producer and non-producer human lymphoblastoid cell lines to superinfection with EB-virus

Twenty-nine lymphoblastoid lines and one IgE-producing myeloma line of human origin were exposed to Epstein-Barr virus (EBV) concentrates in vitro. The adsorption of the virus to the outer cell membrane was assessed by counting the number of direct membrane fluorescence-positive cells immediately after infection and by the direct radioimmune membrane labelling method. Reference reagents were derived for both tests from the same serum (“Agnes”), containing antibodies against EB-viral envelope and capsid antigens. The intracellular course of infection was followed by counting the number of cells that responded with the development of early antigen (EA) 48 h after infection. The 11 lymphoblastoid lines that produced no EBV-determined membrane and early antigens adsorbed the virus, although there were quantitative differences between them. EA-positive cells appeared in significant numbers in only seven of them, however. Four lines remained EA negative in spite of a relatively good adsorbing capacity. The IgE-producing myeloma line showed neither virus adsorption nor EA development. Eighteen lymphoblastoid lines were “producers”, or “abortive producers”, i.e.a small proportion of the cells continuously generated two or three of the immunofluorescence-detectable viral antigens, MA, EA and VCA. Nine lines failed to adsorb significant virus quantities and showed no certain increase of EA-positive cells. The resistance of these lines to superinfection is probably determined at the level of viral receptors. Five lines showed a relatively good virus adsorption, but this was not followed by any significant increase in the number of EA-positive cells. Four lines showed good adsorption and also responded with a significant increase in the number of EA-positive cells. The same responses can thus be found to EBV-superinfection in producer and non-producer lines, but the producer lines show a strong preponderance of superinfection-resistant lines with an adsorption block at the receptor level.     

Hybridization between a human epithelial line, infectable by Epstein-Barr virus, and Burkitt lymphoma lines: membrane properties, superinfectability, inducibility and tumorigenicity

The human epithelial line U, which is partially infectable with EBV, was hybridized with the EBV-genome carrying Burkitt lymphoma lines P3HR-1 and Daudi. Authenticity of the hybrids U-Put and U-Dut was established by isoenzyme studies. Although the two hybrids carried the EBV genome derived from the lymphoma parent, being 100% positive for Epstein-Barr-virus-associated nuclear antigen (EBNA), they resembled the U parent in many respects: they were deficient for membrane immunoglobulins and Fc receptors, and had a lower concentration of EBV-C3 receptors than either parent. Unlike the P3HR-1 parent, U-Put hybrid was nonpermissive for both the EBV cycle antigens, early antigen (EA) and viral capsid antigen (VCA). The inducing agent 12-O-tetra-decanoyl-phorbol-13-acetate (TPA) caused distinct viral early antigen synthesis (EA) in U-Put, lower, however, than that of the parental P3HR-1. U-Dut was completely nonpermissive and noninducible for early and viral capsid antigens. Thus, even an epithelial parent infectable by EBV restricted, although not completely, expression of EBV antigens, with the exception of EBNA. It has been suggested that EBNA is an autonomous function of the viral genome, independent of host cell control; the latter regulates expression of antigens related to viral cycle. The hybrids U-Put and U-Dut resembled the U parent also in regard to growth in soft agar and tumorigenicity in nude mice, although in this respect the lymphoma parental properties were not completely eclipsed.     

Re-exposure of human lymphoblastoid cell lines to Epstein-Barr virus

Human lymphoblastoid lines of various origins which harbour Epstein-Barr virus (EBV)-specific nucleic acid were re-exposed to EBV. Following infection, cells of the non-virus-producing lines, Raji and S 95, predominantly synthesized EBV-specific early antigens (EA), whereas only a small percentage of cells revealed viral capsid antigens (VCA). In Raji cells, the number of VCA-producing cells was paralleled by the percentage of virus-specific DNA-synthesizing cells. In S 95 cells, however, viral DNA-synthesizing cells exceeded the number of VCA-producing cells by a factor of more than 10. Induction of EA in Raji cells was dose-dependent and inversely related to cell growth. Irradiation of the virus by ultraviolet light prior to infection led to reduced infectivity. This reduction seemed to follow single-hit kinetics. Raji cells, previously re-exposed to EBV, showed reduced EA induction after re-infection with EBV, as compared to Raji control cells not previously exposed. Of 10 lines which spontaneously synthesize EBV-specific antigens, seven lines proved to be refractory to re-infection, whereas three were as susceptible as the Raji and S 95 controls. From three of the refractory lines infectious virus could be recovered from the culture medium prior to infection. These results permit the following interpretations: (1) the response of human lymphoblastoid cells after re-infection with EBV results from the infecting virus and not from stimulation of endogenous genomes; (2) cells demonstrating EA synthesis ultimately die; (3) re-exposure to EBV increases the resistance to re-infection of the surviving cells; and (4) cell lines producing infectious EBV are refractory to re-infection. It is suggested that the spontaneous synthesis of infectious virus favours the selection of resistant cells.     

Epstein-Barr virus-induced malignant lymphoma in a white-lipped marmoset

One of six white-lipped marmosets inoculated with cell-free B95-8 virus developed diffuse malignant lymphoma. Epstein-Barr virus (EBV) DNA was detected in a pathologically enlarged mandibular lymph node by DNA-DNA hybridization. The affected animal at the time of killing had EBV antibody titers of 1:320 for viral capsid antigen (VCA) and 1:20 for early antigen (EA) while all non-diseased animals had less than or equal to 1:80 VCA antibody and no detectable EA antibody. This is the first report of lymphoma development in a white-lipped marmoset following EBV inoculation.