Recovery of transforming EBV from non-producer cells after superinfection with non-transforming P3HR-1 EBV.

Cells of the Raji and NC37 lines can be induced by chemical inducers, such as BrdUrd and IdUrd, or the tumor-promoter TPA to EA-expression only, but do not reveal any VCA synthesis. After superinfection by nontransforming P3HR-1 EBV, however, a varying percentage of the cell population shows VCA synthesis and releases infectious viral particles. The recovered virus differs biologically from P3HR-1 EBV since it transforms human umbilical cord blood lymphocytes into EBNA-positive lymphoblastoid cell lines. Cells of these established lines are susceptible to renewed infection by P3HR-1 EBV which results in EA induction and VCA synthesis. Only cells of one line, NC37-R1, spontaneously produce VCA and EBV particles, which reveal transforming properties and do not induce EA upon superinfection of Raji cells. Infection of P3HR-1 EBV-converted BJA-B cells also leads to EA and VCA induction and the release of viral particles. In contrast to particles recovered from Raji and NC37 cells, no transforming activity was detectable in these virus preparations. According to these data, we propose that viral genomes persisting within Raji and NC37 cells are defective and become complemented by the superinfecting P3HR-1 virus.     

The interaction of non-transforming Epstein-Barr virus (EBV) with cell-associated EBV DNA in superinfected lymphoblastoid cell lines

Two human lymphoblastoid cell lines were established by the transformation of human cord-blood lymphocytes with transforming Epstein-Barr virus (EBV). One cell line (HLB-R1) was established with EBV obtained after the superinfection of Raji cells with HR-1 EBV and the other (HLB-Bl) was established from B95-8 EBV-infected human cord-blood lymphocytes. Both the HLB-R1 and HLB-B1 lines were susceptible to superinfection with HR-1 EBV. We found that EBV DNA was replicated in the superinfected cell lines and that transforming EBV was produced in both the HLB-B1 and HLB-R1 cells. The average titer of transforming EBV obtained in the HR-1 EBV superinfected HLB-B1 and HLB-R1 cell lines was 10(4) transforming units (TU)/ml, whereas the average titers of transforming EBV obtained by the superinfection of Raji cells was 10(1) TU/ml. Epstein-Barr virus capable of inducing early antigen (EA) in superinfected Raji cells (lytic virus) was not detected in any transforming virus preparation. Restriction enzyme digestion patterns of virus DNA isolated from HR-1 and B95-8 cells, as well as from superinfected cells, were compared. The EBV DNA that was replicated in the superinfected HLB-R1 and HLB-B1 cell lines showed a more complex pattern. Our data suggest that recombination between input HR-1 EBV DNA and latent cell-associated EBV DNA occurs. Presumably this recombination results in a change in the biological properties of the newly synthesized virus.     

Persistence of transforming and nontransforming Epstein-Barr virus in high passages of P3HR-1 cell lines

At least three laboratories have reported that the P3HR-1 line, which had originally produced transforming Epstein-Barr virus (EBV), now produces only the nontransforming variant. Studies to determine whether these findings were universal or a consequence of specific cell lines or culture conditions were undertaken in P3HR-1 cultures of identical HLA types from five sources. All of the EBV preparations derived from cell lines cultured at 32, 34, and 35 degrees C transformed cord blood lymphocytes, whereas virus propagated at 37 degrees C did not usually transform. Furthermore, indirect immunofluorescence revealed that a monoclonal antibody directed against transforming EBV membrane glycoprotein bound to 10-12% of the P3HR-1 cells that had been continuously propagated at 34 degrees C, but the antibody did not bind to the same cells cultured at 37 degrees C. Although virus expression was completely repressed in transformed cord blood cells, transforming virus could be rescued by superinfection with nontransforming P3HR-1 EBV. Cells transformed with P3HR-1 virus induced poorly differentiated lymphomas in athymic nude mice after seven or eight passages. Whether all P3HR-1 cells have the potential to produce detectable quantities of transforming virus remains to be determined.     

The transforming prototype of Epstein-Barr virus (B95-8) is also a lytic virus

The B95-8 isolate of the Epstein-Barr virus (EBV) has been described as a non-lytic transforming virus. We have performed experiments in order to determine if the B95-8 EBV is capable of super-infecting and replicating in EBV-genome-positive non-producer lymphoblastoid cells. Using concentrates of B95-8 EBV, prepared from 6 different B95-8 cell lines treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), we demonstrated that virus concentrates could transform human or cotton-top tamarin B-lymphocytes and also lytically replicate in Raji cells, inducing EBV antigens and infectious virus. While the virus obtained from B95-8 super-infected Raji cells was able to transform cord-blood lymphocytes (CBLs) and super-infect Raji cells, transformation was abortive, with cell cultures only growing for up to 6 weeks. Transformation titers of the B95-8 virus concentrates ranged from 10(5) to greater than 10(8) transforming units/ml; early antigen (EA) induction ranged from 1% to 50% after superinfection of Raji cells, depending on the virus stock used, as determined by immunofluorescence. Southern blot analysis was carried out on the DNA prepared from B95-8 cells and virion DNA. The results were consistent with the published EcoRI restriction pattern for B95-8 EBV. The issue of whether the B95-8 cells produce virions with a dual biological phenotype or, rather, 2 biologically distinct viruses, is addressed.     

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.     

Induction of the EBV cycle in B-lymphocyte-derived lines is accompanied by increased natural killer (NK) sensitivity and the expression of EBV-related antigen(s) detected by the ADCC reaction

Human B-lymphocyte-derived lines were forced to enter the EBV-cycle by superinfection with the P3HR-l substrain of EBV or sodium butyrate treatment. The induced cells were used as targets for natural killing (NK) and EBV-specific, antibody-dependent cellular cytotoxicity (ADCC). Two Burkitt lymphoma lines, Raji and Daudi, and one normal adult derived lymphoblastoid cell line, NAD-7, were comparable in their ADCC-sensitivity after induction, but only the Burkitt lymphoma-derived lines showed a major increase in NK-sensitivity. The superinfection-induced membrane change, responsible for both NK and ADCC sensitivity, is an early function of the viral cycle, correlated with the appearance of early antigens (EA). Indirect evidence indicates that the NK and ADCC target sites are different but this problem requires further investigation. Sodium butyrate induced an increased NK sensitivity and EBV-related ADCC sensitivity in the Burkitt lymphoma-derived P3HR-l line. Lymphocyte effectors from different donors showed great differences in their NK and ADCC activity. Optimal ADCC could be demonstrated with effectors that were intermediate in their NK-activity.     

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.     

Expression of the Epstein-Barr virus genome in a nasopharyngeal carcinoma epithelial tumor cell line

An epithelial tumor cell line was recently established from a biopsy specimen of a nasopharyngeal carcinoma (NPC), and designated HONE-I. Uncloned (parental) HONE-I and HONE-I clone (C)-40 cells were found to contain latent Epstein-Barr virus (EBV). Expression of the latent EBV genome in HONE-I C-40 cells has been examined. It was possible to detect a small percentage of cells spontaneously synthesizing EBV early antigen (EA) and virus capsid antigen (VCA) by immunofluorescence (IF). In addition, the EBV nuclear antigens (EBNA-I and EBNA-2), as well as the EBV latent membrane protein (LMP) were detected in the HONE-I cells. Attempts were made to induce the latent EBV genome in these cells with iododeoxyuridine (IUdR). We observed a significant increase in the number of EA/VCA-positive cells, an increase in EBV DNA, the synthesis of virus particles, and the rescue of infectious virus after treatment of HONE-I C-40 cells with IUdR. The HONE-I C-40 cells should facilitate studies of the expression and regulation of the EBV genome in NPC epithelial tumor cells, which have not previously been available.     

Superinfection of epithelial hybrid cells (D98/HR-1, NPC-KT, and A2L/AH) with Epstein-Barr virus and the relationship to the C3d receptor

Three Epstein-Barr virus (EBV) genome-positive epithelial/hybrid cell lines (D98/HR-1, NPC-KT, and A2L/AH) were superinfected with EBV derived from P3HR-1 (HR-1), NPC-KT, and B95-8 cells. The HR-1 virus is lytic and induces early antigen in superinfected Raji cells; the virus is not capable of transforming B-lymphocytes. Virus preparations from NPC-KT cells have both transforming and early antigen-inducing properties, while B95-8 virus can only transform B-lymphocytes. It was possible to demonstrate EBV antigens after superinfection of D98/HR-1 cells with both HR-1 virus and NPC-EBV. The NPC-KT hybrid cells, which were originally prepared by fusing human adenoid epithelial cells (Ad-AH) and EBV genome-positive NPC explanted epithelial cells, were susceptible to superinfection with HR-1 virus but not to NPC-EBV. The A2L/AH hybrid cells, which were prepared by fusion between Ad-AH cells and lymphocytes transformed by B95-8 virus, could not be superinfected with any of the virus preparations. In order to further investigate the nature of the EBV receptor as it relates to other cell membrane components, we examined cell surface markers on Ad-AH, NPC-KT, A2L/AH, and D98/HR-1 cells using monoclonal antibodies and by rosette formation. We found that the NPC-KT, A2L/AH, and Ad-AH cell lines express the OKB2 antigen and that the common acute lymphoblastic leukemia antigen is expressed on the A2L/AH cells. We also found that NPC-KT parental cells and a clone of NPC-KT cells express erythrocyte antibody complement b and erythrocyte antibody complement d, as determined by rosette formation, but were negative for C3b and C3d when monoclonal antibodies against these two markers were used. The D98/HR-1 cells were also confirmed to be negative for C3b and C3d. The data suggest that the C3d receptor may be part of the EBV receptor but that the C3d receptor, by itself, is not the only receptor to which EBV can bind.     

Somatic cell hybrids between human lymphoma lines. IV. Establishment and characterization of a P3HR-1/Daudi hybrid.

A new human B-lymphoma hybrid line designated DIP-1 was derived from the fusion of the Burkitt lines Daudi and P3HR-1. Cytogenetic analyses proved hybridity and showed that the hybrid was nearly complete from the chromosomal point of view. Hybridity was also confirmed by isozyme marker tests. The Daudi-derived IgM-kappa ring was fully exposed on DIP-1, together with the P3HR-1-derived human beta-2-microglobulin. Expression of C3 receptors was intermediate between the two parents and EBV receptors were some what reduced in comparison with both. The spontaneous low-level EBV antigen (EA and VCA) production of the parents was maintained and amplified in the hybrid. The hybrid, like its Daudi parent, was highly inducible with IdUrd. In P3HR-1 virus super-infection experiments, the Daudi parent was more permissive than the BU-P3HR-1 parent. The hybrid was intermediate, resembling the previously studied Raji/Daudi hybrid and contrasting with Raji/Namalwa and Raji/BJAB hybrids. Virus DNA replication patterns after P3HR-1 virus superinfection resembled the antigen pattern. The implication of these findings for the understanding of virus-host cell relationships and the regulation of the viral cycle is discussed The findings are meant to imply that genetically determined isozyme markers are autonomously expressed, as in other systems. Differentiation-related markers and EBV-cycle-related characteristics are autonomously expressed in some cases (surface immunoglobulin, beta-2-microglobulin, spontaneous EBV-production, IdUrd inducibility) and appear to be under a restrictive control in others (EA inducibility by P3HR-1 virus superinfection, C3 and EBV receptors, viral DNA replication).     

Induction of lytic Epstein-Barr virus (EBV) infection in EBV-associated malignancies using adenovirus vectors in vitro and in vivo

The consistent presence of EBV genomes in certain tumor types (in particular, AIDS-related central nervous system lymphomas and nasopharyngeal carcinomas) may allow novel, EBV-based targeting strategies. Tumors contain the latent (transforming) form of EBV infection. However, expression of either of the EBV immediate-early proteins, BZLF1 and BRLF1, is sufficient to induce lytic EBV infection, resulting in death of the host cell. We have constructed replication-deficient adenovirus vectors expressing the BZLF1 or BRLF1 immediate-early genes and examined their utility for killing latently infected lymphoma cells in vitro and in vivo. We show that both the BZLF1 and BRLF1 vectors efficiently induce lytic EBV infection in Jijoye cells (an EBV-positive Burkitt lymphoma cell line). Furthermore, lytic EBV infection converts the antiviral drug, ganciclovir (GCV), into a toxic (phosphorylated) form, which inhibits cellular as well as viral DNA polymerase. When Jijoye cells are infected with the BZLF1 or BRLF1 adenovirus vectors in the presence of GCV, viral reactivation is induced, but virus replication is inhibited (thus preventing the release of infectious EBV particles); yet cells are still efficiently killed. Finally, we demonstrate that the BZLF1 and BRLF1 adenovirus vectors induce lytic EBV infection when they are directly inoculated into Jijoye cell tumors grown in severe combined immunodeficiency mice. These results suggest that induction of lytic EBV infection in tumors, in combination with GCV, may be an effective strategy for treating EBV-associated malignancies.     

Epstein-Barr virus strain-specific differences in transformed cell lines demonstrated in growth characteristics, induction of viral antigens and ADCC susceptibility

Paired lymphoid cell lines were established by transformation with both B95-8 (B) and QIMR-WIL (Q) EBV strains, of cells either from cord blood or from peripheral blood or spleens of patients with leukemias or lymphomas. The morphologies of the transformed cell clumps differed consistently between B-transformed lines (B-lines) and Q-transformed lines (Q-lines) even after 1 year in culture. When the B- and Q-lines were compared for superinfection with P3HR-1 EBV, B-lines had a higher frequency of EA induction than did the Q-lines. The shape of dose-response curves for superinfection indicated that a much higher multiplicity of infection by P3HR-1 EBV was required for EA expression in Q-lines than in B-lines. This difference in superinfection was independent of the EBV receptor concentration on the two types of lines and reflected apparent control of the level of EA induction by the resident EBV genome of the transformed line. Transforming EBV could be rescued by P3HR-1 EBV superinfection of both B- and Q-lines originating from cells of patients with Hodgkin's disease and hairy cell leukemia but not from cord blood. The cord blood lines transformed with virus produced spontaneously from these B- or Q-lines, showed that the cell lines contained antigen-determining information of the resident genome in the original B- or Q-lines, respectively. Superinfected B-lines were more susceptible to ADCC with anti-EBV-positive sera than were superinfected Q-lines. These experiments demonstrate that distinct biology. differences exist in paired cell lines transformed by different EBV strains.     

Heterogeneity of Epstein-Barr virus derived from P3HR-1 cells.

Infection of cells of the EBV-free human B-lymphoma lines BJAB and Ramos resulted in conversion of these cells to EBV-genome carriers expressing EBNA. EBV isolates from P3HR-1 cells induced a heterogeneous EBNA pattern: both a faintly granular pattern and brilliant EBNA-expression were observed. The two types of EBNA-expressing cells could be separated upon cloning. Brilliantly EBNA-expressing cells always segregated varying percentages of EBNA-negative cells. An EBNA-negative subclone derived from these cells was devoid of detectable EBV DNA. Nucleic acid hybridization experiments failed to reveal a correlation between the intensity of EBNA expression and the number of EBV genome equivalents per cell. EBV genome-containing cells had an average of 14-fold more cells showing EA synthesis after superinfection by P3HR-1 virus, when compared with EBNA-negative cells infected under identical conditions. Studies on the kinetics of EA induction in EBNA-positive and EBNA-negative cells indicate that complementation is required for the induction of EA after superinfection.     

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.     

Induction of epstein-barr virus by a new tumor promoter,teleocidin, compared to induction by TPA

The effect of teleocidin, a new, naturally occurring tumor promoter, on induction of Epstein-Barr virus (EBV), was compared with that of a known tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Early antigen (EA) and/or capsid antigen (VCA) of EBV was induced in the EBV genome-carrying cell lines C-6 and P3HR-I cells by teleocidin, its effect being maximal at a concentration of 12.5 ng/ml. The production of infectious EBV from P3HR-I cells was enhanced by teleocidin maximally at a concentration of 0.5 to 2.5 ng/ml. The outgrowth of EBV-transformed cells from peripheral lymphocytes of seropositive healthy donors was also enhanced by teleocidin at a concentration of 0.02 to 0.5 ng/ml. TPA tested simultaneously in all experiments exhibited the same activities as teleocidin, and was effective at similar concentrations. Teleocidin enhanced both EA and VCA synthesis in P3HR-I cells additively with n-butyrate, but not with TPA. This suggests that teleocidin and TPA have a common mechanism of action, although their chemical structures are different.     

Comparative studies of Epstein-Barr virus strains from Ghana and the United States.

A high incidence of oropharyngeal excretion of Epstein-Barr virus (EBV) has been observed in African children with Burkitt's lymphoma (BL) (48%) and matched controls (45%). This compares with an incidence of 77% in American patients with infectious mononucleosis (IM) and 13% in age-matched controls. Cross-neutralization tests between EBV strains derived from BL and IM patients and their sera failed to detect differences in the major neutralizing antigenic components. Cord-blood lymphocytes transformed by American EBV expressed only early viral functions (EBV nuclear and soluble complement-fixing antigens) and produced no detectable transforming activity. By contrast, cord-blood lymphocytes transformed by African EBV strains contained 0.2-0.3% of cells with EBV capsid and early antigen and produced EBV with transforming activity. These cells contained twice as many copies of EBV homologous DNA as the cells transformed by American EBV strains.     

Epstein-Barr virus and Herpesvirus saimiri: sensitivity to interferons and interferon inducers

The in vitro sensitivity of oncogenic herpesviruses, Epstein-Barr virus (EBV), and Herpesvirus saimiri (HVS) to human interferon produced by normal human leukocytes (Le), lymphoblastoid cell lines (LYI), and diploid fibroblasts (Fi) was studied. Four virus strains were used: HVS S295C, the highly oncogenic HVS S396-O, the transforming B95-8 strain of EBV, and the nontransforming P3HR1 strain of EBV. All interferons were active when applied to the cells after absorption of HVS and P3HR1-EBV, although different amounts were required to achieve 50% inhibition of HVS-induced cytopathic effect or EBV-induced early antigen (EA) expression. Transformation of human umbilical cord blood lymphocytes (HCBL) by the B95-8 strain of EBV was prevented only by Le and LYI. In these experiments, the most effective inhibitor of the oncogenic herpesviruses was Le, and the least effective was Fi. The effect of polynucleotides poly(I).poly(C) and the complex of poly(I).poly(C) with poly-L-lysine and carboxymethylcellulose on HVS and EBV was also studied. Their inhibitory action was proportionate to the ability of herpesvirus-infected cells to produce interferon. Thus owl monkey kidney cells, which produce relatively high levels of interferon, required nanogram quantities of polynucleotides to become resistant to HVS. Transformation of HCBL by B95-8-EBV was also prevented by poly(I).poly(C). In Raji cells superinfected with P3HR1-EBV, polynucleotides failed to stimulate interferon, and higher EBV-induced EA expression was observed. The percentage of P3HR1 and Raji cells spontaneously expressing EBV-associated antigens remained unchanged after exposure to either interferon or polynucleotides.     

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.     

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.     

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.