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.     

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.     

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.     

In vitro cellular immunity to Epstein-Barr virus in normal human subjects

Leukocyte migration inhibition was employed to study cell-mediated immunity (CMI) to Epstein-Barr virus (EBV) in 29 normal subjects. Anti-EBV capsid antibody titres in these subjects ranged from 0 to 1:160. Extracts of seven human lymphoblastoid cell lines carrying EBV were evaluated as the source of test antigen. Of these cell lines studied, only P₃HR-1 proved suitable as a source of EBV antigen for use in the test. Undiluted tissue-culture supernatant fluids of the EBV-carrying cell lines P₃HR-1 and SH-T₁ caused only weak or equivocal migration inhibition in normal subjects; others tested were without effect. Antigen controls consisted of extracts of the Raji cell line, which contains the EBV genome but produces no EBV antigen, and cultured human cells with no known association with EBV, all of which failed to induce migration inhibition. Cellular controls consisted of EBV sero-negative adult leukocytes and foetal cord blood leukocytes which did not respond to P₃HR-1 extract, although two of the five cord sera contained anti-EBV capsid antibody. The migration inhibition response was antigen-dose-dependent and was not affected by anti-EBV antibody capable of neutralizing the infectivity of EBV. These results show that CMI to EBV is present in normal individuals who have antibody to EBV capsid antigen.     

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.     

Relationship between Epstein-Barr virus (EBV)-production and the loss of the EBV receptor/complement receptor complex in a series of sublines derived from the same original Burkitt's lymphoma.

Virus production, EBV (P3HR-1 substrain) superinfectability, IdUrd inducibility, EBV receptor and complement (C3) receptor expression were assessed in two independently maintained jijoye lines, the derived P3HR-1 clone that releases a growth inhibitory and cytopathic, non-transforming viral mutant, and in non-producer sublines derived from the P3HR-1 line by the spontaneous cessation of virus production. Both jijoye lines were superinfectable, inducible, and carried EBV and C3 receptors. Virus-producing P3HR-1 cells and recently derived non-producer sublines lacked EBV-receptors and C3 receptors, could not be superinfected, but were IdUrd inducible. Two long-passaged, non-producer sublines of P3HR-1 reexpressed EBV and C3 receptors to an equal degree (different in the two sublines). EBV-superinfectability became partially reestablished in the subline with the higher expression of EBV and C3 receptors. These findings support the hypothesis that the EBV-receptor/C3 receptor negativity of the producer P3HR-1 sublines and their recent non-producer derivatives is due to negative selection by the growth-inhibitory, cytopathic P3HR-1 virus variant. The closely linked disappearance and reappearance of EBV-receptors and complement receptors gives further support to the idea that these two receptors are either identical or closely linked constituents of the cell membrane.     

Further studies on inhibition of Epstein-Barr virus release by bovine serum: partial purification and characterization of the inhibitor

The antivirus release factor (AVRF) was extracted from adult bovine serum and was partially purified in three steps by 1) ammonium sulfate precipitation, 2) Sephadex G-200 chromatography, and 3) DEAE-Sephadex chromatography, procedures that together increased specific activity 160 times. In molecular sieve chromatography, AVRF was found in fractions close to IgM. The AVRF activity was sensitive to KIO4 (0.04 M) but not to trypsin (0.1%), UV irradiation, ethyl ether, and heating at 56 degrees C for 30 minutes. The AVRF neither neutralized infectivity of Epstein-Barr virus (EBV) from the P3HR-1 cell line nor inhibited intracellular virus yields at concentrations that inhibited release of virus into culture fluid. Cellular release of two other strains of EBV (QIMR-WIL and B95-8) was also inhibited by AVRF. Electron microscopic examination of P3HR-1 cells cultured in the presence of AVRF revealed peculiar interacytoplasmic vacuoles containing many mature virus particles with tail-like structures.     

Column separation of viral capsid antigen (VCA)-positive cells from VCA-negative cells in an Epstein-Barr virus (EBV)-producing lymphoid line,.

Virus producing, VCA (viral capsid antigen)-positive cells could be selectively removed from Epstein-Barr virus (EBV)-carrying, virus-producer P3HR-1 cultures by two different methods of column passage. In the first, the virus-producing cells were covered with human EBV antibody and subsequently passaged through anti-human-Ig columns. In the second methods, untreated P3HR-1 cells were allowed to pass through columns of EBV receptor-positive cell lines or, as controls, EBV receptor-positive cell lines or, as controls, EBV receptor-negative cells. The majority of the VCA-positive cells were selectively removed by both techniques. The second method involves the attachment of EBV-producer cells, known to accumulate viral envelope material in their plasma membrane, to EBV receptors. In view of recent evidence indicating an association between EBV and complement receptors, human and mouse complement were tested for their ability to block this attachment. Fresh mouse and human complement regularly exerted blocking activity, whereas heat-inactivated human serum did not block. Heat-inactivated mouse serum did block occasionally, but the effect was more irregular than with fresh mouse serum. Trypsin treatment of the EBV-receptor colum abolished its ability to retain VCA-positive cells.     

Productive Epstein-Barr viral infection of the human lymphoblastoid cell line 6410 with release of early antigen inducing and transforming virus.

The human lymphoblastoid cell line 6410 was superinfected with P3HR-1 derived Epstein-Barr virus. Subsequent to the first cycle of infection, characterized by early (EA) and virus capsid (VCA) antigen synthesis, the culture, designated 6410-EBV, continued to synthesize VCA. Further immunofluorescence and electron microscopic examination over 18-24 months showed the 6410-EBV culture to be productively infected with EBV. Virus was recovered, in quantity, from the culture fluids and assayed for ability to induce EA synthesis in Raji cells and to transform human umbilical cord lymphocytes. The virus was found to possess both properties, in contrast to P3HR-1 virus which only induces EA. HLA and karyologic analyses of P3HR-1, 6410 and 6410-EBV cultures showed relatedness of 6410-EBV to 6410 cells and dissimilarity to P3HR-1. The biologic activity data coupled with the cytologic analyses confirm a productive infection of the target cells. The reason for differences in biologic activity between the infecting (P3HR-1) and progeny virus is unresolved. It may be speculated that the endogenous EBV genome of 6410 cells was activated and gave rise to a different strain of EBV or to a mixed progeny-parent population as a result of dual infection. Alternatively, the parent strain of virus may have been modfied as a result of interaction with the new host cell.     

Properties of a baboon lymphotropic herpesvirus related to Epstein-Barr virus.

Three lymphoblastoid cell lines were established from splenic lymphocytes of a lymphomatous baboon (Papio hamadryas) by co-cultivation of the lymphocytes with X-irradiated cells of marmoset or baboon lymphoblastoid cell cultures; the baboon splenic lymphocytes failed to grow when cultured alone. A herpesvirus, associated with each cell line, was identified by immunofluorescence, molecular hybridization and electron microscopy. Antigenic comparison with Epstein-Barr virus (EBV) showed that the baboon herpesvirus and EBV shared cross-reacting viral capsid antigens (VCA): 20 of 20 (100%) anti-VCA (EBV)-positive human sera and 55 of 62 (89%) baboon sera reacted with the baboon lymphoblastoid cells and baboon sera stained EBV VCA in P3HR-1 and EB-3 cells. No nuclear antigen, as assayed by anti-complement immunofluorescence tests, was detected in baboon lymphoblastoid cells when human or baboon anti-VCA positive sera were used. Baboon anti-VCA-positive sera also failed to stain EBV nuclear antigens (EBNA) in Raji or P3HR-1 cells. Preliminary molecular hybridization studies showed only approximately 40% homology between viral DNA of baboon cell lines and DNA of EBV derived from P3HR-1 cells.     

A quantitative analysis of the susceptibility of human leukocytes to transformation by epstein-barr virus

Susceptibility of lymphocyte-enriched cell fractions isolated from human umbilical cord blood and adult peripheral blood to transformation by the B95–8 strain of Epstein-Barr virus (EBV) was investigated quantitatively. Minimum multiplicity of input of virus (50% transforming dose) per cell (MOI) necessary to induce maximum level transformation of cord cells ranged from 0.02 to 0.2. The frequency of initially transformed cells (fraction of transformable cells) in the cord cell samples from two different individuals was estimated to be 2.6 to 6.2%. In this system, the appearance of cells positive for EBV-associated nuclear antigen (EBNA) paralleled the growth curve of transformed cells. About 70% of the latter were EBNA-positive. In adult cell preparations from two individuals, 1.8 and 0.03%, respectively, of the cells were transformable indicating larger individual variations in sensitivity to EBV than in cord cells. The EBV susceptibility was also determined by the transforming efficiency (TE) expressed as the negative log of the virus dilution which induces transformation in 50% of cell cultures infected at an MOI of 0.2. From the TE value, a minimum MOI which induces transformation could be calculated. Also by this test it was shown that the EBV susceptibility of adult cells was not only lower but also much more variable between individuals than that of cord cells. There was no correlation between the susceptibility of cells and the titer of anti-EBV antibody in donors' sera. In cultures of mixed cord cells and adult cells known to have low EBV susceptibility, the minimum MOI increased in proportion to the amount of adult cells.     

Re-evaluation of a transforming strain of epstein-barr virus from the burkitt lymphoma cell line,Jijoye

The biological properties of Epstein-Barr virus (EBV) from a Burkitt lymphoma cell line, Jijoye, were examined. The synthesis of virus capsid antigen (VGA) and early antigen (EA) in Jijoye cells was markedly enhanced by shift-down of the temperature of incubation from 37°C to 33°C. Cultures of Jijoye cells at 33°C released a large amount of transforming EBV (10^5.2 of 50% transforming doses/ml) into the culture fluid. However, no infectious virus was produced in all cultures at 37°C during the course of this study. The EBV (Jijoye EBV) from Jijoye line was found to possess only transforming activity, but not EA-inducing activity. Jijoye EBV lacks adsorbing capacity to Jijoye cells, in contrast to P3HR-l EBV which can adsorb to Jijoye cells. The Jijoye cells were highly susceptible to superinfection with P3HR-l EBV as demonstrated by the induction of EA, VGA and infectious EBV. The EBV induced by the P3HR-I EBV superinfection of Jijoye cells has also transforming activity but neither EA-inducing activity nor adsorbing capacity to Jijoye cells.     

Lymphoproliferative disease in a cotton-top marmoset after inoculation with infectious mononucleosis-derived Epstein-Barr virus.

Injection of concentrated EBV derived from cells of the Kaplan line of infectious mononucleosis (IM) origin resulted in malignant lymphoproliferation in one out of three cotton-top marmosets 6 weeks after inoculation. Two additional animals receiving the same isolate after incubation with an antibody-containing human serum did not develop tumors. Inoculation of concentrated virus derived from the P3HR-1 line of Burkitt origin did not lead to lymphoproliferations in five marmosets. Three of these received non-neutralized, and two received neutralized P3HR-1 virus. The tumor obtained with the Kaplan isolate revealed characteristics of a lymphosarcome. It contained EBV-specific DNA. In addition, EBV-synthesizing lymphoblastoid lines were established from a tumorous lymph-node, as well as from the spleen of the diseased marmoset. Virus recovered from these lines transformed lymphocytes derived from spleens of healthy marmosets. The tumor-bearing animal developed low levels of anti-VCA antibodies during the course of tumor growth. These data demonstrate the oncogenic potential of EBV directly derived from cells of IM origin.     

Correlated expression of a B-lymphocyte-specific glycoprotein (gp27/35) and the EBV receptor/C3 receptor complex in sublines from the same Burkitt lymphoma.

A series of virus-producer and non-producer sublines, derived from the Burkitt lymphoma line Jijoye and its P3HR-1 clone, were previously found to differ in the expression of EBV receptors and, in parallel, C3 receptors. The differences could be related to an "internal selection" caused by the cytopathic P3HR-1 virus variant, favouring the growth of receptor-negative cells. We have now analyzed the same lines for the expression of a B-lymphocyte-specific glycoprotein (gp27/35). A close parallellism was found between the expression of the EBV receptor-complement receptor complex and the glycoprotein, measured by quantitative absorption. The results favour a relationship between gp27/35 and EBV/C3 receptor expression.     

Heterogeneity of Epstein-Barr virus originating from P3HR-1 cells. I. Studies on EBNA induction.

Infection of EBV-negative human B-lymphoma cells of the lines BJAB and Ramos with EBV from P3HR-1 or B95-8 cells resulted in gradual conversion of these cells to EBNA synthesis. Whereas B 95-8 virus-infected cells exhibited a uniform brilliant EBNA fluorescence, two distinct fluorescence patterns were observed in P3HR-1 virus-converted BJAB and Ramos cells, a faint granular and a brilliant fluorescence, with predominance of the faint granular pattern. Cloning of P3HR-1 virus-converted BJAB cells resulted in 20 clones, 11 of them showing the heterogenous parental pattern, six revealing exclusively faint granular EBNA staining, and three with brilliantly stained nuclei, containing also a varying percentage of EBNA-negative cells. Further subcloning of one of the latter clones resulted in 26 subclones with brilliant EBNA expression, always segregating a significant percentage of EBNA-negative cells and one entirely EBNA-negative subclone. Reassociation kinetics did not reveal striking differences in the genome content of clones showing exclusively the faint granular or the brilliant type of EBNA expression. The EBNA-negative clone did not contain detectable amounts of EBV-DNA. Upon superinfection of the converted clones by the parental P3HR-1 virus, a significant increase in EA induction was noted when compared to non-converted BJAB and Ramos cells. This accounted in particular for cells with faint granular EBNA expression. These data support previous interpretations (Fresen and zur Hausen, 1976), suggesting the existence of at least two populations of EBV molecules within P3HR-1 cells. The reason for the apparently labile association of P3HR-1 EBV genomes inducing the brilliant EBNA flourescence in BJAB cells still remains obscure. The possible existence of a "helper" effect, exerted by the faint granular EBNA-inducing virus in stabilizing the persistence of the former, is discussed.     

Differences in EBV receptor concentration between in vitro EBV-converted lymphoma sublines reflect biological differences between the converting viral substrains.

We have compared the EBV-receptor concentration of two originally EBV-negative human B-cell lymphoma lines, after in vitro conversion with the transforming B95-8 or the cytopathic P3HR-1 EB-viral substrain, respectively, into permanent EBV-carrying sublines. Receptors were measured by the quantitative EBV-absorption bioassay of Sairenji and Hinuma (1973). EBV receptor concentration of all P3HR-1 virus-converted sublines was significantly reduced, in comparison with the B95-8 virus-converted sublines. This suggests that cells with a low receptor concentration are more likely to survive the initial infection with the P3HR-1 viral harvest. The results further confirm the biological differences between the two EBV substrains.     

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).     

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.     

Colonies of EBNA-positive cells in soft agar from peripheral leukocytes of infectious mononucleosis patients.

Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA)-positive lymphoblastoid cells grew as colonies in soft agar after seeding of leukocytes from the peripheral blood of four patients with infectious mononucleosis serologically determined to be caused by EBV. In individual cases more colonies were obtained from blood specimens during the acute phase of the disease than during the convalescent phase. Incorporation of human umbilical cord serum, which contained neutralizing antibody to EBV, into the agar medium did not reduce the number of colonies developing. Our observations indicate that colony-forming cells were originally present in the blood samples, and that they were not infected and subsequently transformed in vitro. Cells from less than 20% of the EBNA-positive colonies grew to form lymphoblastoid cell lines, which were EBNA-positive and had B lymphocyte surface markers. However, the majority (over 80%) of the EBNA-positive colonies failed to form immortalized cell lines. No colonies were obtained from 91 blood samples from healthy young adults and from five patients with an IM-like disease unrelated to EBV infections. The present results strongly suggest that already transformed cells or cells very easily transformed by EBV are present in the blood of IM patients.     

Enhancement of replication of Epstein-Barr virus DNA in human lymphoblastoid cell lines by N-methyl-N''-nitro-N-nitrosoguanidine

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a potent carcinogenthat alkyiates nucleic acids. Interaction of MNNG with humanlymphoblastoid cell fines carrying Epstein-Barr virus (EBV)was studied. Treatment of virus-producing cells (P3HR-1) withMNNG resulted in an {small tilde}3-fold increase in EBV genomecopies per cell as determined by cRNA - DNA hybridization. Thiseffect was not observed in a non-virus-producer line (Raji).Dose-response studies indicated that the optimum concentrationwas between 0.5 µg and 2 µg/ml. This same dose rangewas most effective in inhibiting cell proliferation both ofP3HR-1 and Raji cells. Concomitant treatment of P3HR-1 cellswith MNNG and 12-O-tetradecanoyl-phorbol-13-acetate gave anadditive increase to 9-fold of the number of EBV genome copiesper cell. Pretreatment of Raji cells with MNNG followed by superinfectionwith P3HR-1 virus resulted in a 35% enhancement of EBV DNA replicationas analyzed by density centrifugation. In contrast, Raji cellssuperinfected with MNNG-treated EBV showed a marked reductionin EBV DNA replication which indicates that the lesions producedin the viral genome by the drug interfered with the infectiouspotential of the virus.