Epstein-Barr Virus Genome Copy Number Research Articles

Updated guidelines for chronic active Epstein-Barr virus disease.

Chronic active Epstein-Barr virus disease (CAEBV), formerly named chronic active Epstein-Barr virus infection, is characterized by systemic inflammation and clonal proliferation of Epstein-Barr virus (EBV)-infected T or NK cells. As CAEBV is a potentially life-threatening illness, appropriate diagnosis and therapeutic interventions are necessary for favorable clinical outcomes. Substantial evidence regarding the pathogenesis and treatment of CAEBV has been accumulated since previous guidelines for the diagnosis of CAEBV were proposed. To reflect this evidence, we updated the guidelines for the diagnosis and treatment of CAEBV to improve clinical management of the disease. The details of the updated guidelines are presented in this report. Diagnosis of CAEBV now requires confirmation of a high copy number of EBV genome and EBV-infected T or NK cells. An EBV DNA load ≥ 10,000IU/mL in whole blood is proposed as the diagnostic cutoff value for CAEBV in this updated guideline. A standard treatment approach for CAEBV has not been established, and hematopoietic stem cell transplantation (HSCT) is considered the only curative treatment. Chemotherapy can be administered to control disease activity before HSCT.

The copy number of Epstein-Barr virus latent genome correlates with the oncogenicity by the activation level of LMP1 and NF-κB

A tumor model that Epstein-Barr virus (EBV) latent infection facilitated the tumorigenicity was previously established using the Maxi-EBV system. In the present approach, EBV-lost cell clones demonstrated significantly decreased tumorigenesis. On the other hand, the LMP1 gene in Maxi-EBV genome was replaced by that of nasopharyngeal carcinoma origin. The resultant cell line, 293-1/NL showed much lower malignancy than the original 293-EBV. The result was opposite to our expectation. The change of 293 sublineage cells for EBV harboring also got similar result. To seek the underlying reason, the copy number of EBV genome in all the cell lines was detected. The result indicated that 293-EBV contained about 4.5-fold higher EBV copies than 293-1/NL did. Parallel EBV genomes led to relatively stable copies in different 293 sublineages, suggesting the viral genome structure is a factor for the sustainability of EBV's copy number. Moreover, the LMP1 transcription in high copy-containing cells showed abnormally high level. Furthermore, the main LMP1-driven pathway, transcription factor NF-κB, was highly activated in high-copy cells. Here we first manifest by experimental model that the copy number of EBV latent genome correlates with the viral pathogenesis, which depends on the activation level of LMP1 and NF-κB. Overall, both the presence and amount of EBV genome are crucial for the viral oncogenicity.

Cordycepin is a novel chemical suppressor of Epstein-Barr virus replication.

Cordyceps species are known to produce numerous active components and are used for diverse medicinal purposes because of their varied physiological activities, including their ability to protect the liver from damage as well as their anticancer, antidepressant, anti-inflammatory, hypoglycemic, antimicrobial effects. Cordycepin, an adenosine derivative, differs from adenosine in that its ribose lacks an oxygen atom at the 3' position. Several research groups have reported that cordycepin has antiviral activity against several viruses including influenza virus, plant viruses, human immunodeficiency virus(HIV), murine leukemia virus, and Epstein-Barr virus (EBV). In this study, we identify the epigenetic mechanisms by which cordycepin exerts its anti-gammaherpesvirus effects. We show that cordycepin possesses antitumor and antiviral activity against gastric carcinoma and EBV, respectively. A comparison of the CD50 values of cordycepin and its analogs showed that the lack of a 2'-hydroxyl group in cordycepin was critical for its relatively potent cytotoxicity. Cordycepin treatment decreased the rate of early apoptosis in SNU719 cells by up to 64%, but increased late apoptosis/necrosis by up to 31%. Interestingly, cordycepin increased BCL7A methylation in SNU719 cells by up to 58% and decreased demethylation by up to 37%. Consistent with these changes in methylation, cordycepin treatment significantly downregulated most EBV genes tested. Under the same conditions, cordycepin significantly decreased the frequency of Q and F promoter usage, and H3K4me3 histone enrichment was significantly reduced at several important EBV genomic loci. Extracellular and intracellular EBV genome copy numbers were reduced by up to 55% and 30%, respectively, in response to 125 μM cordycepin treatment. Finally, cordycepin significantly suppressed the transfer of EBV from LCL-EBV-GFP to AGS cells, indicating that EBV infection of gastric epithelial cells was inhibited. These results suggest that cordycepin has antiviral and antitumor activities against gammaherpesviruses and host cells latently infected with virus.

Host genetic variants and gene expression patterns associated with Epstein-Barr virus copy number in lymphoblastoid cell lines.

Lymphoblastoid cell lines (LCLs) are commonly used in molecular genetics, supplying DNA for the HapMap and 1000 Genomes Projects, used to test chemotherapeutic agents, and informing the basis of a number of population genetics studies of gene expression. The process of transforming human B cells into LCLs requires the presence of Epstein-Barr virus (EBV), a double-stranded DNA virus which through B-cell immortalisation maintains an episomal virus genome in every cell of an LCL at variable copy numbers. Previous studies have reported that EBV alters host-gene expression and EBV copy number may be under host genetic control. We performed a genome-wide association study of EBV genome copy number in LCLs and found the phenotype to be highly heritable, although no individual SNPs achieved a significant association with EBV copy number. The expression of two host genes (CXCL16 and AGL) was positively correlated and expression of ADARB2 was negatively correlated with EBV copy number in a genotype-independent manner. This study shows an association between EBV copy number and the gene expression profile of LCLs, and suggests that EBV copy number should be considered as a covariate in future studies of host gene expression in LCLs.

Protein kinase inhibitors that inhibit induction of lytic program and replication of Epstein–Barr virus

Signaling pathways mediating Epstein–Barr virus (EBV) reactivation by Ag-bound B-cell receptor (BCR) were analyzed using a panel of 80 protein kinase inhibitors. Broad range protein kinase inhibitors Staurosporine, K252A, and PKC-412 significantly reduced the EBV genome copy numbers measured 48h after reactivation perhaps due to their higher toxicity. In addition, selected inhibitors of the phosphatidylinositol-3-kinase (PI3K), protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways, glycogen synthase kinase 3β (GSK-3β), platelet-derived growth factor receptor-associated tyrosine kinase (PDGFRK), and epidermal growth factor receptor-associated tyrosine kinase (EGFRK) significantly reduced the EBV genome copy numbers. Of those, only U0126 and Erbstatin analog, which inhibit MAPK pathway and EGFRK, respectively, did not inhibit viral reactivation assessed by expression of the EBV early protein, EA-D. None of the tested compounds, except for K252A, affected the activity of the EBV-encoded protein kinase in vitro. These results show that EBV reactivation induced by BCR signaling is mainly mediated through PI3K and PKC, whereas MAPK might be involved in later stages of viral replication.

Development of a High-Throughput Screen for Inhibitors of Epstein-Barr Virus EBNA1

Latent infection with Epstein-Barr virus (EBV) is a carcinogenic cofactor in several lymphoid and epithelial cell malignancies. At present, there are no small-molecule inhibitors that specifically target EBV latent infection or latency-associated oncoproteins. EBNA1 is an EBV-encoded sequence-specific DNA binding protein that is consistently expressed in EBV-associated tumors and required for stable maintenance of the viral genome in proliferating cells. EBNA1 is also thought to provide cell survival function in latently infected cells. In this work, the authors describe the development of a biochemical high-throughput screening (HTS) method using a homogeneous fluorescence polarization (FP) assay monitoring EBNA1 binding to its cognate DNA binding site. An FP-based counterscreen was developed using another EBV-encoded DNA binding protein, Zta, and its cognate DNA binding site. The authors demonstrate that EBNA1 binding to a fluorescent-labeled DNA probe provides a robust assay with a Z factor consistently greater than 0.6. A pilot screen of a small-molecule library of ~14,000 compounds identified 3 structurally related molecules that selectively inhibit EBNA1 but not Zta. All 3 compounds had activity in a cell-based assay specific for the disruption of EBNA1 transcription repression function. One of the compounds was effective in reducing EBV genome copy number in Raji Burkitt lymphoma cells. These experiments provide a proof of concept that small-molecule inhibitors of EBNA1 can be identified by biochemical HTS of compound libraries. Further screening in conjunction with medicinal chemistry optimization may provide a selective inhibitor of EBNA1 and EBV latent infection.

Viral DNA contamination is responsible for Epstein-Barr virus detection in cytotoxic T lymphocytes stimulated in vitro with Epstein-Barr virus B-lymphoblastoid cell line.

Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (LCLs) are used to prepare human EBV-specific T lymphocytes (EBV-CTL) in vitro. Within an LCL, up to 5-7% the cells release infectious EBV, and this has fostered safety concerns for therapeutic applications because of the exposure of T cells to EBV. The release of infectious viruses can be prevented by ganciclovir, but this drug may seriously affect LCL growth. In the wake of these concerns, the present work was designed to compile safety data on EBV-CTL preparation for the purpose of submission to a regulatory agency. We showed that further to supernatant exclusion, the number of EBV genome copies (EBVc) associated with the EBV-CTL always made up a constant proportion of the EBVc number detected in the culture supernatant. In addition, such was the case whether infectious virus could be produced by the LCL or not, suggesting that the EBV signal detected was due to a DNA contamination rather than an infection. Furthermore, we demonstrated that the number of EBVc associated with the EBV-CTL was highly sensitive to DNAse treatment, and finally that EBVc could no longer be detected after the EBV-CTL had been amplified in the absence of LCL. Consequently, during in vitro EBV-CTL preparation, either T cells cannot be infected or they die rapidly after EBV infection.

Discovery of Selective Inhibitors Against EBNA1 via High Throughput In Silico Virtual Screening

BackgroundEpstein-Barr Virus (EBV) latent infection is associated with several human malignancies and is a causal agent of lymphoproliferative diseases during immunosuppression. While inhibitors of herpesvirus DNA polymerases, like gancyclovir, reduce EBV lytic cycle infection, these treatments have limited efficacy for treating latent infection. EBNA1 is an EBV-encoded DNA-binding protein required for viral genome maintenance during latent infection.MethodologyHere, we report the identification of a new class of small molecules that inhibit EBNA1 DNA binding activity. These compounds were identified by virtual screening of 90,000 low molecular mass compounds using computational docking programs with the solved crystal structure of EBNA1. Four structurally related compounds were found to inhibit EBNA1-DNA binding in biochemical assays with purified EBNA1 protein. Compounds had a range of 20–100 µM inhibition of EBNA1 in fluorescence polarization assays and were further validated for inhibition using electrophoresis mobility shift assays. These compounds exhibited no significant inhibition of an unrelated DNA binding protein. Three of these compounds inhibited EBNA1 transcription activation function in cell-based assays and reduced EBV genome copy number when incubated with a Burkitt lymphoma cell line.ConclusionsThese experiments provide a proof-of-principle that virtual screening can be used to identify specific inhibitors of EBNA1 that may have potential for treatment of EBV latent infection.

Epstein-Barr Virus Episome Stability Is Coupled to a Delay in Replication Timing

The temporal regulation of DNA replication is thought to be important for chromosome organization and genome stability. We show here that Epstein-Barr virus (EBV) genomes replicate in mid- to late S phase and that agents that accelerate replication timing of EBV reduce viral genome stability. Hydroxyurea (HU) treatment, which is known to eliminate EBV episomes, shifted EBV replication to earlier times in the cell cycle. HU treatment correlated with hyperacetylation of histone H3 and loss of telomere repeat factor 2 (TRF2) binding at the EBV origin of plasmid replication (OriP). Deletion of TRF2 binding sites within OriP or short hairpin RNA depletion of TRF2 advanced the replication timing of OriP-containing plasmids. Inhibitors of class I histone deacetylases (HDACs) increased histone acetylation at OriP, advanced the replication timing of EBV, and reduced EBV genome copy number. We also show that HDAC1 and -2 form a stable complex with TRF2 at OriP and that HU treatment inhibits HDAC activity. We propose that the TRF2-HDAC complex enhances EBV episome stability by providing a checkpoint that delays replication initiation at OriP.

Detection of specific lytic and latent transcripts can help to predict the status of Epstein-Barr virus infection in transplant recipients with high virus load.

Epstein-Barr virus (EBV), a member of the family Herpesviridae, is widely spread in the human population and has the ability to establish lifelong latent infection. In immunocompetent individuals the virus reactivation is usually harmless and unnoticeable. In immunocompromised patients productive infection or type III latency may lead to EBV-associated post-transplant lymphoproliferative disorder (PTLD). The aim of our research was to investigate the utility of PCR-based methods in the diagnosis and monitoring of EBV infections in bone marrow transplant recipients. Thirty-eight peripheral blood leukocyte samples obtained from 16 patients were analysed, in which EBV DNA was confirmed by PCR. We used semi-quantitative PCR to estimate the viral load and reverse-transcription PCR (RT-PCR) to differentiate between latent and productive EBV infection. In 14 patients we confirmed productive viral infection. We observed a correlation between higher number of EBV genome copies and the presence of transcripts specific for type III latency as well as clinical symptoms.

Analysis of Epstein-Barr virus gene expression upon phorbol ester and hydroxyurea treatment by real-time quantitative PCR

Epstein-Barr virus (EBV) has the potential to undergo latent and lytic pathways during infection. However, expression of many of the viral genes during the lytic-latent transition remains unclear. In this study, we investigated the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and hydroxyurea (HU), two commonly used modulators of EBV life cycle, on the expression profiles of the entire genome of EBV persistent infected in B95-8 cells. After treatment with TPA for 48 h, the copy number of EBV genome in the cells increased about 2.5 fold, whereas HU treatment resulted in a reduction to approximately two-thirds of the original level. Except a small set of genes, the amounts of EBV mRNA are generally less abundant than that of beta-actin. The expression of a large fraction of the 80 EBV genes was found to be activated after TPA treatment with a noticeable increase of 19 and 21 fold, respectively in BSLF1 and BBLF4. In contrast, treatment of the B95-8 cells with HU, a nucleotide synthesis inhibitor, dramatically suppressed the expression of EBV lytic genes. In summary, we have demonstrated that real-time quantitative PCR is a reliable method to monitor the influence of drug-treatment in EBV genes regulation. Our results also provide a basis for further investigation on how the virus coordinates its own gene expression during latent-lytic pathway transition.

Risk Factors for Hodgkin's Lymphoma by EBV Status and Significance of Detection of EBV Genomes in Serum of Patients with EBV-Associated Hodgkin's Lymphoma

Epstein–Barr virus (EBV) is associated with around one-third of Hodgkin's lymphoma (HL) cases and this association is believed to be causal. In these EBV-associated cases, there is a clonal EBV infection within tumors, and EBV genomes and gene products are detectable in Hodgkin and Reed-Sternberg (HRS) cells. The proportion of EBV-associated HL in any population varies with age, sex, ethnicity and histologic subtype. Two population-based epidemiologic studies have examined risk factor profiles in HL with cases stratified according to EBV status. For EBV-associated HL cases, there is a small peak in incidence in young adults (15 – 24 years) and a second larger peak in older adults. By contrast, HL that is not associated with EBV (EBV-negative HL) accounts for the major part of the young adult incidence peak after which the incidence of this disease entity then declines. Prior infectious mononucleosis (IM) is associated with an increased risk of developing HL, and there is a specific, probably causal, association between previous IM and young adult EBV-associated HL. We therefore believe that the small peak in the incidence of EBV-associated HL in young adults is real and related to late infection by EBV. EBV-associated HL in childhood and young adults, therefore, appears to follow primary infection by the virus. At the time of diagnosis, EBV-associated HL patients have an increased frequency of circulating EBV-infected cells compared to patients with EBV-negative HL and normal controls. The EBV is present in memory B cells and most probably reflects increased viral replication at another site, such as the oropharynx. EBV genomes are detectable in the serum and plasma of EBV-associated HL cases. The origin of EBV genomes in serum/plasma differs in different disease states; in HL viral genomes are present as naked DNA and are probably shed from tumors. EBV genome copy number in serum/plasma may provide an indication of tumor burden and may prove to be a useful marker for monitoring HL patients. The etiology of EBV-negative HL remains unknown and, while the involvement of an infectious agent may be suspected, none has yet been identified. Overall, epidemiologic studies support the idea that HL can be divided into two etiologic subgroups on the basis of EBV status and suggest that EBV-associated cases can be further divided into three groups related to age at diagnosis.

Quantitative Analysis of Cell-free Epstein-Barr Virus Genome Copy Number in Patients with EBV-associated Hemophagocytic Lymphohistiocytosis

To determine whether the EBV genome content in serum or plasma reflects clinical features and outcome in EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), we quantified the cell-free EBV genome copy number by real-time PCR in 38 patients with EBV-HLH, and compared this to the values from 15 patients with infectious mononucleosis (IM). The median (range) cell-free EBV genome copy number at diagnosis was 3.0 × 10 3 (undetectable m 5.5 × 10 7 ) copies/ml in EBV-HLH, which was significantly higher than the 6.6 × 10 1 (undetectable m 1.0 × 10 3 ) copies/ml in IM (P =0.0008). We serially analyzed cell-free EBV genome copy number in 10 cases of EBV-HLH up to 4 months from diagnosis. In four patients who achieved remission, the EBV genome became undetectable soon after starting therapy. In the remaining six patients who responded poorly to therapy, the EBV genome copy number in the serum or plasma remained at high levels except for one case. In addition, we confirmed that the EBV genome became undetectable after hematopoietic stem cell transplantation in 4 EBV-HLH cases. These results suggest that the quantitative analysis of cell-free EBV genome copy number is useful for evaluating disease activity and for predicting the response to therapy in EBV-HLH.

Rapid reconstitution of Epstein-Barr virus–specific T lymphocytes following allogeneic stem cell transplantation

Epstein-Barr virus (EBV)-specific CD8 T lymphocytes are present at remarkably high frequencies in healthy EBV(+) individuals and provide protection from EBV-associated lymphoproliferative diseases. Allogeneic peripheral blood stem cell transplantation (allo-PBSCT) is a commonly used therapy in which T-cell surveillance for EBV is temporarily disrupted. Herein, human leukocyte antigen (HLA) class I tetramers were used to investigate the reestablishment of the EBV-specific CD8 T-cell repertoire in patients following allo-PBSCT. CD8(+) T cells specific for lytic and latent cycle-derived EBV peptides rapidly repopulate the periphery of matched sibling allo-PBSCT patients. The relative frequencies of T cells specific for different EBV peptides in transplantation recipients closely reflect those of their respective donors. Investigation of patients at monthly intervals following unmanipulated allo-PBSCT demonstrated that the frequency of EBV-specific T cells correlates with the number of EBV genome copies in the peripheral blood and that expansion of EBV-specific T-cell populations occurs even in the setting of immunosuppressive therapy. In contrast, patients undergoing T-cell-depleted or unrelated cord blood transplantation have undetectable EBV-specific T cells, even in the presence of Epstein-Barr viremia. The protective shield provided by EBV-specific CD8 T cells is rapidly established following unmanipulated matched sibling allo-PBSCT and demonstrates that HLA class I tetramers complexed with viral peptides can provide direct and rapid assessment of pathogen-specific immunity in this and other vulnerable patient populations. (Blood. 2000;96:2814-2821)

Monitoring of epstein-barr virus DNA load in peripheral blood by quantitative competitive PCR.

A competitive quantitative PCR (Q-PCR) assay combined with simple silica-based DNA extraction was developed for monitoring of Epstein-Barr virus (EBV) DNA load in unfractionated peripheral blood. The Q-PCR is based on competitive coamplification of a highly conserved 213-bp region of the EBNA-1 open reading frame with an internal standard (IS), added in a known concentration. The IS has the same amplicon length and base composition as the wild-type (WT) EBNA-1 amplicon but differs in 23 internally randomized bases. Competitive coamplification yields two PCR products that are quantified by enzyme immunoassay or by electrochemiluminescence detection, with probes specific for the 23 differing internal nucleotides. The Q-PCR has a sensitivity of 10 copies of either WT or IS plasmid DNA. The Q-PCR was validated by quantification of known amounts of plasmid containing the WT EBNA-1 target. Furthermore, we determined EBV genome copy numbers in different cell lines. For EBV quantification in clinical samples, DNA was isolated from lysed whole blood by silica-affinity purification. Forty-six percent of healthy donor peripheral blood samples were positive by Q-PCR. In most of these samples, viral load was less than 2,000 EBV copies/ml of blood. In peripheral blood samples from two AIDS-related non-Hodgkin's lymphoma patients, elevated EBV loads (up to 120,000 copies/ml) were observed, which decreased upon therapy. In Burkitt's lymphoma patients, up to 4,592,000 EBV genome copies/ml of blood were detected. In conclusion, the EBNA-1-based Q-PCR assay provides a reproducible, accurate, and easy method for studying the relationship between EBV load and clinical parameters.

Use of quantitative competitive PCR to measure Epstein-Barr virus genome load in the peripheral blood of pediatric transplant patients with lymphoproliferative disorders.

A quantitative competitive PCR (QC-PCR) assay for Epstein-Barr virus (EBV) has been developed to provide accurate measurement of EBV genome load in pediatric transplant recipients at risk for developing posttransplant lymphoproliferative disorder (PTLD). The assay quantifies between 8 and 5,000 copies of the EBV genome in 10(5) lymphocytes after a 30-cycle amplification reaction. For 14 pediatric patients diagnosed with PTLD, the median EBV genome load was 4,000, and 13 of the 14 patients had values of >500 copies per 10(5) lymphocytes. Only 3 of 12 control transplant recipients not diagnosed with PTLD had detectable viral genome loads (median value, 40). This median was calculated by using the highest value obtained by PCR testing on each of these patients posttransplantation. PCR values of >500 copies per 10(5) lymphocytes appear to correlate with a diagnosis of PTLD. By a modified protocol, the EBV genome copy number in latently infected adults was estimated to be <0.1 copy per 10(5) lymphocytes.

Expression of the Epstein-Barr Virus (EBV) latent membrane protein is tightly regulated, independently of EB nuclear antigen 2 and of EBV integration or copy number

In vivo, Epstein-Barr virus (EBV) is associated with human tumours and with lymphoproliferations in immunosuppressed patients. In vitro, EBV induces unlimited growth of normal B-lymphocytes, a phenomenon known as immortalization. A limited number of viral genes is expressed during this phenomenon and their relative role concerning the deregulation of cellular proliferation is still unclear. At present, the nuclear antigen EBNA2 and the membrane protein LMP are the two EBV proteins considered to be implicated in the immortalization process. Moreover, many data support the hypothesis that EBNA2 is the major inducer of LMP expression by transactivation; however, in some instances, expression of the two proteins is not correlated, suggesting the existence of complex interactions between EBV and its host-cell that influence viral gene regulation. In an attempt to study thoroughly these EBNA2/LMP interactions, it is important to evaluate whether EBNA2 is or is not a major inducer of LMP expression, and which other parameters can influence LMP expression. By analysing two sets of B-lymphoma lines either infected in vitro with EBV or stably transfected with EBNA2, we have demonstrated that (1) LMP expression can be absolutely independent of EBNA2 expression, (2) the level of LMP expression is very tightly regulated, and is independent of EBV genome status (integrated or episomal) and copy number. Our findings provide compelling evidence that LMP expression has to be related to that of cellular factors that remain to be identified.

Epstein-Barr virus transformation of human B lymphocytes despite inhibition of viral polymerase.

Epstein-Barr virus transformed human lymphocytes despite the presence of up to 500 microM acyclovir [9-(2-hydroxyethoxymethyl)guanine], a viral DNA polymerase inhibitor. The transformed cells contained multiple Epstein-Barr virus genome copy numbers. Functional viral DNA polymerase is probably not required for cell transformation and the initial amplification of the viral genome.

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 carcinogen that alkylates nucleic acids. Interaction of MNNG with human lymphoblastoid cell lines carrying Epstein-Barr virus (EBV) was studied. Treatment of virus-producing cells (P3HR-1) with MNNG resulted in an approximately 3-fold increase in EBV genome copies per cell as determined by cRNA--DNA hybridization. This effect was not observed in a non-virus-producer line (Raji). Dose-response studies indicated that the optimum concentration was between 0.5 micrograms and 2 micrograms/ml. This same dose range was most effective in inhibiting cell proliferation both of P3HR-1 and Raji cells. Concomitant treatment of P3HR-1 cells with MNNG and 12-O-tetradecanoylphorbol-13-acetate gave an additive increase to 9-fold of the number of EBV genome copies per cell. Pretreatment of Raji cells with MNNG followed by superinfection with P3HR-1 virus resulted in a 35% enhancement of EBV DNA replication as analyzed by density centrifugation. In contrast, Raji cells superinfected with MNNG-treated EBV showed a marked reduction in EBV DNA replication which indicates that the lesions produced in the viral genome by the drug interferred with the infectious potential of the virus.

Relationship between Epstein-Barr Virus Genome Number, Amount of Nuclear Antigen, and Early Antigen Inducibility in Diploid and Tetraploid Lymphoma Cells of Related Origin

In a collection of near-diploid and near-tetraploid Raji sublines and somatic Raji/Raji hybrids a linear relationship was found between the number of Epstein-Barr virus (EBV) genome copies and the relative amount of EBV-determined nuclear antigen per cell. Inducibility of the viral cycle by iododeoxyuridine and by P3HR-1 virus superinfection coulb be related to the number of resident EBV genome copies per haploid target cell. The implications of these findings are discussed.