Analysis of epstein-Barr virus (EBV) of P3HR-1: isolation of EBV with EBNA induction ability in human cord lymphocytes and without EA induction ability in Raji cells

A subline of P3HR-1 cells was isolated through a prolonged (over 1 year) propagation of the cells at a non-EBV-productive condition followed by cell cloning procedures. Cloned cells thus obtained, designated DHR1, produced EBV when brought back to the EBV-productive condition. Restriction enzyme analysis of the viral DNA revealed that DHR1 EBV is composed of an apparently homogeneous EBV population, and it displays a similar but not identical genome organization compared with HR-1 EBV. The characteristic biological properties of DHR1 EBV included the ability to induce EBV-associated nuclear antigen (EBNA) in human cord lymphocytes and the inability to induce EBV-associated early antigen (EA) in Raji cells. These are in striking contrast to the behavior of the parental HR-1 EBV. Thus, P3HR-1 cultures after a period of nonproductivity reinitiated the production of virus with an apparently homogeneous and unique population of EBV distinguishable from the original HR-1 virus.     

Monoclonal antibody specific for capsid antigen of Epstein-Barr virus

A hybrid cell line (Cl-5l) producing an anti-capsid antibody was obtained by fusion of mouse myeloma cells with spleen cells from mice immunized with purified P3HR-1 Epstein-Barr virus (EBV). Immunofluorescence showed that the Cl-5l antibody reacted with the cytoplasm and the nucleus of P3HR-1 and B95-8 cells, but not with Raji, BJAB, Molt-4, and superinfected Raji cells in the presence of cytosine arabinoside (Ara-C). The viable P3HR-1 and B95-8 cells were not stained nor was the viral infectivity neutralized. The Cl-5l antibody immunoprecipitated 123,000 and 120,000 dalton polypeptides of P3HR-1 and B95-8 cells, respectively, and both were sensitive to phosphonoacetic acid. Specific reactions were not evident with extracts of Raji cells and superinfected Raji cells in the presence of Ara-C. An analysis of the purified virus particles showed that this antibody recognized a capsid component of EBV.     

Differences in Epstein-Barr virus (EBV) receptors expression on various human lymphoid targets and their significance to EBV-cell interaction

This study was aimed at quantitating, by means of fluorescence-activated cell sorter (FACS), EBV binding to different types of target cells, and at learning about a possible relation between EBV receptor density and the fate of cell-surface bound virus. We used fluoresceinated virus preparations of two strains of EBV (B95-8: lymphocyte transforming strain; P3HR-1: non-transforming strain) to analyze quantitatively the expression and density of EBV receptors on different human lymphoid cell lines and on B lymphocytes from both EBV-seropositive and -seronegative donors. FACS analysis was also used as a tool to approximate the cell surface area of the different lymphoid cells examined. Our results indicate that: (a) after accounting for the difference in cell surface dimensios, the fluorescence intensity of EBV-bound Raji (a B line) cells was three to four times higher per unit area than that of EBV-bound fresh B lymphocytes from an EBV-seropositive donor; (b) Molt-4 (a T line) cells bound about 21-fold less P3HR-1 EBV and 6-fold less B95-8 EBV than Raji cells per unit area; (c) B lymphocytes from EBV-seronegative adult donors bound only about one third as much virus as B cells from seropositive individuals; (d) two B lymphocyte sub-populations can be identified in the peripheral blood in regard to their ability to bind EBV, regardless of the EBV antibody status of the donor; (e) the EBV receptor on Molt-4 cells appears structurally different from the one found on Raji cells since EBV binding to Molt-4 cells was not blocked by a monoclonal antibody (OKB7) specific to the complement receptor (CR2). Further, in contrast to Raji cells, Molt-4 expressed a differential binding activity for each of the two EBV strains used. Taken together, the important differences observed in regard to EBV attachment to various targets also appear to relate to the fate of cell-surface bound virus: i.e., virus penetration might be determined, at least in part, by the density of EBV receptors on the target cell surface; thus the receptor density may play a major role in viral infection.     

A simple method for the production of specific antiserum to the second nuclear antigen (EBNA-2) of Epstein-Barr virus (EBV)

A simple technique for raising specific antiserum to the native molecule of Epstein-Barr virus (EBV)-encoded nuclear antigen-2 (EBNA-2) from Raji cells is described. The procedure involves the use of immunoblotting to identify the EBNA-2 polypeptide followed by subsequent excision from an SDS-gel and immunization of experimental animals. The anti-EBNA-2 antiserum recognized a single polypeptide of 86-87 kDa on immunoblots prepared from extracts of EBV-positive Raji or B95-8 cells, while it did not react with any proteins form P3HR-1 or Daudi cells, which carry EBNA-2-defective virus. The method might be applicable to other systems where isolation of purified proteins for immunization is either difficult or unfeasible.     

Increased sensitivity to natural killing in Raji cells is due to effector recognition of molecules appearing on target cell membranes following EBV cycle induction.

Previous studies demonstrated that NK resistant Epstein-Barr virus (EBV) carrying human lymphoblastoid cell lines become sensitive to NK cell-mediated destruction following induction of the viral cycle by superinfection with the P3HR-1 substrain of EBV or chemicals. In the present report we analysed the cellular membrane changes that were related to the development of sensitivity to NK activity in Raji cells with metabolic inhibitors. NK sensitivity does not develop in P3HR-1 superinfected Raji cells that are cultured in the presence of the RNA synthesis inhibitor actinomycin D and drops to half the amount usually detected in superinfected cells that are grown with the protein synthesis inhibitor cyclohexamide. In experiments with cyclohexamide blocks removed after 24 h, the target cell sensitivity to NK returns to normal levels. Control Raji cells cultured with the same inhibitors for up to 72 h do not develop any sensitivity to NK cell activity. These findings suggest that the development of sensitivity to NK destruction in Raji cells following superinfection is due to the addition to the cell membrane of a virally promoted molecule(s) that requires active RNA and protein synthesis.     

Epstein-Barr virus DNA synthesized in superinfected Raji cells.

Raji and P3HR-1 are established Burkitt lymphoma-derived cell lines that carry the Epstein-Barr virus (EBV) genome. Superinfection of the Raji cell line, a non-virus-producer, with virus from P3HR-1 cells results in the synthesis of several thousand copies of EBV-DNA per cell with attendant inhibition of synthesis and breakdown of Raji cell DNA. The DNA synthesized in superinfected Raji cells has been characterized. Raji cells infected with P3HR-1 virus and labeled with 32P 10 hr after infection synthesized only viral DNA. As much as 90% of the 55 S, 32P-labeled material was localized in the nucleus of superinfected cells after a 10-hr labeling period. The viral DNA purified from superinfected cells had the same buoyant density as the DNA isolated from P3HR-1 virions and after purification could be recovered with a specific activity exceeding 106 cpm/μg in an amount which approached 10 wg/107 infected Raji cells. The viral DNA from superinfected cells reassociated with the DNA from Raji or P3HR-1 cells and with the DNA from virus but did not reassociate with DNA from cell line 698 (a lymphoblastoid cell line lacking the EBV genome). Nuclei isolated from superinfected Raji cells incorporated label from deoxythymidine triphosphate into an acid-insoluble product, most of which had a buoyant density identical to that of DNA from P3HR-1 virions. Digestion of the DNA from superinfected cells with the restriction endonuclease EcoRI produced a number of fragments with molecular weights which ranged from less than 1 million to approximately 30 million when analyzed by electrophoresis on agarose gels. All of the fragments produced by digestion of DNA from virus were present in the digest of DNA from superinfected Raji cells.     

Induction of the Epstein-Barr virus (EBV) cycle in latently infected cells by n-butyrate.

n-Butyrate was found to increase the number of virus producer cells to a dramatic extent in the Epstein-Barr virus (EBV)-carrying P3HR-1 and B95-8 lines. Induction was also seen in the nonproducer Raji and the low producer Daudi lines, but at a mucch lower level. The virus containing supernatant of the butyrate treated P3HR-1 cells induced preferentially EBNA in EBV-negative Ramos target cells, whereas the spontaneously produced virus induced predominantly EA in Raji indicator cells. This suggests a possible difference in the biological properties of the butyrate induced vs the prototype virus. In addition to providing a convenient method to obtain a high yield of viral-DNA and virus antigen-producing cells in the severely restricted EBV system, the findings raise interesting questions on the mechanism of EBV induction, and its possible relationship to the known differentiation inducing ability of n-butyrate.     

Infection of mouse lymphocytes by Epstein-Barr virus. II. Stimulation of cellular DNA synthesis by EBV in the absence of EBNA induction and cell transformation

Epstein-Barr virus (EBV) of the transforming and nontransforming strains induced transient stimulation of cellular DNA synthesis during lytic infection of normal mouse lymphocytes. In contrast to human B lymphocytes, the action of both EBV strains in mouse cells was additive. The nontransforming P3HR-1 virus had no cytotoxic effect on mouse lymphocytes. The EBV-infected, stimulated mouse lymphocytes did not express EB virus-determined nuclear antigen and do not grow into immortalized cell lines. The cells expressed EBV-determined early and virus capsid antigens. These results suggest that the stimulation of cellular DNA synthesis by EBV is independent of EBNA synthesis and cell transformation.     

Effect of 12-O-tetradecanoyl-phorbol-13-acetate on the replication of Epstein-Barr virus. I. Characterization of viral DNA.

The tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), induces replication of Epstein-Barr virus (EBV) DNA in a virus-producing human lymphoblastoid cell line, P3HR-1, but not in a nonproducer cell line, Raji. A 6-fold increase in EBV genome copies per P3HR-1 cell parallels the increase in percentage of cells synthesizing viral capsid antigen. In situ cytohybridization with EBV-specific cRNA shows that most of the TPA-treated population participates in the virus-productive cycle. In Raji cells there is abortive induction with an increase in cells showing early antigen from <0.01% to approximately 10%, but there is no increase in EBV genome copies per cell. The optimal TPA concentration for induction of viral DNA replication is 10 ng/ml. EBV DNA synthesized in Raji cells superinfected by virus prepared from TPA-induced P3HR-1 cells is increased approximately 15-fold above that of Raji cells superinfected with control virus. The buoyant density of EBV DNA isolated from virus from TPA-induced cells or of DNA from Raji cells superinfected with TPA-induced and control virus is identical; viral DNA from all sources had the same S value. The XhoI restriction endonuclease digestion patterns of TPA-induced viral DNA and control viral DNA were the same as the viral DNA recovered from Raji cells superinfected with TPA-induced and control virus. Some differences were noted in the molar ratios of some of the fragments.     

Effects of cis-dichloro-diammine-platinum (II) (cis-DDP) on Epstein-Barr virus induction and cell differentiation

Morphological changes were induced by cis-dichlorodiammine platinum (II) (cis-DDP) in two Epstein-Barr virus (EBV) transformed cell lines: the productive P3HR-1 and the nonproductive Raji cell line. In P3HR-1 cells cis-DDP induced synthesis of viral antigens, viral particles and morphological changes characteristic for virus replication. In Raji cells, the virus replicative cycle was not induced and virus-specific morphological changes were limited to the sporadic appereance of some very early alterations in cell morphology. However, in Raji cells, but not in P3HR-1 cells, up to 20 per cent of the cell population exhibited differentiation-related changes towards plasma cell morphology. The most advances stage of cell differentiation detected was classified as plasmablast. Cis-DDP also induced some changes associated with the cytostatic effect of the drug. In the treated cell population cytokinesis was inhibited and frequently multinuclear cells appeared; moreover, extensive degenerative changes were observed.     

Induction and biological characterization of the Epstein-Barr virus (EBV) carried by the Jijoye lymphoma line

In contrast to all other EBV isolates tested, the P3HR-1 line, a clonal derivative of the Burkitt lymphoma line Jijoye, releases nontransforming, cytopathic virus, which induces an abortive cycle in superinfected, EBV-receptor-positive lymphoid cell lines. We have examined the question whether P3HR-1 virus is a unique mutant or if the original, Jijoye-associated virus had already similar biological characteristics,. Jijoye cells were induced with sodium butyrate and with anti-IgM, respectively. Transforming virus was recovered, capable of inducing EBNA in cord blood cells. Both human cord blood and marmoset lymphocytes were immortalized by Jijoye virus. Both derived lines showed a typical lymphoblastoid (LCL) phenotype, including agarose clonability. It is concluded that P3HR-1 virus is a mutant of the original transforming Jijoye virus. Moreover, the results suggest that the phenotypic properties of the transformed lines are determined at the cellular, not the viral genome level.     

Mechanism of infection by Epstein-Barr virus. II. Comparison of viral DNA from HR-1 and superinfected Raji cells by restriction enzymes.

Virus DNA (RS virus DNA) was directly isolated from Raji cells superinfected with Epstein-Barr virus derived from P3HR-1 cells and compared with original superinfecting virus DNA from P3HR-1 cells (HR-1 virus DNA) in agarose-gel electrophoresis after digestion with various restriction enzymes. EcoR-1 digestion of RS virus DNA produced 15 fragments identical to those from HR-1 virus DNA. However, two fragments, EcoR1 No. 6 (10 × 10⁶ daltons) and EcoR1 No. 11 (4.6 × 10⁶ daltons), observed in HR-1 virus DNA were not detected in RS virus DNA from superinfected Raji cells. In addition, the EcoR1 No. 4 (13.5 × 10⁶ daltons) fragment of RS virus DNA showed a molar ratio of 2 whereas HR-1 virus DNA produced the same fragment with a molar ratio of 1. Electrophoresis patterns of virus DNA digested with Hind III, Bam H-I, Hpa I, and Sal I were also examined. In general, both types of virus DNA produced similar patterns after gel electrophoresis, with minor differences in molar ratios after being treated with the restriction enzymes suggesting that RS virus DNA obtained by superinfection of Raji cells is basically identical to HR-1 virus DNA but may contain a population of DNA a little more heterogenous than HR-1 virus DNA.     

Expression of the Epstein-Barr virus (EBV) receptor on the surface of cells infected with EBV derived from nasopharyngeal carcinoma

An Epstein-Barr virus (EBV) genome-positive epithelial hybrid cell line, NPC-KT, derived from the fusion of primary nasopharyngeal carcinoma cells with a human epithelial cell line of adenoid origin and a subline of EBV genome-positive Ramos cells, Ramos/NPC, converted after infection with NPC-KT EBV have been previously described (Takimoto et al., 1984; Takimoto et al., 1987). The NPC-KT cells produce virus (NPC virus) with both transforming and lytic properties. In this study, NPC-KT and Ramos/NPC cells were examined for the presence of the EBV receptor as measured by the capacity to absorb radio-labelled P3HR-1 and NPC viruses. It was determined that only P3HR-1 virus can attach to NPC-KT cells. Also, the relative concentration of NPC virus receptors on Ramos/NPC cells was found to be significantly reduced when compared to EBV genomenegative Ramos cells, whereas the relative concentration of receptors for P3HR-1 virus was similar to parental Ramos cells. The results suggest that there are differences at least in part of the receptors for P3HR-1 and NPC viruses.     

Effect of n-butyrate on superinfection of Raji lymphoblastoid cell line by Epstein-Barr virus

The effect of n-butyrate on superinfectability of virus-nonproducer Raji cells by the P3HR-1 strain of Epstein-Barr virus (EBV) was investigated. n-Butyrate is known to be a potent inducer of virus antigen synthesis in virus-producer cell lines and of cell differentiation in virus nonproducers. The drug inhibited the growth of Raji cells but did not interfere markedly with cell viability. It induced a low rate of early antigen (EA) synthesis in about 1-2% of noninfected Raji cells. While the number of superinfectable cells remained relatively constant after treatment with butyrate, an increase in antigen positivity was noted in untreated cells. This relative decrease in sensitivity to superinfection in butyrate-treated Raji cells was more pronounced in cultures that had been treated with the drug for 48 or 72 hr as compared to those treated for 24 hr. A blocking of the treated cells in the certain cell-cycle phase and their drug-induced differentiation towards plasma cells might have been involved in the phenomenon described.     

Effect of inhibition of DNA synthesis on Epstein--Barr virus induction by tumor promoters

The two lymphoblastoid lines Raji and P3HR-1, both derived from Burkitt's lymphomas, are readily inducible for EBV antigens by the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA). P3HR-1 cells produce large quantities of EBV particles, whereas induction in Raji cells is exclusively abortive, leading to early antigen (EA) synthesis only. Inhibition of DNA synthesis at the time of induction by cytosine arabinoside (Ara-C), or by pretreating these cells with fluorodeoxyuridine (FdU), provided the following results: Simultaneous treatment of the cells with Ara-C and TPA did not block EA or VCA induction and VCA-positive cells synthesized DNA even in the presence of the inhibitor. FdU pretreatment of the cells for 3 days prior to induction also did not block DNA synthesis completely and VCA was expressed following induction of P3HR-1 cells. Pretreatment of the cells with Ara-C, however, abolished DNA synthesis completely. Even under these conditions TPA induced EA whereas VCA induction was efficiently suppressed. These results show that early events in virus induction do not require cellular DNA synthesis.     

Infection of human epithelial cells by Epstein-Barr virus (EBV). II. Biochemical characterization of EBV-determined proteins synthesized in epithelial cells

Primary cultures of epithelial cells were grown from tonsils of patients with diseases not related to EBV. The cells were implanted with EBV receptors and exposed to EBV of the transforming (B95-8, AG-876) and nontransforming (P3HR-1) strains. The EBV-infected and control cells were pulsed with [35S]methionine at 18-24 h after infection, and cell extracts were prepared for immunoprecipitation with anti-EBV sera and analysis by gel electrophoresis and autoradiography. About 20 EBV-determined proteins ranging from 22 to 185 kDa were detected in P3HR-1 virus-infected epithelial cells. Only a few polypeptides were detected in extracts of cells infected with AG-876 virus while no EBV-specific proteins were immunoprecipitated from extracts of B95-8 virus-infected cells. These results demonstrate that the system of EBV receptor-implanted normal human epithelial cells can be used for direct biochemical analysis of EBV infection in the epithelial tissue.     

Radioimmune precipitation study comparing the Epstein-Barr virus membrane antigens expressed on P3HR-1 virus-superinfected Raji cells to those expressed on cells in a B-95 virus-transformed producer culture activated with tumor-promoting agent (TPA)

The Epstein-Barr virus (EBV)-induced membrane antigen (MA) complex was compared in Raji cells abortively infected with P3HR-1 virus and in a cell line producing transforming EBV. To enhance MA production in the producer cell line, the cells were exposed to 40 ng/ml tumor-promotor agent (TPA) for 72–96 hr. Such treatment increased the percentage of MA-positive cells from 10 to 30–50% over this time period. Both types of cultures were surface labeled by three different but highly sensitive techniques. The cells were then solubilized with Triton X-100 and the radiolabeled EBV-specific membrane proteins were precipitated by sera containing high antibody titers to MA. Analysis of these immune precipitates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified only one antigen of 90,000 molecular weight common to both the superinfected Raji cells and TPA-activated producer culture. In addition, four additional antigens at 300,000, 250,000, and 170,000 molecular weights were found with two of the labeling procedures to be present on the superinfected Raji cells while the TPA-activated producer culture contained only one additional high molecular weight antigen of 320,000. To determine if this difference denoted a possible strain difference or, alternatively, reflected a difference in membrane antigen production between abortive versus permissive infections, TPA-activated cells from the parent culture producing P₃HR-1 virus were also examined. Three of these proteins identified in abortively infected cells were identified in the membranes of this producer culture. The data suggest, therefore, that these differences in membrane components might reflect differences between various EBV strains.     

Expression of Epstein-Barr-virus-associated membrane antigen in Raji cells superinfected with two different virus strains.

Papain treatment of Epstein-Barr virus (EBV; (P3HR-1 strain)-superinfected Raji cells removed the initially adsorbed membrane antigen (MA) positive material from the cell membranes. MA positive cells appeared again after 20 hr in culture, reaching a maximum level at about 30 hr. Puromycin, cycloheximide, and actinomycin D prevented the appearance of MA, whereas cytosine arabinoside had no effect. These results suggest that MA is synthesized de novo by P3HR-1 virus-infected cells and can be detected after 20 hr. The effects of the different metabolic inhibitors are in line with the concept that MA synthesis is an early function of the viral genome. Parallel EA induction tests showed that papain treatment had no effect on the frequency of EA positive cells compared to buffer-treated cells.A comparison between P3HR-1 and B95-8 virus strains with equal EBNA-inducing capacity showed that B95-8 virus was deficient with regard to its ability to induce MA in Raji cells, as judged by direct immunofluorescence.     

Effect of the host cell on the maintenance and replication of Epstein-Barr virus.

We have studied the role the host cell plays in controlling the expression of the Epstein-Barr virus (EBV) by using Burkitt somatic cell hybrids of human and mouse cells. Mouse/Burkitt somatic-cell hybrids were shown to contain a repressed EBV genome that was inducible with iododeoxyuridine. Electron microscopic examination of human/Burkitt hybrid cells (D98/HR-1 and D98/Raji) and Burkitt lymphoblastoid cells in which EBV was replicating showed an enhancement of virus replication concomitant with an enhancement of EBV-specific cytopathologic effect in D98/HR-1 and D98/Raji cells when compared to Burkitt lymphoblastoid cells. When the stability of the EBV genome in human/Burkitt hybrid cells was studied, it was found that the EBV genome in hybrids of the nonproducer Burkitt cells (Raji) was less stable over time than hybrid cells of producer cells (HR-1). The data obtained in this study support the concept that the cell in which the EBV genome resides plays a major role in the maintenance, expression, and replication of EBV.     

Heterogeneity of Epstein-Barr virus. II. Induction of early antigens (EA) by complementation.

Cells of Epstein-Barr virus (EBV)-negative human B lymphoma lines BJA and Ramos were converted into EBV genome carriers by virus isolates from P3HR-1 and B 95-8 cells (Fresen and zur Hausen, 1976). Cloning of P3HR-1 virus-converted BJA cells resulted in clones with two different Epstein-Barr nuclear antigen (EBNA) patterns: a faint granular EBNA staining and clones with a brilliant EBNA expression (Fresen et al., 1977). The latter always segregated EBNA-negative cells from which one EBNA-negative subclone (B1-28) was isolated. Induction of early antigens (EA) was studied by infecting parental lines (BJA and Ramos), converted lines (BJA-HR1K, BJA-B 95-8, Ramos-HR1K, Ramos-B 95-8), the BJA-HR1K clones A5 (faint granular EBNA expression) and B1-19 (brilliant EBNA expression), the EBNA-negative subclone B1-28, and Raji cells with EBV from P3HR-1 and B 95-8 cells, respectively. The following results were obtained: (1) EA induction by P3HR-1 virus is enhanced on the average 14-fold in EBV genome-harboring cells when compared to genome-negative lines. (2) B 95-8 virus induces EA only in P3HR-1 virus-converted cells and to a small extent also in Raji cells. A significant EA induction occurs in the A5 clone of BJA-HR1K, whereas the brilliantly EBNA-expressing B1-19 clone is not induced. B 95-8 virus-converted cells cannot be induced by B 95-8 virus. (3) EA induction following infection of EBV genome-carrying cells is directly proportional to the dilution of the infecting virus. In EBV genome-free cells, EA induction is reduced by the square of the dilution factor. These results imply that resident genomes complement superinfecting genomes in EA induction by EBV and that two different populations of genomes (present in P3HR-1 virus isolates) are required for EA induction following infection of B lymphoblasts.