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.     

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.     

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.     

Effects of n-Butyrate and phorbol ester (TPA) on induction of Epstein-Barr virus antigens and cell differentiation

Summary N-Butyrate, an effective inducer of synthesis of Epstein-Barr virus (EBV) antigens in virus-producer P3HR-1 cells, has recently been shown (2) to induce morphological differentiation towards plasma cell in nonproducer Raji cells. The effects of n-butyrate and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on both EBV-antigen induction and cell differentiation in two virus-nonproducer lymphoblastoid cell lines, Raji and NC37, were now studied. The following observations were made (1). On its own either drug induced 1–2 per cent of cells to EBV-early-antigen positivity in both lines; their mixture induced 35 and 15 per cent positive cells in Raji and NC37 respectively (2). In Raji, n-butyrate induced about 80 per cent of cells to differentiate to plasmablast or plasma cell morphology, whereas TPA only induced the early stages of differentiation in 8 per cent of cells; a mixture of both inducers produced a similar effect as TPA alone. The addition of TPA alone or butyrate-TPA mixture led to some cellular alterations resembling virus-specific changes in virus-producer cell lines. In NC37, either drug alone or their mixture drove 13 per cent of cells to differentiate into plasmablasts or earlier stages of differentiation. In the presence of TPA protrusions and loops were seen on cell surfaces.Evidently, the stage of differentiation at which B-lymphoblastoid cell lines have been arrested can be changedin vitro. However, cell-line dependent and inducer-dependent differences in the differentiation response were apparent.With 5 Figures     

Pattern of Epstein-Barr virus (EBV)-specific proteins synthesized during primary lytic infection of mouse lymphocytes by EBV

Normal mouse lymphocytes were implanted with EBV receptors and exposed to the virus of P3HR-1 strain. 5% of the cells expressed early (EA) and viral capsid (VCA) antigens as assayed by immunofluorescence 24 h after the infection. Only 0.1% of cells expressed nuclear-like antigen (EBNA) 48 h post-infection. When labelled metabolically with [35S]methionine, extracted, immunoprecipitated with EBV-positive sera, and analyzed by SDS-gel electrophoresis and autoradiography, about 20 EBV-determined proteins ranging from 19 to 165 kd were detected. Their pattern and relative quantitative expression differed from those in P3HR-1 virus superinfected Raji cells. Polypeptides of approximate molecular size 78, 72, 65, 48 and 26.5 kd were predominant in EBV-infected mouse lymphocytes. In contrast, 130, 98, 59, 50.5 and 36 kd proteins were predominant in the induced Raji cells. Our results demonstrate that rodent lymphocytes can be used for the direct biochemical analysis of EBV-translational products during primary lytic infection in normal 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.     

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.     

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.     

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.     

Differential effect of phosphonoacetic acid on early antigen synthesis in two Epstein-Barr virus producer cell lines.

The effect of phosphonoacetic acid (PAA) on the expression of Epstein-Barr virus (EBV)-determined antigens in producer (P3HR-1 and B95-8) lymphoblastoid cell lines (LCLs) and iododeoxyuridine (IUDR)-treated nonproducer cell lines (Raji and F137) was investigated. PAA effectively inhibited spontaneous early antigen (EA) synthesis in the P3HR-1 LCL only, while it had no effect on EA production in the B95-8 cell line nor on the IUDR-induced EA in nonproducer LCLs. The inhibition of spontaneous EA in P3HR-1 cell was dose and time dependant, and removal of PAA from cultures and subsequent propagation of these cells in PAA-free medium did not restore spontaneous EA production. The data reported show that EA is synthesized by at least two mechanisms in P3HR-1 cells, one of which is sensitive to PAA, thus suggesting that viral DNA synthesis is required for spontaneous EA synthesis only in the P3HR-1 cells; however, in these same cells, viral DNA synthesis would not be required for the IUDR-induced EA production.     

Induction of replication of Epstein-Barr virus DNA by 12-O-tetradecanoyl-phorbol-13-acetate. II. Inhibition by retinoic acid and 9-(2-hydroxyethoxymethyl) guanine

An Epstein-Barr virus (EBV)-producing cell line (P3HR-1) is readily inducible for EBV DNA replication by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The effects of an antiherpesvirus drug, Acyclovir (ACV) [9-(2-hydroxyethoxymethyl)-guanine], and a tumor promoter antagonist, retinoic acid (RA), were tested on EBV DNA replication in P3HR-1 cells. The activation of P3HR-1 cells by TPA greatly stimulates [³H]thymidine incorporation into the viral DNA as determined by analysis on cesium chloride gradients. This effect is completely inhibited by RA. However, RA has no inhibitory effect on the spontaneously induced viral DNA replication that occurs in P3HR-1 cells not treated with TPA. In contrast, ACV inhibits both the spontaneously induced and TPA-induced EBV DNA replication. The discriminatory effect of ACV and RA on viral DNA replication in P3HR-1 cells is further demonstrated by cRNA-DNA hybridization with EBV-specific cRNA.     

Epstein-Barr virus-induced early polypeptides in Raji and NC37 cells activated by diterpene ester TPA in combination with N-butyrate

Epstein-Barr virus (EBV)-induced early polypeptides in activated nonproducer Raji and NC37 cells, and such polypeptides induced in producer P3HR-1 cells were studied for comparison. The major difference was that the two polypeptides with molecular weights of 140,000 (140K) and 120,000 (120K) which are presumably involved in viral DNA synthesis in the activated producer cells, were not detectable in the activated nonproducer cells.     

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.     

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.     

Multiplicity-dependent induction of viral capsid antigen in Raji cells superinfected with Epstein-Barr virus

A quantitative analysis of Epstein-Barr virus (EBV)-induced early antigen (EA) and viral capsid antigen (VCA) syntheses was carried out in Raji cells superinfected with purified, concentrated P3HR-1 EBV. When the cells were exposed to the virus and assessed by immunofluorescence and immunoprecipitation, EA induction occurred significantly (17%) but not VCA (less than 1%), at a low-input multiplicity of infection (MOI) of 10 EBV DNA copies/cell. In contrast, at a high MOI of 500 EBV DNA copies/cell, the majority of cells were positive for both EA (82%) and VCA (61%). The latter VCA synthesis was accompanied by the replication of EBV DNA. Kinetic studies showed that EA induction was directly proportional to the dilution of the infecting virus, while VCA was made following three-hit kinetics. The implications of these results are discussed in relation to the heterogeneous nature of P3HR-1 EBV and a possible role of EA in VCA synthesis.     

Expression of altered ribonucleotide reductase activity associated with the replication of the Epstein-Barr virus.

The expression of the Epstein-Barr virus (EBV) genome can be regulated in the epithelial/Burkitt hybrid (D98/HR-1) cell line and Raji lymphoblastoid nonproducer cell line by induction with 5-iododeoxyuridine (IUdR) or by superinfection with EBV. Extracts of control and induced D98/HR-1 and Raji cells were assayed for ribonucleotide reductase activity in the presence and absence of hydroxyurea (HU). Enzyme activity of control D98/HR-1 and Raji cells was inhibited by greater than 70%, by HU at both low (2 × 10^?4 M) bot high (5 × 10^?4 M) concentrations; however, the reductase activities of IUdR-induced D98/HR-1 cells, superinfected Raji cells, and IUdR-induced Raji cells were resistant to both levels of HU, and enzyme activities of 85 to +100% of control values were obtained in all cases. Under conditions that allowed only partial expression of the EBV genome (before removal of IUdR; early time after superinfection), very significant levels (70–85°k) of HU-resistant enzyme activity were obtained in the presence of 2 × 10^?4 M HU, whereas only 30 to 45% of control reductase activity was observed at the high HU concentration. Mixing experiments employing combinations of various D98/HR-1 control and induced cell extracts indicated that the HU-resistant reductase activity present in induced 1)98/HR-1 cells was due to the presence of an altered enzyme activity and not due to some nonenzymatic factor(s). Additional experiments, in which HU was preincubated with control and induced cell extracts, showed that the HU-resistant ribonucleotide reductase in extracts of induced D98/HR-1 cells was not due to inactivation of the inhibitor by an enzyme or factor present in these cells. These findings of an altered ribonucleotide reductase activity associated with EBV replication, considered in light of similar findings for equine herpesvirus type 1 and herpes simplex virus types 1 and 2, suggest that alteration of this enzyme activity may be a feature of herpesvirus replication.     

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.