Persistent herpes simplex virus infections established in two Burkitt lymphoma derived cell lines.

Examination of P3HR-I cells (Epstein-Barr virus [EBV] producer) persistently infected with the MAL strain of herpes simplex virus type I (HSV-I) suggested that only a few cells were actively producing a virus indistinguishable from HSV-I (MAL) despite the presence of immunofluorescent HSV-I antigens associated with the majority of cells. EBV-specific immunofluorescence was not altered in HSV-I persistently infected P3HR-I cells. HSV-I persistently infected cells, labelled for 72 h with 14C-thymidine, incorporated approx. 8% of the label into cell associated HSV-I DNA as resolved by caesium chloride gradients. Values greater than 8% of the total were suggested by hybridization of gradient fractions with 3H-HSV-I DNA. To determine whether the establishment of HSV persistent infections in Burkitt lymphoma derived cells was a general phenomenon, six strains of HSV-I (MAL, KOS, Patton, Syn R, BF and SYN V) and two strains of type 2 (333 and MS) were used to infect the P3HR-I and Raji (EBV non-producer) cell lines derived from Burkitt lymphomas. In P3HR-I cells, persistent infections were established with all strains of HSV-I but not with HSV-2. In Raji cells, persistent infections were established with all strains of HSV-I, except Syn V, and with both strains of HSV-2. No external support was required to maintain these infections.     

Analysis of restriction endonuclease fragments of intracellular Epstein-Barr virus DNA and isoenzymes indicate a common origin of the Raji, NC-37, and F-265 human lymphoid cell lines

Circular Epstein-Barr virus DNA molecules isolated from different virus-transformed cell lines usually show similar but distinct restriction enzyme cleavage patterns. It is shown here that EBV DNA from the cell line Raji, which is of African Burkitt lymphoma origin, has the same cleavage pattern on digestion with either EcoRI, HindIII, or BamHI as EBV DNA from two more recently established human lymphoid cell lines, NC-37 and F-265, which are presumed to be of nontumor origin. On the other hand, these three EBV DNA isolates are clearly different from DNA of the standard B95-8 strain of the virus as judged by the same criteria. A restriction enzyme cleavage map of the circular EBV DNA from Raji cells has been made to provide a basis for the comparison. The isoenzyme patterns of 14 different marker enzymes were also investigated for the Raji, NC-37, and F-265 lines and found to be closely similar. These data indicate that the NC-37 and F-265 lines are not independent isolates of human lymphoid cell lines.     

Identification of a novel EBV-induced membrane glycoprotein of 43 kDa with H667 MAb.

Using purified B95-8 Epstein-Barr virus (EBV), a MAb designated H667 was produced. We demonstrated by indirect membrane immunofluorescence (IF) on six EBV producer cell lines and by immunoelectron microscopy that H667 reacted with a membrane antigen. H667 recognized a 43-kDa EBV protein (p43) as determined by immunoblotting using purified EBV from the six producer cell lines. Phosphonoacetic acid treatment of B95-8 cells was associated with the disappearance of p43, indicating that it was a late antigen. This antigen was shown to be a glycoprotein by incorporation of [14C]glucosamine and was shown to contain an N-asparagine-linked glycosyl group by its sensitivity to tunicamycin. It was named gp43. The H667 MAb inhibited B95-8 EBV cord blood lymphocyte transformation only when a low inoculum was used but failed to inhibit EA induction in Raji cells by P3HR1 EBV. Human sera reactivity against the gp43 antigen was studied. By the immunoblotting method, using H667 immunoaffinity chromatography-purified gp43, we showed that 70.9% of the human sera tested had antibodies directed against gp43. By IF blocking tests, we found that only 12.5% of the sera tested were reactive, indicating that the epitope corresponding to the H667 MAb was not the most immunogenic gp43 epitope.     

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.     

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.     

Phenotypic and genotypic characteristics of new euploid-diploid lymphoblastoid B cell lines EBV+, normal human bone marrow derived, spontaneously overgrown in vitro

The present study describes the phenotypic and genotypic features of seven individual growth transformed, euploid-diploid EBV+ human B cell lines arisen spontaneously in vitro. The lines, obtained under general and standard culture conditions (un-manipulated), from seven individual bone marrow samples of 18 healthy young adults, Caucasian, of both sexes, display many traits of normal B cells and represent a mixture of EBV infected latently (latency type III) and producer cells (5-16% VCA+ by immunofluorescence) releasing seven individual different viral strains [Fruscalzo et al., 2001. DNA sequence heterogeneity within the Epstein-Barr virus family of repeats in the latent origin of replication. Gene 265, 165-173] similar to the B95-8 genotype as shown by results of Southern blot of BamHI-digested DNA fragment. These tests were planned to characterize more fully this panel of new bone marrow cell lines sharing normal B cell traits.     

Effect of the diterpene ester TPA on Epstein-Barr virus antigen- and DNA synthesis in producer and nonproducer cell lines

Diterpene esters like TPA (12-O-tetradecanoylphorbol-13-acetate) which are effective tumor promoters in mice, induce the synthesis of Epstein-Barr virus capsid antigen (VCA) in producer lines and of the early antigen complex (EA) in nonproducer lines. In three lymphoblastoid lines which produce nontransforming (P3HR-1) or transforming virus (B95-8, QIMR-WIL) and in two nonproducer lines (Raji, NC 37) the role of TPA on viral DNA synthesis was studied. At different times after addition of TPA the percentage of VCA- and EA-positive cells was determined and the amount of viral DNA per cell estimated by measuring the reassociation kinetics of labeled EV DNA in the presence of cellular DNA. In virus-producing lines the number of VCA-positive cells increased about 3- to 5-fold from 7–10% to 30–40%, whereas the amount of viral DNA per cell increased 10- to 20-fold. Thus, TPA not only increases the number of viral antigen-producing cells, but also enhances viral DNA replication. In nonproducer lines the amount of EBV DNA per cell was constant despite efficient EA induction by TPA. This indicates that the induction of EA is not linked to the replication of viral DNA.     

Activation of B lymphocytes by Epstein-Barr virus/CR2 receptor interaction

Epstein-Barr virus (EBV) infects and transforms human B lymphocytes. The virus receptor was shown to be identical to the complement receptor CR2. The consequences of EBV/CR2 receptor interaction on B lymphocyte activation were analyzed by infection of B cells with the transforming (B95-8) and the nontransforming (P3HR1) virus strains and with UV-inactivated B95-8 virus. Similar to mitogens and antibodies against surface IgM and CR2 receptor, transforming and nontransforming EBV induced the release of leukocyte migration inhibitory factor (LIF). LIF production occurred early after infection and was not affected by irradiation of the B95-8 virus with doses of UV-light which prevented the expression of viral functions. B lymphocytes infected with UV-inactivated virus responded to T cell-derived B cell growth factors. The results demonstrate that binding of EBV to the CR2 receptor activates the B cells. Progression of the infected cells in the activation pathway required helper cell-derived factors or signals provided by the intact viral genome. The nontransforming P3HR1 virus induced a low level of RNA synthesis and increase of cell size and major histocompatibility complex class I antigen expression. Only the transforming virus induced expression of EBV nuclear antigens and cellular DNA synthesis.     

Copy number and location of Epstein-Barr Viral genomes in neonatal human lymphocytes transformed after separation by size and treatment with mitogens

We wished to learn whether genome amplification was a regular event which followed in vitro immortalization by Epstein-Barr virus. We also explored the hypothesis that the physiologic state of neonatal human lymphocytes, at the time of exposure to EB virus, influences the number or the physical state of the viral genomes in the resultant transformed cells. Purified B lymphocytes from four fresh umbilical cords were fractionated by velocity sedimentation on continuous Ficoll-Hypaque gradients into large and small cells. Part of each size fraction pool was treated either with lipopolysaccharide (LPS) from Escherichia coli (E. coli) or with a mitogen derived from a bacteriophage lysate of Staphylococcus aureus (SPL); the remainder was untreated. The frequency of transformation was invariably higher in the small cell fraction than in the large cells. LPS treated cells were 1.6-fold more sensitive to transformation, while SPL-treated cells did not transform at a higher frequency. A series of 41 cell lines established following infection with very low multiplicities of the B95-8 strain of EBV (10^?3 to 10^?5) were examined for the amount and physical state of the EBV DNA. High-molecular-weight DNA from the established cell lines was fractionated on neutral CsCl density gradients and the viral DNA sequences were localized by nucleic acid hybridization. The median number was approximately 32 EBV DNA copies per cell; this estimate was not detectably influenced by cell size or treatment with LPS or SPL before viral infection. Moreover, the physical state of the EBV DNA was similar among the different cell lines. Most of the viral DNA occurred as nonintegrated, covalently closed circular DNA. In addition, viral sequences with properties of integrated DNA were present, and different cell lines contained different relative amounts of such sequences.     

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.     

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.     

Analysis of early and late Epstein-Barr virus associated polypeptides by immunoprecipitation.

The Epstein-Barr virus-producing cell lines P3HR-1 and B95-8 and the nonproducer cell lines Raji clone No. 7 and NC37 were induced to viral antigen synthesis by the tumor promoter TPA and then analyzed by immunoprecipitation with human sera for early and late virus-associated polypeptides. After labeling of producer cells for a 4-day period with [³⁵S]methionine, two polypeptides with molecular weights of 140,000 and 150,000 were identified reacting predominately with virus capsid antigen (VCA⁺) sera. Analysis of purified Epstein-Barr virus demonstrated that the 140,000 polypeptide presumably represents an envelope protein while the 150,000 polypeptide is a nucleocapsid protein. In 4-hr radioactively labeled producer cells an additional polypeptide with a molecular weight of 130,000 was found to be immunoreactive with VCA⁺ sera. Immunoprecipitation of [³⁵S]methionine-labeled cell extracts from nonproducer cells resulted in the specific precipitation of two polypeptides with molecular weights of 85,000 and 35,000 which most likely represent early EBV-associated proteins. Producer cells exhibit three additional apparently early EBV-associated polypeptides with molecular weights of 120,000, 18,000, and 16,000. None of these polypeptides could be detected in EBV genome-negative Ramos cells after TPA treatment.     

An EBNA-negative, EBV-genome-positive human lymphoblast cell line in which superinfecting EBV DNA is not maintained

A human B-lymphoid cell line, designated TG8, which does not express detectable levels of the EBV (Epstein-Barr virus)-associated nuclear antigen (EBNA), yet carries an average of one to two plasmid copies of the P3HR-1 EBV genome has been identified. TG8 can be superinfected by B95-8 EBV, resulting in up to 60-70% of the population becoming EBNA-positive and 20-30% of the incoming EBV genomes becoming circular by 48 hr postinfection. Neither EBNA expression nor the superinfecting viral DNA is maintained in the population. It is concluded that (1) superinfection of this EBV-genome-positive lymphoblast cell line leads to detectable EBNA expression and circularization of the incoming viral genome and (2) the incoming viral genome and detectable EBNA expression are selectively lost, whereas the endogenous viral plasmid DNA is maintained.     

Complementation of adenovirus early region 1a and 2a mutants by Epstein-Barr virus immortalized lymphoblastoid cell lines.

Human B-lymphocytes may be infected by both adenoviruses and the Epstein-Barr virus (EBV). Some of the immediate early and early proteins in the two viruses are similar in function even though their primary structures are different. As these viruses might infect the same B-cells in man, we asked if complementation could take place. The adenovirus mutant H5ts125 has a thermolabile DNA-binding protein and is defective in DNA replication at 39 degrees. Several EBV-transformed human lymphoblastoid cell lines and a tamarin cell line B95-8 were infected with H5ts125 and incubated at either the nonpermissive or the permissive temperatures. Adenoviral DNA replication and assembly of new virions were observed at both temperatures, suggesting complementation by the resident EBV gene products. The adenovirus E1a region is deleted in the mutant d1312. Complementation of this mutant was only obtained in the EBV producer B95-8 cells. Immortalization by EBV was apparently not sufficient for effective complementation. This supports an earlier observation that one of the EBV early proteins (MS-EA) behaves like adenovirus E1a and can transactivate the E4 promoter in a CAT assay. The complementation of mutant adenoviruses in EBV-transformed lymphocytes may help the rescue of new adenovirus serotypes in immunosuppressed patients.     

Characterization of an EBV-like virus from African green monkey lymphoblasts

A lymphoblastoid cell line (AGM-2206) has been established from the peripheral blood of an African green monkey. Approximately 1% of these cells showed fluorescence for EBV capsid antigens (VCA), when examined with human EBV-VCA-positive sera by indirect immunofluorescence. Titration of human and monkey sera on AGM-2206 and P3HR-1 cells revealed a partial cross-reactivity between capsid antigens of the AGM virus and human EBV. In addition, cross-reactivity within the EA complex is suggested by reactivity of about 20% of monkey sera with human EBV-EA. Neither human nor monkey sera reacted with an EBNA-like antigen in AGM lymphoblasts. Cleavage patterns of AGM-EBV DNA with EcoRI and HindIII restriction endonucleases showed differences when compared to human EBV DNA isolated from B 95-8, P3HR-1, and QIMR-WIL cells. Hybridization of labeled EBV DNA from B 95-8 cells to fragments of AGM-EBV DNA blotted onto nitrocellulose filters resulted in some hybridization to a number of AGM-EBV DNA bands.     

Characterization of an Epstein-Barr virus nuclear antigen 2 variant (EBNA 2B) by specific sera

The Jijoye Epstein-Barr virus (EBV) strain is characterized by a substitution of 1.8 kb in the C-terminal part of EBNA 2 gene compared to B95-8 or M-ABA virus. Protein immunoblot analysis using human sera against EBNA 2 indicated that an immunological variant to the EBNA 2 of B95-8 (type A) is encoded by the Jijoye virus (type B). In order to generate a specific EBNA 2B antiserum the NaeI/NsiI DNA fragment of the Jijoye virus containing 237 bp of the C-terminus from the EBNA 2B gene was cloned in an E. coli expression vector (pME3). The resulting fusion protein contained 79 C-terminal amino acids of the viral protein and a 37,000 Da part of the bacterial anthranilate synthase. Rabbit antisera generated against this fusion protein reacted specifically with two proteins of 73,000 and 77,000 Da from Jijoye cells and three other cell lines carrying type B virus, while no proteins could be identified in the type B cell line BL 29. In addition, using these sera directed against the pME3 fusion protein, no reaction could be observed with the EBNA 2A protein from the B95-8 and several other cell lines containing type A virus.     

Production of human monoclonal antibodies against Epstein-Barr virus-specific antigens by the virus-immortalized lymphoblastoid cell lines

The possible production of human monoclonal antibodies against Epstein-Barr virus (EBV) was assessed through the EBV immortalization technique. When individual lymphocyte samples from 50 clinical patients and healthy donors were immortalized by EBV, 4 lymphoblastoid lines yielded antibodies to EBV antigens. These positive lines were cloned and each line yielded cultures that secreted monoclonal antibodies against either viral capsid antigen (VCA) or membrane antigen (MA) component. Above all, a clonal line TAKA-SP-8 produced 5 micrograms MA antibody/10(6) cells/ml for more than 12 months. The culture fluid specifically immunoprecipitated a single polypeptide with a size of 93K from both P3HR-1 and B95-8 cell extracts. FUKA-SP-3, on the other hand, secreted 5 micrograms VCA antibody/10(6) cells/ml for at least 8 months. This antibody recognized two polypeptides with sizes of 123K and 120K, from P3HR-1 and B95-8 cell extracts, respectively. When B95-8 and P3HR-1 EBV were treated with the human MA monoclonal, both nuclear antigen (EBNA) synthesis and early antigen (EA) induction were strongly inhibited. All EBV antibody-producing cultures were exclusively achieved from splenic lymphocytes of patients with autoimmune diseases, but not from other donors.     

Real-time quantitative PCR for assessment of antiviral drug effects against Epstein-Barr virus replication and EBV late mRNA expression

This study assesses the ability of quantitative real-time PCR to measure the effects of virus DNA polymerase inhibitors on EBV DNA and late mRNAs syntheses in EBV-producing cell lines. In-house real-time quantitative PCRs were used to measure EBV DNA (thymidine kinase) and mRNAs (BLLF1 gene/gp350/220, BVRF2 gene/protease) in P3HR-1 and B95-8 cells induced for EBV production by PMA and exposed to ganciclovir, cidofovir and foscarnet. The calculated 50% effective concentrations (EC(50)) for viral DNA replication inhibition in P3HR-1 cells after 7 days of drug exposure were 0.28+/-0.06, 0.29+/-0.01 and 13.6+/-0.17 microg/mL for ganciclovir, cidofovir and foscarnet, respectively. The EC(50) for B95-8 cells were 0.44+/-0.02, 0.70+/-0.06 and 46.8+/-0.5 microg/mL, respectively. The quantitation of the late viral mRNAs showed a decrease of 79-89% in the mRNA amount after 4 days of antiviral treatment. Nevertheless, a substantial amount of mRNA still remained detectable after drug exposure. The real-time PCR is an improvement in the attempt to simplify EBV DNA-quantitation for antiviral assays. The quantitation of late mRNA does not appear as more informative than DNA quantitation for the assessment of the DNA polymerase inhibitor activity, but it may be useful to assess the antiviral activity of drugs acting by another mechanism.     

Attempts to induce interferon production by IdUrd induction and EBV superinfection in human lymphoma lines and their hybrids

Inducers of the Epstein-Barr virus (EBV) cycle, 5-iodo-2-deoxyuridine (IdUrd), phorbol myristate acetate (TPA) and sodium butyrate were tested for their ability to induce EBV-determined antigens, early antigen (EA) and virus capsid antigen (VCA), and to stimulate interferon (IF) production in a variety of EBV-carrying lymphoid cell lines. IdUrd and TPA induced IF production to various extents in the different lines, whereas sodium butyrate did not. There was no relationship between induction of the EBV cycle and production of IF; the two appear to be independent characteristics. Superinfection with the transforming B95-8 virus substrain of EBV induced IF production, whereas superinfection with the abortively cytopathic, non-transforming P3HR-1 substrain had little or no IF-inducing effect, in spite of its highly potent effect on virus antigen (particularly EA) synthesis. Analysis with specific antisera against IF showed that IF preparations produced by three different lymphoid cell lines in response to IdUrd treatment were composed of a mixture of the Le and F antigenic types, with the latter forming a minor species. In contrast, no detectable F interferon was present in spontaneously produced IF preparations from Namalwa cells, or after induction with B95-8 virus.