Human neonatal lymphocytes immortalized after microinjection of Epstein-Barr virus DNA.

Epstein-Barr virus (EBV) is a highly efficient acute transforming agent in human cells, provided that the intact virus is used. To investigate the ability of viral DNA alone to transform cells, we introduced the EBV genome into human lymphocytes. After microinjection of EBV DNA into neonatal B lymphocytes, we established a cell line that in early passages contained multiple viral fragments. This cell line retained sequences from the short, unique (Us) region of the EBV genome and sequences from EcoRI-E. The viral sequences were not expressed; however, the cells expressed a 2.3-kilobase polyadenylated message homologous to the c-fgr oncogene, a cellular locus believed to be activated by EBV infection [M. S. C. Cheah, T. J. Ley, S. R. Tronick, and K. C. Robbins, Nature (London) 319:238-240.]. The cell line was monoclonal with rearrangement at the immunoglobulin locus and had a reciprocal translocation t(1;7)(p34;q34) and a deletion of sequences within the locus for the beta chain of the T-cell receptor. The close proximity of the translocation to the chromosomal loci for c-fgr on chromosome 1 and the T-cell receptor beta chain on chromosome 7 suggests that structural alteration of these genes was critical to this transformation event.     

Latent membrane protein 1 of Epstein-Barr virus plays an important role in the serum starvation resistance of Epstein-Barr virus-immortalized B lymphocytes

We have previously shown that SNU-1103, which is a latency type III Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line (LCL) that was developed from a Korean cancer patient, resists serum starvation-induced G(1) arrest. In this study, we examined the role of latent membrane protein-1 (LMP-1) in serum starvation resistance, since LMP-1 is known to be essential for EBV-mediated immortalization of human B lymphocytes. The LMP-1 gene from SNU-1103 was introduced into the EBV-negative BJAB cell line, and shown to be associated with resistance to G(1) arrest during serum starvation. Western blot analyses of the LMP-1-transfected cells revealed several protein alterations as compared to vector-transfected control cells. The expression of key cell-cycle regulatory proteins was affected in the G(1) phase: the expression of cyclin D3, CDK2, p27, and E2F-4 was up-regulated, and the expression of cyclin D2, CDK6, p21, and p103 was down-regulated during serum starvation. These results imply that of the several EBV viral genes expressed in EBV-negative B lymphoma cells, LMP-1 mediates resistance to serum starvation-induced G(1) arrest. However, we cannot rule out the possibility that other EBV genes are also involved in the cell-cycle progression of the EBV-transformed LCL during serum starvation, since the altered protein expression profile of the LMP-1 transfectants was distinct from that of the SNU-1103 cells that expressed all of the EBV viral proteins.     

Host cell and EBNA-2 regulation of Epstein-Barr virus latent-cycle promoter activity in B lymphocytes.

The six latent-cycle nuclear antigens (EBNAs) of Epstein-Barr virus (EBV), whose genes share 5' leader exons and two promoters (Cp and Wp), are differentially expressed by cells of the B lineage. To examine the possibility that EBNA gene expression is regulated through selective use of Cp and Wp, we monitored the activity of promoter-chloramphenicol acetyltransferase (CAT) gene constructs transfected into EBV-positive and EBV-negative B lymphocytes and Burkitt's lymphoma cells. Wp was a much stronger promoter than Cp in EBV genome-negative B-cell lines and was used exclusively in primary B cells. When B cells were infected with transforming EBV, Cp became the stronger promoter. This switch was not observed when B cells were infected with an immortalization-deficient virus, P3HR-1, which lacks the EBNA-2 open reading frame and expresses a mutant leader protein (EBNA-LP). Cp function was transactivated when EBV-negative or P3HR-1-infected B cells were cotransfected with Cp and a 12-kb fragment of DNA (BamHI-WWYH) that spanned the P3HR-1 deletion. This activity was mapped to the EBNA-2 gene within WWYH; constructs expressing EBNA-LP did not induce Cp function, and the deletion of 405 bp from the EBNA-2 open reading frame abolished transactivation. This research demonstrates host cell and EBNA-2 regulation of latent-cycle promoter activity in B lymphocytes, a mechanism with implications for persistence of EBV-infected lymphoid cells in vivo.     

A role for homologous recombination proteins in cell cycle regulation

Eukaryotic cells respond to DNA breaks, especially double-stranded breaks (DSBs), by activating the DNA damage response (DDR), which encompasses DNA repair and cell cycle checkpoint signaling. The DNA damage signal is transmitted to the checkpoint machinery by a network of specialized DNA damage-recognizing and signal-transducing molecules. However, recent evidence suggests that DNA repair proteins themselves may also directly contribute to the checkpoint control. Here, we investigated the role of homologous recombination (HR) proteins in normal cell cycle regulation in the absence of exogenous DNA damage. For this purpose, we used Chinese Hamster Ovary (CHO) cells expressing the Fluorescent ubiquitination-based cell cycle indicators (Fucci). Systematic siRNA-mediated knockdown of HR genes in these cells demonstrated that the lack of several of these factors alters cell cycle distribution, albeit differentially. The knock-down of MDC1, Rad51 and Brca1 caused the cells to arrest in the G2 phase, suggesting that they may be required for the G2/M transition. In contrast, inhibition of the other HR factors, including several Rad51 paralogs and Rad50, led to the arrest in the G1/G0 phase. Moreover, reduced expression of Rad51B, Rad51C, CtIP and Rad50 induced entry into a quiescent G0-like phase. In conclusion, the lack of many HR factors may lead to cell cycle checkpoint activation, even in the absence of exogenous DNA damage, indicating that these proteins may play an essential role both in DNA repair and checkpoint signaling.     

Studies on host-virus genome relationship in Epstein-Barr virus immortalized lymphoblastoid cell lines

Five human lymphoblastoid cell lines immortalized in vitro with the B95-8 EBV strain, chosen to have a low number of copies of EBV genome, were examined to detect variations in electrophoretic mobility of viral restriction fragments and in the karyotype. Patterns of mobility detected with different viral probes are always the same as those obtained with fragments from purified virus-plasmidic DNA, with one exception. This "non-plasmidic" pattern occurs with a probe containing the termini of the linear virion DNA and consists in an increase of the molecular weight and in the appearance of more than one band. Cytogenetic studies carried on the same cell populations used as source of DNA, early after immortalization, showed a diploid modal chromosome number and no G banding rearrangements.     

A bicistronic Epstein-Barr virus mRNA encodes two nuclear proteins in latently infected, growth-transformed lymphocytes.

EBNA2 is a nuclear protein expressed in all cells latently infected with and growth transformed by Epstein-Barr virus (EBV) infection (K. Hennessy and E. Kieff, Science 227:1230-1240, 1985). The nucleotide sequence of the EBNA2 mRNA (J. Sample, M. Hummel, D. Braun, M. Birkenbach, and E. Kieff, Proc. Natl. Acad. Sci. USA 83:5096-5100, 1986) revealed that it begins with a 924-base open reading frame that has an unusual potential translational initiation site (CAAATGG). This open reading frame is followed by 138 nucleotides with only one highly unlikely translational initiation site (TACATGC), which would translate a pentapeptide before the next stop codon. The last part of the mRNA is the open reading frame which encodes EBNA2. In this paper, we demonstrate that the 924-base open reading frame translates a 40-kilodalton protein in vitro or in murine cells transfected with the EBNA2 cDNA under control of the murine leukemia virus long terminal repeat. A protein of identical size was detected in EBV-transformed, latently infected human lymphocyte nuclei by using antibody specific for the leader open reading frame expressed in bacteria. Therefore, this is a rare example of a mRNA which translates two proteins from nonoverlapping open reading frames. Since the protein encoded by the leader of the EBNA mRNA is expressed in all nuclei of a latently infected cell line, it was designated EBNA-LP. EBNA-LP localizes to small intranuclear particles and differs in this respect from EBNA1, EBNA2, or EBNA3. EBNA-LP is not expressed in an EBV-transformed marmoset lymphocyte cell (B95-8) or in one EBV-infected Burkitt tumor cell line (Raji) but is expressed in three other Burkitt tumor cell lines (Namalwa, P3HR-1, and Daudi).     

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.     

Measles virus infection of B lymphocytes permits cellular activation but blocks progression through the cell cycle.

Measles virus infection of unstimulated B lymphocytes suppresses both proliferation and differentiation into immunoglobulin-secreting cells. However, mitogenic stimulation of these infected cells results in cell volume enlargement, rapid RNA synthesis, and the expression of cell surface activation antigens 4F2, HLA-DS, and transferrin receptor. The cellular genes c-myc and histone 2B are induced during early G1 and S phase of the cell cycle, respectively, and viral RNA synthesis can be detected during this interval. However, total RNA synthesis is decreased at 48 h after stimulation, and the histone 2B RNA steady-state level at 48 h is fivefold less than that in uninfected cells. This sequence of events defines an arrest in the G1 phase of the cell cycle in measles virus-infected B cells.     

Human plasma accelerates immortalization of B lymphocytes by Epstein-Barr virus

Abstract.   Objective : Serum and plasma contain species-specific factors that modulate cell population growth and function, and that are required for proliferation of most cell cultures. Foetal calf serum (FCS) is the most common source of these growth factors. We studied the effect of human plasma (HP) on the immortalization process of B lymphocytes by Epstein–Barr virus (EBV) was studied. Materials and methods : The effect of HP as compared to FCS was done through assessment of cell proliferation. Results : It was found that HP (autologous and non-autologous plasma) is more effective than FCS in generating lymphoblastoid cell lines, regardless of EBV status of the donors: 65% of HP-supplemented cultures developed into lymphoblastoid cell lines by 7–14 culture days, as compared to 16% of cultures with FCS. In addition, 6% of HP-supplemented cultures did not achieve becoming lymphoblastoid cell lines by day 35 in comparison to 94% of cultures with FCS. The higher proliferative effect of HP was not altered by heat inactivation or filtration. HP maintained its proliferative activity at 4 °C over 8 months, thus indicating that HP contains a stable growth factor(s), which accelerates B-lymphocyte immortalization. Conclusion : The results support other studies that recommend the use of autologous plasma for tissue culture, mainly in the case of autologous transplantation. Furthermore, the use of HP allows preparation of lymphoblastoid cell lines from a small amount of peripheral blood in a shorter period of time and with a higher rate of success.     

Antibodies binding granulocyte-macrophage colony stimulating factor produced by cord blood-derived B cell lines immortalized by Epstein-Barr virus in vitro

We detected natural antibodies (auto-Abs) binding human granulocyte-macrophage colony stimulating factor (GM-CSF) in umbilical cord blood (CB) (23 of 94 samples screened) and peripheral blood of women at the end of pregnancy (6 of 42 samples tested). To demonstrate that Abs detected in CB were produced by the fetus, CB mononuclear cells were infected with Epstein-Barr virus in vitro. Ten cell lines producing constitutively anti-recombinant human GM-CSF (rhGM-CSF) Abs were isolated and characterized. These cells displayed a male karyotype, an early activated B cell phenotype, coexpressed surface IgM and IgD, and secreted only IgM with prevailing lambda clonal restriction. Specific cell surface binding of biotinylated rhGM-CSF and high-level anti-rhGM-CSF IgM Ab production were typical features of early cell cultures. In late cell passages the frequency of more undifferentiated B cells increased. Serum Abs of either maternal or fetal origin or Abs produced in culture did not affect the granulocyte and macrophage colony stimulating activity of rhGM-CSF from bone marrow progenitors in soft agar, suggesting that the Abs produced were nonneutralizing.     

Epstein-Barr virus DNA is amplified in transformed lymphocytes.

Leukocytes isolated from two adult donors who lacked detectable antibodies to antigens associated with Epstein-Barr virus were exposed to an average of 0.02 to 0.1 DNA-containing particles of Epstein-Barr virus per cell and immediately clones in agarose. Within about 30 generations all transformed cell clones contained between 5 and 800 copies of viral DNA per cell. Only 1 in 10(4) to less than 1 in 10(5) of the cells of each clone release virus, and the frequency of release did not correlate with the average number of copies of viral DNA in the cells of each clone. One clone that had an average of five copies of viral DNA per cell was recloned, and the average number of copies in four of six subclones increased 15-to 50-fold while the subclones were being propagated sufficiently to study them. These results indicate that Epstein-Barr virus DNA can undergo amplification relative to cell DNA at different times after it transforms cells.     

An efficient method for the rapid establishment of Epstein-Barr virus immortalization of human B lymphocytes

Abstract. Several methods have been developed for the immortalization of B lymphocytes by Epstein-Barr virus (EBV). We developed an efficient method which reduces the time from culture initiation to immortalization and cryopreservation. Two infections of EBV to lymphocytes, and the use of phorbol ester-induced EBV stock significantly improved immortalization efficiency and reduced the time between initiation and immortalization and cryopreservation. The resulting cell bank was used to produce DNA for genetic studies focusing on the genes involved in immune and autistic disorders.     

A novel interleukin-12 p40-related protein induced by latent Epstein-Barr virus infection in B lymphocytes.

We have isolated a cDNA encoding a novel hematopoietin receptor family member related to the p40 subunit of interleukin-12 and to the ciliary neurotrophic factor receptor, whose expression is induced in B lymphocytes by Epstein-Barr virus (EBV) infection. This gene, which we have designated EBV-induced gene 3 (EBI3), encodes a 34-kDa glycoprotein which lacks a membrane-anchoring motif and is secreted. Despite the absence of a membrane-anchoring motif and of cysteines likely to mediate covalent linkage to an integral membrane protein, EBI3 is also present on the plasma membrane of EBV-transformed B lymphocytes and of transfected cells. Most newly synthesized EBI3 is retained in the endoplasmic reticulum in an endoglycosidase H-sensitive form associated with the molecular chaperone calnexin and with a novel 60-kDa protein. EBI3 is expressed in vivo by scattered cells in interfollicular zones of tonsil tissue, by cells associated with sinusoids in perifollicular areas of spleen tissue, and at very high levels by placental syncytiotrophoblasts. EBI3 expression in vitro is induced in EBV-negative cell lines by expression of the EBV latent infection membrane protein-1 and in peripheral blood mononuclear cells by pokeweed mitogen stimulation. EBI3 maps to chromosome 19p13.2/3, near genes encoding the erythropoietin receptor and the cytokine receptor-associated kinase, Tyk2. EBI3 synthesis by trophoblasts and by EBV-transformed cells and similarities to interleukin-12 p40 are compatible with a role for EBI3 in regulating cell-mediated immune responses.     

Pathways of activation of the Epstein-Barr virus productive cycle.

The promoter for the 2.8-kb RNA of Epstein-Barr virus encoding BZLF1 and BRLF1 was identified and shown to be activated by both BZLF1 and BRLF1 but not by 12-O-tetradecanoylphorbol-13-acetate. Site-directed mutagenesis suggests that two binding sites for BZLF1 within the promoter contribute to the transactivation by BZLF1. The early kinetics of induction of the 2.8- and 1.0-kb RNAs encoding BZLF1 and BRLF1 in Akata cells treated with anti-immunoglobulin indicate that both RNAs appear within 60 min. The results indicate some likely pathways of activation of Epstein-Barr virus productive cycle gene expression.