Epstein-Barr virus nuclear protein EBNA3C is required for cell cycle progression and growth maintenance of lymphoblastoid cells

Epstein-Barr virus (EBV) infection converts primary human B cells into continuously proliferating lymphoblastoid cell lines (LCLs). To examine the role of EBV nuclear antigen (EBNA) 3C in the proliferation of LCLs, we established LCLs infected with an EBV recombinant that expresses EBNA3C with a C-terminal fusion to a 4-hydroxytamoxifen (4HT)-dependent mutant estrogen receptor, E3C-HT. In the presence of 4HT, LCLs expressed the E3C-HT protein and grew like WT LCLs. When E3C-HT EBV-infected LCLs were transferred to medium without 4HT, E3C-HT protein slowly disappeared, and the LCLs gradually ceased growing. WT EBNA3C expression from an oriP plasmid transfected into E3C-HT LCLs protected the LCLs from growth arrest in medium without 4HT, whereas expression of EBNA3A or EBNA3B did not. The expression of other EBNA proteins and of LMP1, CD21, CD23, and c-myc was unaffected by EBNA3C inactivation. However, EBNA3C inactivation resulted in the accumulation of p16INK4A, a decrease in the hyperphosphorylated form of the retinoblastoma protein, and a decrease in the proportion of cells in S or G2/M phase. These results indicate that EBNA3C has an essential role in cell cycle progression and the growth maintenance of LCLs.     

The EBV nuclear antigen 1 (EBNA1) enhances B cell immortalization several thousandfold

The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is one of the earliest viral proteins expressed after infection and is the only latent protein consistently expressed in viral-associated tumors. EBNA1's crucial role in viral DNA replication, episomal maintenance, and partitioning is well examined whereas its importance for the immortalization process and the tumorgenicity of EBV is unclear. To address these open questions, we generated, based on the maxi-EBV system, an EBNA1-deficient EBV mutant and used this strain to infect primary human B cells. Surprisingly, lymphoblastoid cell lines (LCL) emerged from these experiments, although with very low frequency. These cell lines were indistinguishable from normal LCLs with respect to proliferation and growth conditions. A detailed analysis indicated that the entire viral DNA was integrated into the cellular genome. At least 5 of the 11 latent EBV proteins were expressed, indicating the integrity of the EBV genome. EBNA1-positive and DeltaEBNA1-EBV-LCLs were injected into severe combined immunodeficient (SCID) mice to examine their tumorgenicity in comparison. Both groups supported tumor growth, indicating that EBNA1 is not mandatory for EBV's oncogenic potential. The results shown provide genetic evidence that EBNA1 is not essential to establish LCLs but promotes the efficiency of this process significantly.     

Recombinant Epstein-Barr virus with small RNA (EBER) genes deleted transforms lymphocytes and replicates in vitro.

Strains of Epstein-Barr virus (EBV) with deletions of the small RNA (EBER) genes were made by homologous recombination using the EBV P3HR-1 strain, which has undergone deletion of the essential transforming gene that encodes the EBV nuclear antigen, EBNA-2, and a DNA fragment that was wild type at the EBNA-2 locus but from which the EBER genes had been deleted. Even though the EBER and EBNA-2 genes are separated by 40 kilobases, selection for transforming P3HR-1 recombinants that required a restored EBNA-2 gene resulted in 20% cotransfer of the EBER deletion. EBER-deleted recombinants transformed primary B lymphocytes into lymphoblastoid cell lines (LCLs), which were indistinguishable form LCLs transformed by wild-type EBV in their proliferation, in latency-associated EBV gene expression, and in their permissiveness for EBV replication cycle gene expression. EBER-deleted virus from infected LCL clones could infect and growth-transform primary B lymphocytes. These procedures should be applicable to the construction of other EBV recombinants within 40 kilobases of the EBNA-2 gene. The EBER-deleted EBV recombinants should be useful in further evaluating the role of EBERs in EBV infection.     

Transforming growth factor beta-1 (TGF-β1) released by an Epstein-Barr virus (EBV) positive spontaneous lymphoblastoid cell line from a patient with Kostmann's congenital neutropenia inhibits the growth of normal committed haemopoietic progenitors in vitro

Summary This study reports the characterization of a spontaneous lymphoblastoid cell line (LCL) raised from the peripheral blood of a patient with Kostmann's congenital neutropenia. The LCL was composed of EBV-infected polyclonal B cells and displayed surface markers and pattern of growth in vitro typical of normal LCLs. The supernatant of the LCL contained a colony inhibiting activity (CIA) that decreased the cloning efficiency of normal committed haemopoietic progenitors and was identified as immunoreactive transforming growth factor β1 (TGF-β1) by neutralization experiments with a specific antiserum. Control studies with a panel of LCLs spontaneously derived from the peripheral blood of patients seropositive for Epstein-Barr virus (EBV) infections showed that 5/30 LCLs produced a CIA. This CIA was not identifiable as TGF-β1 but rather was due to the combined effects of tumour necrosis factor α (TNFα). tumour necrosis factor β (TNFβ) and interferon α (IFNα), that were present in the LCL supernatants. The hypothesis that the B cells latently infected by EBV in vivo and possibly expanded as a consequence of the infection may have contributed to the inhibition of the patient granulopoiesis by releasing TGF-β1 will be discussed.     

Plasmacytoid differentiation of Epstein-Barr virus-transformed B cells in vivo is associated with reduced expression of viral latent genes.

The Epstein-Barr virus (EBV)-associated B-cell lymphoproliferative disorders that arise in immunosuppressed individuals are considered to resemble EBV-transformed in vitro lymphoblastoid cell lines (LCLs) with a mature activated B-cell phenotype. In this study of human lymphoproliferative disorders in the severe combined immunodeficiency mouse model, however, we demonstrate that EBV-infected tumor cells are not LCL-like but are predominantly plasmacytoid and that this phenotype correlates with reduced expression of EBV latent genes. B-cell tumors developed within 3-6 weeks after injection of LCLs into severe combined immunodeficiency mice. The tumors and the injected LCLs were analyzed by flow cytofluorometry for B-cell differentiation and activation markers and by ribonuclease protection assay for cellular and viral gene expression. No differences in the expression of CD19 and CD21 were observed. However, a decrease in CD23, CD11a (lymphocyte function-associated antigen LFA-1), and CD58 (LFA-3) expression and an increase in CD38 (a plasma-cell-associated antigen), CD54 (intracellular adhesion molecule ICAM-1), and HLA class I in the tumor cells relative to the LCLs was observed. Two-color flow cytofluorometric analysis showed that the predominant population (> 80%) in LCLs was CD23hi/CD38lo and that the major population in LCL-derived tumors was CD23lo/CD38hi. Cell cycle analysis showed that, in contrast to actively cycling LCLs, the majority of tumor cells had exited the cell cycle and were restricted to G0/G1 phase. Finally, and most important, a reduction in mRNA for the EBV latent genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein (LMP1) was observed in the tumors.     

Soluble factors produced by activated CD4+ T cells modulate EBV latency

Following infection with Epstein-Barr virus (EBV), the virus is carried for life in the memory B-cell compartment in a silent state (latency I/0). These cells do not resemble the proliferating lymphoblastoid cells (LCLs) (latency III) that are generated after infection. It is of fundamental significance to identify how the different EBV expression patterns are established in the latently infected cell. In view of the prompt activatability of CD4(+) T cells in primary EBV infection, and their role in B-cell differentiation, we studied the involvement of CD4(+) T cells in the regulation of EBV latency. Lymphoblastoid cell lines (LCLs) were cocultured with autologous or allogeneic CD4(+) T cells. Activated T cells influenced the expression of two key viral proteins that determine the fate of the infected B cell. EBNA2 was down-regulated, whereas LMP1 was unregulated and the cells proliferated less. This was paralleled by the down-regulation of the latency III promoter (Cp). Experiments performed in the transwell system showed that this change does not require cell contact, but it is mediated by soluble factors. Neutralizing experiments proved that the up-regulation of LMP1 is, to some extent, mediated by IL21, but this cytokine was not responsible for EBNA2 down-regulation. This effect was partly mediated by soluble CD40L. We detected similar regulatory functions of T cells in in vitro-infected lymphocyte populations. In conclusion, our results revealed an additional mechanism by which CD4(+) T cells can control the EBV-induced B-cell proliferation.     

Growth- and tumor-promoting effects of deregulated BCL2 in human B-lymphoblastoid cells.

Human follicular B-cell lymphomas possess a t(14;18) that translocates a putative protooncogene, BCL2, into the immunoglobulin heavy chain locus. The normal BCL2 gene is quiescent in resting B cells, expressed in proliferating, but down-regulated in differentiated B cells. Inappropriately high levels of BCL2-immunoglobulin chimeric RNA are present in t(14;18) lymphomas for their mature B-cell stage. We examined the biologic effects of BCL2 deregulation in human B cells by introducing BCL2 into human B-lymphoblastoid cell lines (LCLs) with retroviral gene transfer. Although deregulated BCL2 expression as a single agent was not sufficient to confer tumorigenicity to LCLs, it consistently produced a 3- to 4-fold increment in LCL clonogenicity in soft agar. In addition, BCL2 deregulation complements the transforming effects of the MYC oncogene in LCLs. BCL2 augmented the clonogenicity of LCLs bearing exogenous MYC and increased the frequency and shortened the latency of tumor induction in immunodeficient mice. These results demonstrate a role for BCL2 as a protooncogene that affects B-cell growth and enhances B-cell neoplasia.