Establishment of latent Epstein-Barr virus infection and stable episomal maintenance in murine B-cell lines

Epstein-Barr virus (EBV) is a strict human pathogen for which no small animal models exist. Plasmids that contain the EBV plasmid origin of replication, oriP, and express EBV nuclear antigen 1 (EBNA1) are stably maintained extrachromosomally in human cells, whereas these plasmids replicate poorly in rodent cells. However, the ability of oriP and EBNA1 to maintain the entire EBV episome in proliferating rodent cells has not been determined. Expression of the two human B-cell receptors for EBV on the surfaces of murine B cells allows efficient viral entry that leads to the establishment of latent EBV infection and long-term persistence of the viral genome. Latent gene expression in these cells resembles the latency II profile in that EBNA1 and LMP1 can be detected whereas EBNA2 and the EBNA3s are not expressed.     

Three pathways of Epstein-Barr virus gene activation from EBNA1-positive latency in B lymphocytes.

Previous studies on Epstein-Barr virus (EBV)-positive B-cell lines have identified two distinct forms of virus latency. Lymphoblastoid cell lines generated by virus-induced transformation of normal B cells in vitro, express the full spectrum of six EBNAs and three latent membrane proteins (LMP1, LMP2A, and LMP2B); furthermore, these lines often contain a small fraction of cells spontaneously entering the lytic cycle. In contrast, Burkitt's lymphoma-derived cell lines retaining the tumor biopsy cell phenotype express only one of the latent proteins, the nuclear antigen EBNA1; such cells do not enter the lytic cycle spontaneously but may be induced to do so by treatment with such agents as tetradecanoyl phorbol acetate and anti-immunoglobulin. The present study set out to determine whether activation of full virus latent-gene expression was a necessary accompaniment to induction of the lytic cycle in Burkitt's lymphoma lines. Detailed analysis of Burkitt's lymphoma lines responding to anti-immunoglobulin treatment revealed three response pathways of EBV gene activation from EBNA1-positive latency. A first, rapid response pathway involves direct entry of cells into the lytic cycle without broadening of the pattern of latent gene expression; thereafter, the three "latent" LMPs are expressed as early lytic cycle antigens. A second, delayed response pathway in another cell subpopulation involves the activation of full latent gene expression and conversion to a lymphoblastoidlike cell phenotype. A third response pathway in yet another subpopulation involves the selective activation of LMPs, with no induction of the lytic cycle and with EBNA expression still restricted to EBNA1; this type of latent infection in B lymphocytes has hitherto not been described. Interestingly, the EBNA1+ LMP+ cells displayed some but not all of the phenotypic changes normally induced by LMP1 expression in a B-cell environment. These studies highlight the existence of four different types of EBV infection in B cells, including three distinct forms of latency, which we now term latency I, latency II, and latency III.     

Epstein-barr virus-induced resistance to drugs that activate the mitotic spindle assembly checkpoint in Burkitt's lymphoma cells

Epstein-Barr virus (EBV) is associated with a number of human cancers, and latent EBV gene expression has been reported to interfere with cell cycle checkpoints and cell death pathways. Here we show that latent EBV can compromise the mitotic spindle assembly checkpoint and rescue Burkitt's lymphoma (BL)-derived cells from caspase-dependent cell death initiated in aberrant mitosis. This leads to unscheduled mitotic progression, resulting in polyploidy and multi- and/or micronucleation. The EBV latent genes responsible for this phenotype are expressed from the P3HR1 strain of virus and several viruses with similar genomic deletions that remove the EBNA2 gene. Although EBNA2 and the latent membrane proteins are not expressed, the EBNA3 proteins are present in these BL cells. Survival of the EBV-positive cells is not consistently associated with EBV lytic gene expression or with the genes that are expressed in EBV latency I BL cells (i.e., EBNA1, EBERs, and BARTs) but correlates with reduced expression of the cellular proapoptotic BH3-only protein Bim. These data suggest that a subset of latent EBV gene products may increase the likelihood of damaged DNA being inherited because of the impaired checkpoint and enhanced survival capacity. This could lead to greater genetic diversity in progeny cells and contribute to tumorigenesis. Furthermore, since it appears that this restricted latent EBV expression interferes with the responses of Burkitt's lymphoma-derived cells to cytotoxic drugs, the results of this study may have important therapeutic implications in the treatment of some BL.     

Epstein-barr virus (EBV) nuclear protein 2-induced disruption of EBV latency in the Burkitt's lymphoma cell line Akata: analysis by tetracycline-regulated expression

The Burkitt's lymphoma (BL) cell line Akata retains the latency I program of Epstein-Barr virus (EBV) gene expression and cross-linking of its surface immunoglobulin G (IgG) by antibodies results in activation of viral replication. When EBV nuclear antigen 2 (EBNA2) was artificially expressed by a constitutive expression vector, the Cp EBNA promoter remained inactive and accordingly the latency III program was not induced. In contrast, expression of LMP2A and activity of the Fp lytic promoter were activated. Consistent with this Fp activity, the rate of spontaneous activation of the EBV replicative cycle was increased significantly, suggesting the possibility that EBNA2 can induce EBV replication. The efficiency of anti-IgG-induced activation of the viral replication was reduced in Akata cells expressing EBNA2. To obtain more direct evidence for EBNA2-induced activation of the EBV replicative cycle, this protein was next expressed by a tetracycline-regulated expression system. EBNA2 was undetectable with low doses (<0.5 microgram/ml) of tetracycline, while its expression was rapidly induced after removal of the antibiotic. This induced expression of EBNA2 was immediately followed by expression of EBV replicative cycle proteins in up to 50% of the cells, as shown by indirect immunofluorescence and immunoblot analysis. These results suggest an unexpected potential of EBNA2 to disrupt EBV latency and to activate viral replication.     

Different Patterns of Epstein-Barr Virus Latency in Endemic Burkitt Lymphoma (BL) Lead to Distinct Variants within the BL-Associated Gene Expression Signature

Epstein-Barr virus (EBV) is present in all cases of endemic Burkitt lymphoma (BL) but in few European/North American sporadic BLs. Gene expression arrays of sporadic tumors have defined a consensus BL profile within which tumors are classifiable as “molecular BL” (mBL). Where endemic BLs fall relative to this profile remains unclear, since they not only carry EBV but also display one of two different forms of virus latency. Here, we use early-passage BL cell lines from different tumors, and BL subclones from a single tumor, to compare EBV-negative cells with EBV-positive cells displaying either classical latency I EBV infection (where EBNA1 is the only EBV antigen expressed from the wild-type EBV genome) or Wp-restricted latency (where an EBNA2 gene-deleted virus genome broadens antigen expression to include the EBNA3A, -3B, and -3C proteins and BHRF1). Expression arrays show that both types of endemic BL fall within the mBL classification. However, while EBV-negative and latency I BLs show overlapping profiles, Wp-restricted BLs form a distinct subgroup, characterized by a detectable downregulation of the germinal center (GC)-associated marker Bcl6 and upregulation of genes marking early plasmacytoid differentiation, notably IRF4 and BLIMP1. Importantly, these same changes can be induced in EBV-negative or latency I BL cells by infection with an EBNA2-knockout virus. Thus, we infer that the distinct gene profile of Wp-restricted BLs does not reflect differences in the identity of the tumor progenitor cell per se but differences imposed on a common progenitor by broadened EBV gene expression.     

Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.     

Epstein-Barr virus nuclear antigen 2 trans-activates the cellular antiapoptotic bfl-1 gene by a CBF1/RBPJ kappa-dependent pathway

The human herpesvirus Epstein-Barr virus (EBV) establishes latency and promotes the long-term survival of its host B cell by targeting the molecular machinery controlling cell fate decisions. The cellular antiapoptotic bfl-1 gene confers protection from apoptosis under conditions of growth factor deprivation when expressed ectopically in an EBV-negative Burkitt's lymphoma-derived cell line (B. D'Souza, M. Rowe, and D. Walls, J. Virol. 74:6652-6658, 2000), and the EBV latent membrane protein 1 (LMP1) and its cellular functional homologue CD40 can both drive bfl-1 via an NF-kappaB-dependent enhancer element in the bfl-1 promoter (B. N. D'Souza, L. C. Edelstein, P. M. Pegman, S. M. Smith, S. T. Loughran, A. Clarke, A. Mehl, M. Rowe, C. Gélinas, and D. Walls, J. Virol. 78:1800-1816, 2004). Here we show that the EBV nuclear antigen 2 (EBNA2) also upregulates bfl-1. EBNA2 trans-activation of bfl-1 requires CBF1 (or RBP-J kappa), a nuclear component of the Notch signaling pathway, and there is an essential role for a core consensus CBF1-binding site on the bfl-1 promoter. trans-activation is dependent on the EBNA2-CBF1 interaction, is modulated by other EBV gene products known to interact with the CBF1 corepressor complex, and does not involve activation of NF-kappaB. bfl-1 expression is induced and maintained at high levels by the EBV growth program in a lymphoblastoid cell line, and withdrawal of either EBNA2 or LMP1 does not lead to a reduction in bfl-1 mRNA levels in this context, whereas the simultaneous loss of both EBV proteins results in a major decrease in bfl-1 expression. These findings are relevant to our understanding of EBV persistence, its role in malignant disease, and the B-cell developmental process.     

Mechanisms of EBV-mediated oncogenesis

Epstein-Barr virus (EBV) is the DNA tumor virus, which is known to be relevant to various cancers. EBV maintains latent infection in cancer cells, and there are three types of latent infection (type I-III) according to the patterns of viral latent genes expression. EBV has the ability to transform B cells into immortalized lymphoblastoid cell lines (LCL) showing type III latency, in which all latent genes are expressed. The mechanism of B-cell transformation has provided a model of EBV-associated lymphomas in immunosuppressed individuals. In type I and II latency, the limited numbers of latent genes are expressed. Previous studies have demonstrated the oncogenic functions of latent EBV genes including nuclear antigen EBNA1, membrane protein LMP1 and LMP2A. In addition, we have demonstrated that EBV-encoded small RNA EBERs play a significant role in oncogenesis. Here we summarize recent progresses in the studies on molecular mechanisms of EBV-mediated oncogenesis.