Epstein-Barr virus lytic infection contributes to lymphoproliferative disease in a SCID mouse model

Most Epstein-Barr virus (EBV)-positive tumor cells contain one of the latent forms of viral infection. The role of lytic viral gene expression in EBV-associated malignancies is unknown. Here we show that EBV mutants that cannot undergo lytic viral replication are defective in promoting EBV-mediated lymphoproliferative disease (LPD). Early-passage lymphoblastoid cell lines (LCLs) derived from EBV mutants with a deletion of either viral immediate-early gene grew similarly to wild-type (WT) virus LCLs in vitro but were deficient in producing LPD when inoculated into SCID mice. Restoration of lytic EBV gene expression enhanced growth in SCID mice. Acyclovir, which prevents lytic viral replication but not expression of early lytic viral genes, did not inhibit the growth of WT LCLs in SCID mice. Early-passage LCLs derived from the lytic-defective viruses had substantially decreased expression of the cytokine interleukin-6 (IL-6), and restoration of lytic gene expression reversed this defect. Expression of cellular IL-10 and viral IL-10 was also diminished in lytic-defective LCLs. These results suggest that lytic EBV gene expression contributes to EBV-associated lymphoproliferative disease, potentially through induction of paracrine B-cell growth factors.     

Epstein-Barr Virus Nuclear Antigen 3A Promotes Cellular Proliferation by Repression of the Cyclin-Dependent Kinase Inhibitor p21WAF1/CIP1

Latent infection by Epstein-Barr virus (EBV) is highly associated with the endemic form of Burkitt lymphoma (eBL), which typically limits expression of EBV proteins to EBNA-1 (Latency I). Interestingly, a subset of eBLs maintain a variant program of EBV latency - Wp-restricted latency (Wp-R) - that includes expression of the EBNA-3 proteins (3A, 3B and 3C), in addition to EBNA-1. In xenograft assays, Wp-R BL cell lines were notably more tumorigenic than their counterparts that maintain Latency I, suggesting that the additional latency-associated proteins expressed in Wp-R influence cell proliferation and/or survival. Here, we evaluated the contribution of EBNA-3A. Consistent with the enhanced tumorigenic potential of Wp-R BLs, knockdown of EBNA-3A expression resulted in abrupt cell-cycle arrest in G0/G1 that was concomitant with conversion of retinoblastoma protein (Rb) to its hypophosphorylated state, followed by a loss of Rb protein. Comparable results were seen in EBV-immortalized B lymphoblastoid cell lines (LCLs), consistent with the previous observation that EBNA-3A is essential for sustained growth of these cells. In agreement with the known ability of EBNA-3A and EBNA-3C to cooperatively repress p14^ARF and p16^INK4a expression, knockdown of EBNA-3A in LCLs resulted in rapid elevation of p14^ARF and p16^INK4a. By contrast, p16^INK4a was not detectably expressed in Wp-R BL and the low-level expression of p14^ARF was unchanged by EBNA-3A knockdown. Amongst other G1/S regulatory proteins, only p21^WAF1/CIP1, a potent inducer of G1 arrest, was upregulated following knockdown of EBNA-3A in Wp-R BL Sal cells and LCLs, coincident with hypophosphorylation and destabilization of Rb and growth arrest. Furthermore, knockdown of p21^WAF1/CIP1 expression in Wp-R BL correlated with an increase in cellular proliferation. This novel function of EBNA-3A is distinct from the functions previously described that are shared with EBNA-3C, and likely contributes to the proliferation of Wp-R BL cells and LCLs.     

Epstein-Barr virus nuclear protein 2 mutations define essential domains for transformation and transactivation.

Epstein-Barr virus (EBV) nuclear protein 2 (EBNA-2) is essential for B-lymphocyte growth transformation. EBNA-2 transactivates expression of the EBV latent membrane protein (LMP-1) and also transactivates expression of the B-lymphocyte proteins CD21 and CD23. In order to analyze the functional domains of EBNA-2, we constructed 11 linker-insertion and 15 deletion mutations. Each of the mutant EBNA-2 proteins localized to the nucleus, and each was expressed at levels similar to wild-type EBNA-2. Deletion of both EBNA-2 basic domains was required to prevent nuclear localization, indicating that either is sufficient for nuclear translocation. The mutant EBNA-2 genes were assayed for lymphocyte transformation after recombination with the EBNA-2-deleted P3HR-1 EBV genome and for LMP-1 transactivation following transfection into P3HR-1-infected B-lymphoma cells. Cell lines transformed by recombinant EBV carrying EBNA-2 mutations were assayed for growth properties and LMP-1, CD21, and CD23 expression. The mutational analysis indicates that at least four separate EBNA-2 domains are essential for lymphocyte transformation. Two other domains are necessary for the full transforming phenotype. Two deletion and eight linker-insertion mutations did not reduce transforming activity. Mutations which diminish or abolish lymphocyte transformation also diminish or abolish LMP-1 transactivation, respectively. Cells transformed by recombinant EBV carrying EBNA-2 genes with diminished or normal transforming activity all expressed high levels of LMP-1, CD23, and CD21. These findings suggest that transactivation of these viral and cellular genes by EBNA-2 plays a critical role in lymphocyte transformation by EBV. Furthermore, these results indicate that the transformation and transactivation functions of EBNA-2 may not be separable.     

Epstein-Barr virus nuclear protein 2 is a critical determinant for tumor growth in SCID mice and for transformation in vitro.

Injection of Epstein-Barr virus (EBV)-transformed human lymphoblastoid B cells into immunodeficient SCID mice results in the appearance of rapidly growing, fatal human B-cell tumors. To evaluate the role of EBV nuclear protein 2 (EBNA-2) in this process, we generated lymphoblastoid cell lines transformed by several EBV mutants which were identical except for deletions in the EBNA-2 gene (J. I. Cohen, F. Wang, and E. Kieff, J. Virol. 65:2545-2554, 1991). These cell lines were injected intraperitoneally into SCID mice, and the interval until tumor detection was determined. Cell lines transformed with EBV type 1 (strain W91) or with EBV type 2 (strain P3HR-1) with an inserted type 1 EBNA-2 gene grew at the same rapid rate, indicating the potential importance of EBNA-2 for tumor formation in vivo. Cell lines derived from three different EBV mutants with deletions in the amino half of EBNA-2 produced tumors more slowly than cell lines transformed by wild-type W91 virus. In contrast, a cell line transformed with an EBV mutant with a deletion in the carboxy terminus of EBNA-2 grew more rapidly than cell lines transformed by wild-type virus. EBV mutants with deletions in the amino half of EBNA-2 had had reduced transforming activity in vitro, while the carboxy-terminal EBNA-2 mutant had had transforming activity greater than or equal to that of the wild type. These data indicate that EBNA-2 plays a critical role both for B-cell tumor growth in SCID mice and for B-lymphocyte transformation in vitro.     

Genetic evidence that EBNA-1 is needed for efficient, stable latent infection by Epstein-Barr virus

Replication and maintenance of the 170-kb circular chromosome of Epstein-Barr virus (EBV) during latent infection are generally believed to depend upon a single viral gene product, the nuclear protein EBNA-1. EBNA-1 binds to two clusters of sites at oriP, an 1, 800-bp sequence on the EBV genome which can support replication and maintenance of artificial plasmids introduced into cell lines that contain EBNA-1. To investigate the importance of EBNA-1 to latent infection by EBV, we introduced a frameshift mutation into the EBNA-1 gene of EBV by recombination along with a flanking selectable marker. EBV genomes carrying the frameshift mutation could be isolated readily after superinfecting EBV-positive cell lines, but not if recombinant virus was used to infect EBV-negative B-cell lines or to immortalize peripheral blood B cells. EBV mutants lacking almost all of internal repeat 3, which encode a repetitive glycine and alanine domain of EBNA-1, were generated in the same way and found to immortalize B cells normally. An EBNA-1-deficient mutant of EBV was isolated and found to be incapable of establishing a latent infection of the cell line BL30 at a detectable frequency, indicating that the mutant was less than 1% as efficient as an isogenic, EBNA-1-positive strain in this assay. The data indicate that EBNA-1 is required for efficient and stable latent infection by EBV under the conditions tested. Evidence from other studies now indicates that autonomous maintenance of the EBV chromosome during latent infection does not depend on the replication initiation function of oriP. It is therefore likely that the viral chromosome maintenance (segregation) function of oriP and EBNA-1 is what is required.     

Initiation of DNA replication within oriP is dispensable for stable replication of the latent Epstein-Barr virus chromosome after infection of established cell lines

The 165-kb circularized chromosome of Epstein-Barr virus (EBV) is replicated in latently infected cells once per cell cycle by host proteins during S phase. Replication initiates at multiple sites on latent EBV chromosomes, including within a 1.8-kb region called oriP, which can provide both replication and stabilization for recombinant plasmids in the presence of the EBV-encoded protein, EBNA-1. Replication initiates at or near the dyad symmetry component (DS) of oriP, which depends on multiple EBNA-1 binding sites for activity. To test the importance of the replication function of oriP, the DS was deleted from the viral genome. EBV mutants lacking the DS and carrying a selectable gene could establish latent infections in BL30 cells, in which circular, mutant viral chromosomes were stably maintained. Analysis of replication fork movement using two-dimensional gel electrophoresis showed that the deletion of the DS reduced the initiation events to an undetectable level within the oriP region so that this segment was replicated exclusively by forks entering the region from either direction. A significant slowing or stalling of replication forks that occurs normally at the approximate position of the DS was also eliminated by deletion of the DS. The results confirm the DS as both a replication origin and a place where replication forks pause. Since the replication function of oriP is dispensable at least in certain cell lines, the essential role of EBNA-1 for infection of these cell lines is likely to be that of stabilizing the EBV chromosome by associating with the 30-bp repeats of oriP. The results also imply that in established cell lines, the EBV chromosome can be efficiently replicated entirely from origins that are activated by cellular factors. Presumably, initiation of replication at the DS, mediated by EBNA-1, is important for the natural life cycle of EBV, perhaps in establishing latent infections of normal B cells.     

Cellular target genes of Epstein-Barr virus nuclear antigen 2

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is a key determinant in the EBV-driven B-cell growth transformation process. By activating an array of viral and cellular target genes, EBNA-2 initiates a cascade of events which ultimately cause cell cycle entry and the proliferation of the infected B cell. In order to identify cellular target genes that respond to EBNA-2 in the absence of other viral factors, we have performed a comprehensive search for EBNA-2 target genes in two EBV-negative B-cell lines. This screen identified 311 EBNA-2-induced and 239 EBNA-2-repressed genes that were significantly regulated in either one or both cell lines. The activation of most of these genes had not previously been attributed to EBNA-2 function and will be relevant for the identification of EBNA-2-specific contributions to EBV-associated malignancies. The diverse spectrum of EBNA-2 target genes described in this study reflects the broad spectrum of EBNA-2 functions involved in virus-host interactions, including cell signaling molecules, adapters, genes involved in cell cycle regulation, and chemokines.     

Epstein-Barr virus (EBV) recombinants: use of positive selection markers to rescue mutants in EBV-negative B-lymphoma cells.

The objective of these experiments was to develop strategies for creation and identification of recombinant mutant Epstein-Barr viruses (EBV). EBV recombinant molecular genetics has been limited to mutations within a short DNA segment deleted from a nontransforming EBV and an underlying strategy which relies on growth transformation of primary B lymphocytes for identification of recombinants. Thus, mutations outside the deletion or mutations which affect transformation cannot be easily recovered. In these experiments we investigated whether a toxic drug resistance gene, guanine phosphoribosyltransferase or hygromycin phosphotransferase, driven by the simian virus 40 promoter can be recombined into the EBV genome and can function to identify B-lymphoma cells infected with recombinant virus. Two different strategies were used to recombine the drug resistance marker into the EBV genome. Both utilized transfection of partially permissive, EBV-infected B95-8 cells and positive selection for cells which had incorporated a functional drug resistance gene. In the first series of experiments, B95-8 clones were screened for transfected DNA that had recombined into the EBV genome. In the second series of experiments, the transfected drug resistance marker was linked to the plasmid and lytic EBV origins so that it was maintained as an episome and could recombine with the B95-8 EBV genome during virus replication. The recombinant EBV from either experiment could be recovered by infection and toxic drug selection of EBV-negative B-lymphoma cells. The EBV genome in these B-lymphoma cells is frequently an episome. Virus genes associated with latent infection of primary B lymphocytes are expressed. Expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) and the EBNA-3 genes is variable relative to that of EBNA-1, as is characteristic of some naturally infected Burkitt tumor cells. Moreover, the EBV-infected B-lymphoma cells are often partially permissive for early replicative cycle gene expression and virus replication can be induced, in contrast to previously reported in vitro infected B-lymphoma cells. These studies demonstrate that dominant selectable markers can be inserted into the EBV genome, are active in the context of the EBV genome, and can be used to recover recombinant EBV in B-lymphoma cells. This system should be particularly useful for recovering EBV genomes with mutations in essential transforming genes.     

5-Azacytidine up regulates the expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) through EBNA-6 and latent membrane protein in the Burkitt''s lymphoma line rael.

Nonproductive infection of B lymphocytes by Epstein-Barr virus (EBV) is associated with a highly restricted expression of viral genes. In growth-transformed lymphoblastoid cell lines, the products of these genes include a complex of at least six EBV nuclear antigens (EBNAs) (EBNA-1 through EBNA-6) and one membrane protein (latent membrane protein [LMP]). EBV-carrying Burkitt's lymphoma (BL) biopsies and derived cell lines that have retained a representative phenotype (group I BL lines) express only EBNA-1 (M. Rowe, D. T. Rowe, C. D. Gregory, L. S. Young, P. J. Farrell, H. Rupani, and A. B. Rickinson, EMBO J. 6:2743-2751, 1987). We have found that EBNA-2 through EBNA-6 and LMP can be up regulated by treating the group I BL line Rael with the DNA-demethylating agent 5-azacytidine (5-AzaC). The drug acted in a time- and dose-dependent manner. EBNA-2-positive cells were detected by anti-complement immunofluorescence staining just 12 h after addition of 4 microM 5-AzaC and reached a maximum number at 72 h, when up to 75% of the population was positive. EBNA-2, EBNA-3, EBNA-4, EBNA-4, EBNA-6, and LMP were demonstrated immunoblots starting at 48 h. The EBV-encoded early antigens and viral capsid antigens were also induced but at a lower level. EBNA-2 and the lytic cycle-associated antigens appeared with a different time course and in largely nonoverlapping cell subpopulations, as demonstrated by double fluorescence staining. Thus, EBNA-2 expression was not restricted to lytically infected cells, nor was EBNA-2 required for entry into the lytic cycle. The coding and regulatory sequences of EBNA-2 and LMP were found to be highly methylated in Rael cells and were, as expected, demethylated after 5-AzaC treatment. These findings suggest that DNA methylation may participate in the regulation of growth transformation-associated viral genes in BL cells.     

The Effect of Freeze-Thaw Cycles on Gene Expression Levels in Lymphoblastoid Cell Lines

Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) are a widely used renewable resource for functional genomic studies in humans. The ability to accumulate multidimensional data pertaining to the same individual cell lines, from complete genomic sequences to detailed gene regulatory profiles, further enhances the utility of LCLs as a model system. However, the extent to which LCLs are a faithful model system is relatively unknown. We have previously shown that gene expression profiles of newly established LCLs maintain a strong individual component. Here, we extend our study to investigate the effect of freeze-thaw cycles on gene expression patterns in mature LCLs, especially in the context of inter-individual variation in gene expression. We report a profound difference in the gene expression profiles of newly established and mature LCLs. Once newly established LCLs undergo a freeze-thaw cycle, the individual specific gene expression signatures become much less pronounced as the gene expression levels in LCLs from different individuals converge to a more uniform profile, which reflects a mature transformed B cell phenotype. We found that previously identified eQTLs are enriched among the relatively few genes whose regulations in mature LCLs maintain marked individual signatures. We thus conclude that while insight drawn from gene regulatory studies in mature LCLs may generally not be affected by the artificial nature of the LCL model system, many aspects of primary B cell biology cannot be observed and studied in mature LCL cultures.     

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.     

Influence of Burkitt's lymphoma and primary B cells on latent gene expression by the nonimmortalizing P3J-HR-1 strain of Epstein-Barr virus.

The Epstein-Barr virus (EBV) genes expressed in B lymphocytes immortalized in vitro or in Burkitt's lymphoma (BL) cells infected in vivo have been characterized previously; however, the viral products which are essential for immortalization or for establishment of EBV latency are still not known. To approach this question, we compared the kinetics of expression of EBV nuclear antigens and the two EBV-encoded small RNAs, EBER1 and EBER2, after infection of primary B cells or EBV genome-negative BL cells with either an immortalizing EBV strain (B95-8) or the nonimmortalizing deletion mutant (HR-1). Following infection of primary cells with B95-8 virus, EBV nuclear antigen (EBNA)-2 was expressed first, followed by EBNA-1, -3, and -4 (also called leader protein [LP]) and the two small RNAs. Infection of EBV genome-negative BL cells with the same strain of virus resulted in a similar pattern of gene expression, except that the EBNAs appeared together and more rapidly. EBERs were not apparent in one BL cell line converted by B95-8. The only products detected after infection of primary B lymphocytes with the HR-1 deletion mutant were the EBNA-4 (LP) family and trace amounts of EBER1. Although HR-1 could express neither EBNA-1, EBNA-3, nor EBER2 in primary cells, all these products were expressed rapidly after HR-1 infection of EBV genome-negative BL cell lines. The results indicate that the mutation in HR-1 virus affects immortalization not only through failure to express EBNA-2, a gene which is deleted, but also indirectly by curtailing expression of several other EBV genes whose coding regions are intact in the HR-1 virus and normally expressed during latency. The pattern of latent EBV gene expression after HR-1 infection is dependent on the host cell, perhaps through products specific for the cell cycle or the state of B-cell differentiation.