The nuclear lamina binds the EBV genome during latency and regulates viral gene expression

The Epstein Barr virus (EBV) infects almost 95% of the population worldwide. While typically asymptomatic, EBV latent infection is associated with several malignancies of epithelial and lymphoid origin in immunocompromised individuals. In latently infected cells, the EBV genome persists as a chromatinized episome that expresses a limited set of viral genes in different patterns, referred to as latency types, which coincide with varying stages of infection and various malignancies. We have previously demonstrated that latency types correlate with differences in the composition and structure of the EBV episome. Several cellular factors, including the nuclear lamina, regulate chromatin composition and architecture. While the interaction of the viral genome with the nuclear lamina has been studied in the context of EBV lytic reactivation, the role of the nuclear lamina in controlling EBV latency has not been investigated. Here, we report that the nuclear lamina is an essential epigenetic regulator of the EBV episome. We observed that in B cells, EBV infection affects the composition of the nuclear lamina by inducing the expression of lamin A/C, but only in EBV+ cells expressing the Type III latency program. Using ChIP-Seq, we determined that lamin B1 and lamin A/C bind the EBV genome, and their binding correlates with deposition of the histone repressive mark H3K9me2. By RNA-Seq, we observed that knock-out of lamin A/C in B cells alters EBV gene expression. Our data indicate that the interaction between lamins and the EBV episome contributes to the epigenetic control of viral gene expression during latency, suggesting a restrictive function of the nuclear lamina as part of the host response against viral DNA entry into the nucleus.     

Resveratrol inhibits proliferation and survival of Epstein Barr virus-infected Burkitt's lymphoma cells depending on viral latency program

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenolic natural product, shows chemopreventive properties against several cancers, heart diseases, inflammation, and viral infections. Epstein Barr virus (EBV), a γ-herpesvirus, contributes to the development of several human cancers including Burkitt's lymphoma (BL). In this study, we asked whether treatment with resveratrol would affect the viability of EBV-positive BL cells displaying different forms of latency. We report here that resveratrol, regardless of EBV status, induces caspase-dependent apoptosis by arresting cell-cycle progression in G(1) phase. However, resveratrol strongly induced apoptosis in EBV(-) and latency I EBV(+) cells, whereas latency II and latency III EBV(+) BL cells showed a survival advantage that increased with the extent of the pattern of viral gene expression. Resveratrol-induced cell-cycle arrest and apoptosis occurred in association with induction of p38 MAPK phosphorylation and suppression of ERK1/2 signaling pathway. Moreover, NF-κB DNA-binding activity was inhibited in all BL lines except EBV(+) latency III cells. LMP1 oncogene, which is expressed in latency III phenotype, is involved with the higher resistance to the antiproliferative effect of resveratrol because siRNA-mediated inhibition of LMP1 greatly increased the sensitivity of latency III BL cells as well as that of lymphoblastoid cell lines to the polyphenol. We propose that a combined resveratrol/siRNA strategy may be a novel approach for the treatment of EBV-associated B-cell malignancies in which the viral pattern of gene expression has been defined.     

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.     

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.     

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.     

Host-cell-determined methylation of specific Epstein-Barr virus promoters regulates the choice between distinct viral latency programs.

Epstein-Barr virus (EBV) is capable of adopting three distinct forms of latency: the type III latency program, in which six EBV-encoded nuclear antigens (EBNAs) are expressed, and the type I and type II latency programs, in which only a single viral nuclear protein, EBNA1, is produced. Several groups have reported heavy CpG methylation of the EBV genome in Burkitt's lymphoma cell lines which maintain type I latency, and loss of viral genome methylation in tumor cell lines has been correlated with a switch to type III latency. Here, evidence that the type III latency program must be inactivated by methylation to allow EBV to enter the type I or type II restricted latency program is provided. The data demonstrates that the EBNA1 gene promoter, Qp, active in types I and II latency, is encompassed by a CpG island which is protected from methylation. CpG methylation inactivates the type III latency program and consequently allows the type I or II latency program to operate by alleviating EBNA1-mediated repression of Qp. Methylation of the type III latency EBNA gene promoter, Cp, appears to be essential to prevent type III latency, since EBNA1 is expressed in all latently infected cells and, as shown here, is the only viral antigen required for activation of Cp. EBV is thus a pathogen which subverts host-cell-determined methylation to regulate distinct genetic programs.     

The latent membrane protein 1 of Epstein-Barr virus (EBV) primes EBV latency cells for type I interferon production

Epstein-Barr virus (EBV) latency has been associated with a variety of human cancers. Latent membrane protein 1 (LMP-1) is one of the key viral proteins required for transformation of primary B cells in vitro and establishment of EBV latency. We have previously shown that LMP-1 induces the expression of several interferon (IFN)-stimulated genes and has antiviral effect (Zhang, J., Das, S. C., Kotalik, C., Pattnaik, A. K., and Zhang, L. (2004) J. Biol. Chem. 279, 46335-46342). In this report, a novel mechanism related to the antiviral effect of LMP-1 is identified. We show that EBV type III latency cells, in which LMP-1 is expressed, are primed to produce robust levels of endogenous IFNs upon infection of Sendai virus. The priming action is due to the expression of LMP-1 but not EBV nuclear antigen 2 (EBNA-2). The signaling events from the C-terminal activator regions of LMP-1 are essential to prime cells for high IFN production. LMP-1-mediated activation of NF-kappaB is apparently necessary and sufficient for LMP-1-mediated priming effect in DG75 cells, a human B cell line. IFN regulatory factor 7 (IRF-7) that can be activated by LMP-1 is also implicated in the priming action. Taken together, these data strongly suggest that LMP-1 may prime EBV latency cells for IFN production and that the antiviral property of LMP-1 may be an intrinsic part of EBV latency program, which may assist the establishment and/or maintenance of viral latency.     

Differential methylation of Epstein-Barr virus latency promoters facilitates viral persistence in healthy seropositive individuals

Epstein-Barr virus (EBV) establishes a life-long infection in humans, with distinct viral latency programs predominating during acute and chronic phases of infection. Only a subset of the EBV latency-associated antigens present during the acute phase of EBV infection are expressed in the latently infected memory B cells that serve as the long-term EBV reservoir. Since the EBV immortalization program elicits a potent cellular immune response, downregulation of viral gene expression in the long-term latency reservoir is likely to facilitate evasion of the immune response and persistence of EBV in the immunocompetent host. Tissue culture and tumor models of restricted EBV latency have consistently demonstrated a critical role for methylation of the viral genome in maintaining the restricted pattern of latency-associated gene expression. Here we extend these observations to demonstrate that the EBV genomes in the memory B-cell reservoir are also heavily and discretely methylated. This analysis reveals that methylation of the viral genome is a normal aspect of EBV infection in healthy immunocompetent individuals and is not restricted to the development of EBV-associated tumors. In addition, the pattern of methylation very likely accounts for the observed inhibition of the EBV immortalization program and the establishment and maintenance of a restricted latency program. Thus, EBV appears to be the first example of a parasite that usurps the host cell-directed methylation system to regulate pathogen gene expression and thereby establish a chronic infection.     

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.     

Epstein-barr virus regulates c-MYC, apoptosis, and tumorigenicity in Burkitt lymphoma

Loss of the Epstein-Barr virus (EBV) genome from Akata Burkitt lymphoma (BL) cells is coincident with a loss of malignant phenotype, despite the fact that Akata and other EBV-positive BL cells express a restricted set of EBV gene products (type I latency) that are not known to overtly affect cell growth. Here we demonstrate that reestablishment of type I latency in EBV-negative Akata cells restores tumorigenicity and that tumorigenic potential correlates with an increased resistance to apoptosis under growth-limiting conditions. The antiapoptotic effect of EBV was associated with a higher level of Bcl-2 expression and an EBV-dependent decrease in steady-state levels of c-MYC protein. Although the EBV EBNA-1 protein is expressed in all EBV-associated tumors and is reported to have oncogenic potential, enforced expression of EBNA-1 alone in EBV-negative Akata cells failed to restore tumorigenicity or EBV-dependent down-regulation of c-MYC. These data provide direct evidence that EBV contributes to the tumorigenic potential of Burkitt lymphoma and suggest a novel model whereby a restricted latency program of EBV promotes B-cell survival, and thus virus persistence within an immune host, by selectively targeting the expression of c-MYC.     

Phylogenetic comparison of Epstein-Barr virus genomes

Technologies used for genome analysis and whole genome sequencing are useful for us to understand genomic characterization and divergence. The Epstein-Barr virus (EBV) is an oncogenic virus that causes diverse diseases such as Burkitt’s lymphoma (BL), nasopharyngeal carcinoma (NPC), Hodgkin’s lymphoma (HL), and gastric carcinoma (GC). EBV genomes found in these diseases can be classified either by phases of EBV latency (type-I, -II, and -III latency) or types of EBNA2 sequence difference (type-I EBV, type-II EBV or EBV-1, EBV-2). EBV from EBV-transformed lymphoblastoid cell line (LCL) establishes type-III latency, EBV from NPC establishes type-II latency, and EBV from GC establishes type-I latency. However, other important factors play key roles in classifying numerous EBV strains because EBV genomes are highly diverse and not phylogenetically related to types of EBV-associated diseases. Herein, we first reviewed previous studies to describe molecular characteristics of EBV genomes. Then, using comparative and phylogenetic analyses, we phylogenetically analyzed molecular variations of EBV genomes and proteins. The review of previous studies and our phylogenetic analysis showed that EBV genomes and proteins were highly diverse regardless of types of EBV-associated diseases. Other factors should be considered in determining EBV taxonomy. This review will be helpful to understand complicated phylogenetic relationships of EBV genomes.     

Roles of Cell Signaling Pathways in Cell-to-Cell Contact-Mediated Epstein-Barr Virus Transmission

Epstein-Barr virus (EBV), a human gamma herpesvirus, establishes a life-long latent infection in B lymphocytes and epithelial cells following primary infection. Several lines of evidence indicate that the efficiency of EBV infection in epithelial cells is accelerated up to 10(4)-fold by coculturing with EBV-infected Burkitt's lymphoma (BL) cells compared to infection with cell-free virions, indicating that EBV infection into epithelial cells is mainly mediated via cell-to-cell contact. However, the molecular mechanisms involved in this pathway are poorly understood. Here, we establish a novel assay to assess cell-to-cell contact-mediated EBV transmission by coculturing an EBV-infected BL cell line with an EBV-negative epithelial cell line under stimulation for lytic cycle induction. By using this assay, we confirmed that EBV was transmitted from BL cells to epithelial cells via cell-to-cell contact but not via cell-to-cell fusion. The inhibitor treatments of extracellular signal-regulated kinase (ERK) and nuclear factor (NF)-κB pathways blocked EBV transmission in addition to lytic induction. The blockage of the phosphoinositide 3-kinase (PI3K) pathway impaired EBV transmission coupled with the inhibition of lytic induction. Knockdown of the RelA/p65 subunit of NF-κB reduced viral transmission. Moreover, these signaling pathways were activated in cocultured BL cells and in epithelial cells. Finally, we observed that viral replication was induced in cocultured BL cells. Taken together, our data suggest that cell-to-cell contact induces multiple cell signaling pathways in BL cells and epithelial cells, contributing to the induction of the viral lytic cycle in BL cells and the enhancement of viral transmission to epithelial cells.     

Differences in B cell growth phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt''s lymphoma cells.

Recently established Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cell lines, carrying chromosomal translocations indicative of their malignant origin, have been monitored for their degree of in vitro progression towards a more 'lymphoblastoid' cell surface phenotype and growth pattern, and for their expression of three EBV latent gene products which are constitutively present in all virus-transformed normal lymphoblastoid cell lines (LCLs). BL cell lines which stably retained the original tumour biopsy phenotype on serial passage were all positive for the nuclear antigen EBNA 1 but did not express detectable amounts of two other 'transforming' proteins, EBNA 2 and the latent membrane protein (LMP). This novel pattern of EBV gene expression was also observed on direct analysis of BL biopsy tissue. All three viral proteins became detectable, however, in BL cell lines which had progressed towards a more LCL-like phenotype in vitro. This work establishes a link between B cell phenotype and the accompanying pattern of EBV latent gene expression, and identifies a novel type of EBV:cell interaction which may be unique to BL cells.