Increased B cell survival and preferential activation of the memory compartment by a malaria polyclonal B cell activator

Chronic malaria infection is characterized by polyclonal B cell activation, hyperglobulinemia, and elevated titers of autoantibodies. We have recently identified the cysteine-rich interdomain region 1alpha (CIDR1alpha) of the Plasmodium falciparum erythrocyte membrane protein 1 as a T cell-independent polyclonal B cell activator and Ig binding protein. Here, we show that, although the binding affinity of CIDR1alpha to human IgM and IgG is relatively low, B cell activation still proceeds. CIDR1alpha rescues tonsillar B cells from apoptosis, and increases the proportion of cycling cells. Comparison of the impact on naive and memory B cell compartment indicated that CIDR1alpha preferentially activates memory B lymphocytes. Analysis of the gene expression profiles induced by CIDR1alpha and anti-Ig activation using a cDNA microarray demonstrated a low degree of homology in the signatures imposed by both stimuli. The microarray data correlate with the functional analysis demonstrating that CIDR1alpha activates various immunological pathways and protects B cells from apoptosis. Together, the results provide evidence for a role of malaria in preferentially activating the memory B cell compartment. The polyclonal B cell activation and augmented survival induced by CIDR1alpha is of relevance for understanding the mechanisms behind the increased risk of Burkitt's lymphoma in malaria endemic areas.     

EBV interferes with the sensitivity of Burkitt lymphoma Akata cells to etoposide

Burkitt lymphoma (BL) commonly exhibits Epstein-Barr virus (EBV) positivity associated with latent chronic infection. Models of acute EBV infection have been associated with cellular resistance to apoptosis. However, the effect of latent long-term EBV infection on apoptosis induced by drugs is not well defined. To determine this, we have studied the response of the Akata EBV+ cell line (type I latency) to etoposide, before and after downregulating EBV gene expression. We observed that downregulating EBV nuclear antigen-1 (EBNA-1) expression with siRNAs reverted cellular sensitivity to etoposide. In accordance with this finding, Akata EBV+ cells showed increased sensitivity to etoposide, when compared to the Akata EBV- cells. We also observed that Akata EBV+ cells presented increased apoptosis levels and decreased Bcl-xL mRNA and protein levels, when compared to the Akata EBV- cells. In addition, Akata EBV+ cells contained less endoplasmic reticulum (ER) than EBV- cells. Finally, downregulation of EBV with EBNA-1 siRNAs caused an increase in the expression of Bcl-xL indicating that EBV is responsible for the differences found between the Akata EBV+ and EBV- cell lines.     

Immune activation suppresses initiation of lytic Epstein-Barr virus infection

Primary infection with Epstein-Barr virus (EBV) is asymptomatic in children with immature immune systems but may manifest as infectious mononucleosis, a vigorous immune activation, in adolescents or adults with mature immune systems. Infectious mononucleosis and chronic immune activation are linked to increased risk for EBV-associated lymphoma. Here we show that EBV initiates progressive lytic infection by expression of BZLF-1 and the late lytic genes gp85 and gp350/220 in cord blood mononuclear cells (CBMC) but not in peripheral blood mononuclear cells (PBMC) from EBV-naive adults after EBV infection ex vivo. Lower levels of proinflammatory cytokines in CBMC, used to model a state of minimal immune activation and immature immunity, than in PBMC were associated with lytic EBV infection. Triggering the innate immunity specifically via Toll-like receptor-9 of B cells substantially suppressed BZLF-1 mRNA expression in acute EBV infection ex vivo and in anti-IgG-stimulated chronically latently EBV-infected Akata Burkitt lymphoma cells. This was mediated in part by IL-12 and IFN-gamma. These results identify immune activation as critical factor for the suppression of initiation of lytic EBV infection. We hypothesize that immune activation contributes to EBV-associated lymphomagenesis by suppressing lytic EBV and in turn promotes latent EBV with transformation potential.     

Down-regulation of proteolytic complexes following EBV activation in BL cells

In Burkitt's lymphoma cells, Epstein Barr virus (EBV) latency products interact with the ubiquitin-proteasome system to promote episomal maintenance and immunological evasion while the tripeptidylpeptidase II (TPPII) functions as an alternative protease. In the present study, we have examined the activities and levels of the proteasome and TPPII complex in Raji and in Akata cells after induction of EBV lytic cycle. The results show that the chymotrypsin-like and caspase-like activities of the proteasome were substantially reduced in Raji and Akata cells. Similarly, TPPII activity was diminished in both cell lines but was recovered in Akata cells at longer time after induction. Protein levels of the alpha/beta subunits of the 20S proteasome and TPPII concentration decreased to different extents after EBV activation, whereas the ubiquitin binding S6' subunit of the 19S regulatory complex increased three to fourfold along with the levels of ubiquitin-conjugates. Collectively, these observations demonstrate impairment of two major cellular proteolytic systems at the onset of EBV lytic infection.     

Malignant transformation of Epstein-Barr virus-negative Akata cells by introduction of the BARF1 gene carried by Epstein-Barr virus

Spontaneous loss of the Epstein-Barr virus (EBV) genome in the BL cell line Akata led to loss of tumorigenicity in SCID mice, suggesting an important oncogenic activity of EBV in B cells. We previously showed that introduction of the BARF1 gene into the human B-cell line Louckes induced a malignant transformation in newborn rats (M. X. Wei, J. C. Moulin, G. Decaussin, F. Berger, and T. Ooka, Cancer Res. 54:1843-1848, 1994). Since 1 to 2% of Akata cells expressed lytic antigens and expressed the BARF1 gene, we investigated whether introduction of the BARF1 gene into EBV-negative Akata cells can induce malignant transformation. Here we show that BARF1-transfected, EBV-negative Akata cells activated Bcl2 expression and induced tumor formation when they were injected into SCID mice. In addition, when EBV-positive Akata cells expressing a low level of BARF1 protein were injected into SCID mice, the expression of BARF1, as well as several lytic proteins, such as EA-D, ZEBRA, and a 135-kDa DNA binding protein, increased in tumor cells while no latent LMP1 and late gp220-320 expression was observed in tumor cells. These observations suggest that the BARF1 gene may be involved in the conferral of tumorigenicity by EBV.     

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.     

Epstein-Barr Virus Contributes to the Malignant Phenotype and to Apoptosis Resistance in Burkitt’s Lymphoma Cell Line Akata

In the present study, we established an in vitro system representing the Burkitt’s lymphoma (BL)-type Epstein-Barr virus (EBV) infection which is characterized by expression of EBV-determined nuclear antigen 1 (EBNA-1) and absence of EBNA-2 and latent membrane protein 1 (LMP1) expression. EBV-negative cell clones isolated from the EBV-positive BL line Akata were infected with an EBV recombinant carrying a selectable marker, and the following selection culture easily yielded EBV-infected clones. EBV-reinfected clones showed BL-type EBV expression and restored the capacity for growth on soft agar and tumorigenicity in SCID mice that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. Moreover, it was found that EBV-positive cells were more resistant to apoptosis than were EBV-negative cells. EBV-infected cells expressed the bcl-2 protein, through which cells might become resistant to apoptosis, at a higher level than did uninfected cells. This is the first report that BL-type EBV infection confers apoptosis resistance even in the absence of expression of LMP1 and BHRF1, both of which are known to have an antiapoptotic function. Surprisingly, transfection of the EBNA-1 gene into EBV-negative Akata clones could not restore malignant phenotypes and apoptosis resistance, thus suggesting that EBNA-1 alone was not sufficient for conferring them. Our results suggest that the persistence of EBV in BL cells is required for the cells to be more malignant and apoptosis resistant, which underlines the oncogenic role of EBV in BL genesis.     

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 BZLF1 gene, a switch from latency to lytic infection, is expressed as an immediate-early gene after primary infection of B lymphocytes

We demonstrate here that the Epstein-Barr virus (EBV) BZLF1 gene, a switch from latent infection to lytic infection, is expressed as early as 1.5 h after EBV infection in Burkitt's lymphoma-derived, EBV-negative Akata and Daudi cells and primary B lymphocytes. Since BZLF1 mRNA is expressed even when the cells are infected with EBV in the presence of anisomycin, an inhibitor of protein synthesis, its expression does not require prerequisite protein synthesis, indicating that BZLF1 is expressed as an immediate-early gene following primary EBV infection of B lymphocytes.     

Latent membrane protein 2B regulates susceptibility to induction of lytic Epstein-Barr virus infection

The B-lymphotropic Epstein-Barr virus (EBV) encodes two isoforms of latent membrane protein 2 (LMP2), LMP2A and LMP2B, which are expressed during latency in B cells. The function of LMP2B is largely unknown, whereas LMP2A blocks B-cell receptor (BCR) signaling transduction and induction of lytic EBV infection, thereby promoting B-cell survival. Transfection experiments on LMP2B in EBV-negative B cells and the silencing of LMP2B in EBV-harboring Burkitt's lymphoma-derived Akata cells suggest that LMP2B interferes with the function of LMP2A, but the role of LMP2B in the presence of functional EBV has not been established. Here, LMP2B, LMP2A, or both were overexpressed in EBV-harboring Akata cells to study the function of LMP2B. The overexpression of LMP2B increased the magnitude of EBV switching from its latent to its lytic form upon BCR cross-linking, as indicated by a more-enhanced upregulation and expression of EBV lytic genes and significantly increased production of transforming EBV compared to Akata vector control cells or LMP2A-overexpressing cells. Moreover, LMP2B lowered the degree of BCR cross-linking required to induce lytic EBV infection. Finally, LMP2B colocalized with LMP2A as demonstrated by immunoprecipitation and immunofluorescence and restored calcium mobilization upon BCR cross-linking, a signaling process inhibited by LMP2A. Thus, our findings suggest that LMP2B negatively regulates the function of LMP2A in preventing the switch from latent to lytic EBV replication.     

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.     

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 small RNAs potentiate tumorigenicity of Burkitt lymphoma cells independently of an effect on apoptosis

The tumorigenic potential of the Burkitt lymphoma (BL) cell line Akata is dependent on the restricted latency program of Epstein-Barr virus (EBV) that is characteristically maintained in BL tumors. Within these cells, EBV-mediated inhibition of apoptosis correlates with an up-regulation of BCL-2 levels in concert with a down-regulation in c-MYC expression that occurs under growth-limiting conditions. Here we addressed whether EBV's effects on apoptosis and tumorigenicity are mediated by the EBV small RNAs EBER-1 and EBER-2. Stable expression of the EBERs in EBV-negative Akata BL cells, at levels comparable to those in EBV-positive cells, significantly enhanced the tumorigenic potential of EBV-negative BL cells in SCID mice, but did not fully restore tumorigenicity relative to EBV-positive Akata cells. Furthermore, wild-type or greater levels of EBER expression in EBV-negative Akata cells did not promote BL cell survival. These data therefore suggest that EBV can contribute to BL through at least two avenues: an EBER-dependent mechanism that enhances tumorigenic potential independent of a direct effect on apoptosis, and a second mechanism, mediated by an as-yet-unidentified EBV gene(s), that offsets the proapoptotic consequences of deregulated c-MYC in BL.     

Phosphatidylinositol 3-kinase is a determinant of responsiveness to B cell antigen receptor-mediated Epstein-Barr virus activation

B cell Ag receptor (BCR) cross-linking with anti-Ig Abs efficiently induces activation of latently infected EBV in some B cell lines, but not in others. The present study was aimed at defining the molecular mechanisms that determine the response to BCR-mediated EBV activation. Comparison of Burkitt's lymphoma-derived Akata, Mutu-I, and Daudi cells, which are representative responders and nonresponders to BCR-mediated EBV activation, respectively, indicated that three signaling pathways, phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK), were activated in anti-Ig-treated Akata and Mutu-I cells. However, in anti-Ig-treated Daudi cells PI3K was not activated, ERK was faintly activated, and p38 MAPK was constitutively phosphorylated irrespective of anti-Ig treatment. Restoration of PI3K activity with insulin-like growth factor 1 restored ERK and p38 MAPK pathways, and was accompanied by EBV activation in anti-Ig-treated Daudi cells. In contrast, a specific inhibitor for PI3K, wortmannin, inhibited EBV activation by anti-Ig Abs in Akata and Mutu-I cells. Transfection assays in EBV-negative Daudi cells revealed that PI3K activated a promoter for BZLF1, which is a switch of EBV activation from a latent infection, in the absence of other EBV products suggesting that the BZLF promoter was a target of BCR signaling, and that PI3K was important for BCR-mediated BZLF1 activation. These results indicate that the absence of PI3K impedes the progression of signals through the BCR and becomes a determinant of unresponsiveness to BCR-mediated EBV activation.     

Epstein-Barr virus transforming protein LMP1 plays a critical role in virus production

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), which is critical for EBV-induced B-cell transformation, is also abundantly expressed during the lytic cycle of viral replication. However, the biological significance of this strong LMP1 induction remains unknown. We engineered a bacterial artificial chromosome clone containing the entire genome of Akata strain EBV to specifically disrupt the LMP1 gene. Akata cell clones harboring the episomes of LMP1-deleted EBV were established, and the effect of LMP1 loss on virus production was investigated. We found that the degree of viral DNA amplification and the expression levels of viral late gene products were unaffected by LMP1 loss, demonstrating that the LMP1-deleted EBV entered the lytic replication cycle as efficiently as the wild-type counterpart. This was confirmed by our electron microscopic observation that nucleocapsid formation inside nuclei occurred even in the absence of LMP1. By contrast, loss of LMP1 severely impaired virus release into culture supernatants, resulting in poor infection efficiency. The expression of truncated LMP1 in Akata cells harboring LMP1-deleted EBV rescued the virus release into the culture supernatant and the infectivity, and full-length LMP1 partially rescued the infectivity. These results indicate that inducible expression of LMP1 during the viral lytic cycle plays a critical role in virus production.     

The role of p38 mitogen-activated protein kinase in regulating interleukin-10 gene expression in Burkitt's lymphoma cell lines

In malignant B lymphoma cells interleukin-10 (IL-10) expression is frequently upregulated. This effect is thought to support to the malignant transformation of these cells and to be a potential target for pharmacotherapy. To define better the mechanism for upregulation of the IL-10 gene, we tested the association between IL-10 and p38 mitogen-activated protein kinase (MAPK) in several Epstein-Barr virus (EBV) infected and non-infected Burkitt's lymphoma (BL) cell lines. The all BL cell lines expressed IL-10 and IL-10 receptor mRNAs, and produced IL-10. p38 MAPK was constitutively phosphorylated in the cytoplasm of the BL cell lines. We further analyzed molecular effects of p38 MAPK on IL-10 expression in Akata cells. Exogenous IL-10 lead rapidly to phosphorylation of Jak1 and Tyk2 as transducers of signals of IL-10, and promoted growth of Akata cells in a dose-dependent manner. The phosphorylation of cytoplasmic p38 MAPK in Akata cells was reduced by the serine/threonine kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7). A specific inhibitor of p38 MAPK, SB203580, blocked simultaneously STAT3 DNA-binding activity, and IL-10 mRNA expression, IL-10 production, and then the cell growth was inhibited. These results indicate that the p38 MAPK pathway is functionally linked to IL-10 gene expression and supports the view that the constitutive activation of cytoplasmic p38 MAPK in BL cells is a step in the upregulation of IL-10 gene expression and lymphomagenesis.     

Epstein-Barr virus lytic infection induces retinoic acid-responsive genes through induction of a retinol-metabolizing enzyme, DHRS9

Lytic Epstein-Barr virus (EBV) replication occurs in differentiated, but not undifferentiated, epithelial cells. Retinoic acid (RA) induces epithelial cell differentiation. The conversion of retinol into its active form, retinoic acid, requires retinol dehydrogenase enzymes. Here we show that AGS gastric carcinoma cells containing the lytic form of EBV infection have enhanced expression of a gene (DHRS9) encoding an enzyme that mediates conversion of retinol into RA. DHRS9 expression is also increased following induction of lytic viral infection in EBV-positive Burkitt lymphoma cells. We demonstrate that the EBV immediate-early protein, BZLF1, activates the DHRS9 promoter through a direct DNA binding mechanism. Furthermore, BZLF1 expression in AGS cells is sufficient to activate DHRS9 gene expression and increases the ability of retinol to induce the RA-responsive gene, CYP26A1. Production of RA during the lytic form of EBV infection may enhance viral replication by promoting keratinocyte differentiation.