The lytic cycle of Epstein-Barr virus in the nonproducer Raji line can be rescued by the expression of a 135-kilodalton protein encoded by the BALF2 open reading frame.

In Epstein-Barr virus (EBV)-carrying nonproducer Raji cells, the induction of the viral replicative cycle by chemical treatment is limited to only the early stage and viral DNA synthesis is totally inhibited. We previously showed the absence of two messenger RNAs that are encoded by the BamHI-A fragment of the EBV genome and that correspond to open reading frames BALF2 and BARF1 in chemically induced Raji cells. Since the BALF2 gene encodes a 135-kDa DNA-binding protein which was immunoprecipitated by antibody against ICP8 protein, a key protein in herpes simplex virus replication, we asked whether the lack of productive cycle in Raji cells is due to the absence of expression of the BALF2 gene. We transfected the Raji cell line with the BALF2 gene. After chemical induction, the BALF2-transfected cells expressed not only early antigens but also late antigens. In these cultures, the viral particles were detected by electron microscopy. The expression of late antigens was completely inhibited by arabinofuranosylthymine, which is a specific inhibitor of viral DNA replication. The BALF2 gene might play an essential role in the induction of the EBV-lytic cycle.     

Epstein-Barr virus BALF1 is a BCL-2-like antagonist of the herpesvirus antiapoptotic BCL-2 proteins

Cellular BCL-2 family proteins can inhibit or induce programmed cell death in part by counteracting the activity of other BCL-2 family members. All sequenced gammaherpesviruses encode a BCL-2 homologue that potently inhibits apoptosis and apparently escapes some of the regulatory mechanisms that govern the functions of their cellular counterparts. Examples of these protective proteins include BHRF1 of Epstein-Barr virus (EBV) and KSBcl-2 of Kaposi's sarcoma-associated herpesvirus, also known as human herpesvirus 8. The gamma-1 subgroup of these viruses, such as EBV, encodes a second BCL-2 homologue. We have now found that this second BCL-2 homologue encoded by EBV, BALF1, inhibits the antiapoptotic activity of EBV BHRF1 and of KSBcl-2 in several transfected cell lines. However, BALF1 failed to inhibit the cellular BCL-2 family member, BCL-x(L). Thus, BALF1 acts as a negative regulator of the survival function of BHRF1, similar to the counterbalance observed between cellular BCL-2 family members. Unlike the cellular BCL-2 family antagonists, BALF1 lacked proapoptotic activity and could not be converted into a proapoptotic factor in a manner similar to cellular BCL-2 proteins by caspase cleavage or truncation of the N terminus. Coimmunoprecipitation experiments and immunofluorescence assays suggest that a minimal amount, if any, of the BHRF1 and BALF1 proteins colocalizes inside cells, suggesting that mechanisms other than direct interaction explain the suppressive function of BALF1.     

The Epstein-Barr Virus (EBV) gN Homolog BLRF1 Encodes a 15-Kilodalton Glycoprotein That Cannot Be Authentically Processed unless It Is Coexpressed with the EBV gM Homolog BBRF3

The Epstein-Barr virus (EBV) homolog of the conserved herpesvirus glycoprotein gN is predicted to be encoded by the BLRF1 open reading frame (ORF). Antipeptide antibody to a sequence corresponding to residues in the predicted BLRF1 ORF immunoprecipitated a doublet of approximately 8 kDa from cells expressing the BLRF1 ORF as a recombinant protein. In addition, four glycosylated proteins of 113, 84, 48, and 15 kDa could be immunoprecipitated from virus-producing cells by the same antibody. The 15-kDa species was the mature form of gN, which carried α2,6-sialic acid residues. The remaining glycoproteins which associated with gN were products of the BBRF3 ORF of EBV, which encodes the EBV gM homolog. The 8-kDa doublet seen in cells expressing recombinant gN comprised precursors of the mature 15-kDa gN. Coexpression of EBV gM with EBV gN was required for authentic processing of the 8-kDa forms to the 15-kDa form.     

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.     

trans-acting requirements for replication of Epstein-Barr virus ori-Lyt.

Epstein-Barr virus (EBV) utilizes a completely different mode of DNA replication during the lytic cycle than that employed during latency. The latency origin of replication, ori-P, which functions in the replication of the latent episomal form of the EBV genome, requires only a single virally encoded protein, EBNA-1, for its activity. During the lytic cycle, a separate origin, ori-Lyt, is utilized. Relatively little is known about the trans-acting proteins involved in ori-Lyt replication. We established a cotransfection-replication assay to identify EBV genes whose products are required for replication of ori-Lyt. In this assay, a BamHI-H plasmid containing ori-Lyt was replicated in Vero cells cotransfected with the BamHI-H target, the three EBV lytic-cycle transactivators Zta, Rta, and Mta, and the EBV genome provided in the form of a set of six overlapping cosmid clones. By removing individual cosmids from the cotransfection mixture, we found that only three of the six cosmids were necessary for ori-Lyt replication. Subcloning of the essential cosmids led to the identification of six EBV genes that encode replication proteins. These genes and their functions (either known or predicted on the basis of sequence comparison with herpes simplex virus) are BALF5, the DNA polymerase; BALF2, the single-stranded DNA-binding protein homolog; BMRF1, the DNA polymerase processivity factor; BSLF1 and BBLF4, the primase and helicase homologs; and BBLF2/3, a potential homolog of the third component of the helicase-primase complex. In addition, ori-Lyt replication in this cotransfection assay was also dependent on one or more genes provided by the EBV SalI-F fragment and on the three lytic-cycle transactivators Zta, Rta, and Mta.     

Role of bcl-2 in Epstein-Barr virus-induced malignant conversion of Burkitt's lymphoma cell line Akata

We have demonstrated that Epstein-Barr virus (EBV) confers enhanced growth capability in soft agarose, tumorigenesis in the SCID mouse, and resistance to apoptosis in the Burkitt's lymphoma cell line Akata. Subsequently, we have shown that EBV-encoded small RNAs (EBERs) are responsible for these phenotypes. We constantly observed the upregulation of bcl-2 oncoprotein expression upon EBV infection and expression of EBERs. To test whether these phenotypes were due to the upregulation of bcl-2 expression, we introduced bcl-2 into EBV-negative Akata cells at various levels encompassing the range at which EBV-positive cells expressed it. As cells expressed bcl-2 at higher levels, they became more capable of growing in soft agarose and became resistant to apoptosis. However, clones expressing bcl-2 at a higher level than EBV-positive Akata cells were negative in the tumorigenesis assay in the SCID mouse. On the other hand, introduction of bax into EBV-positive Akata cells reduced the resistance to apoptosis; however, it failed to reduce the growth capability in soft agarose. These data indicate that EBV targets not only bcl-2, but also an unknown pathway(s) to enhance the oncogenic potential of Akata cells.     

Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death.

Epstein-Barr virus (EBV) not only induces growth transformation in human B lymphocytes, but has more recently been shown to enhance B cell survival under suboptimal conditions where growth is inhibited; both effects are mediated through the coordinate action of eight virus-coded latent proteins. The effect upon cell survival is best recognized in EBV-positive Burkitt's lymphoma cell lines where activation of full virus latent gene expression protects the cells from programmed cell death (apoptosis). Here we show by DNA transfection into human B cells that protection from apoptosis is conferred through expression of a single EBV latent protein, the latent membrane protein LMP 1. Furthermore, we demonstrate that LMP 1 mediates this effect by up-regulating expression of the cellular oncogene bcl-2. The interplay between EBV infection and expression of this cellular oncogene has important implications for virus persistence and for the pathogenesis of virus-associated malignant disease.     

Correlation between EBV DNA and rearrangement and expression of Bcl-2 gene in aggressive non-Hodgkin's lymphoma

Previous in vitro studies have shown that bcl-2 expression can be induced by transfection of Epstein-Barr virus (EBV)-negative non-Hodgkin's lymphoma (NHL) cell lines with EBV. This induced expression of bcl-2 is important for the long survival of EBV-positive cells and might be a first step in tumorigenesis. The purpose of the present study was to investigate the possibility of similar correlation between bcl-2 expression and EBV infection in vivo in a cohort of patients with aggressive NHL, who were uniformly evaluated and treated with effective chemotherapy. The 42 patients included were 25-65 years old. None had prior treatment, discordant lymphoma, or human immunodeficiency virus seropositivity. Fresh biopsied samples were obtained and stored frozen for analysis of bcl-2 gene rearrangement major break point and of EBV DNA by PCR. Bcl-2 protein expression was estimated by Western blot, and enzyme immunoassay. With a median follow-up of 30 months, overall survival (OS) and disease-free survival (DFS) were measured to determine the prognostic significance of these variables. Analyzable DNA was present in all samples, 24% demonstrating bcl-2 rearrangement and 33% showing EBV DNA. Patients with bcl-2 gene rearrangement tended to have shorter DFS, and OS than patients without translocation. Bcl-2 protein expression was not correlated to gene rearrangement and had no significant influence on survival. The presence of EBV DNA in NHL had no prognostic significance but was correlated to bcl-2 expression. EBV-positive tumors showed higher bcl-2 expression than EBV-negative tumors did. Our results suggest a role of EBV infection in inducing bcl-2 expression as a survival factor for EBV-positive cells.     

The Epstein-Barr virus pol catalytic subunit physically interacts with the BBLF4-BSLF1-BBLF2/3 complex

The Epstein-Barr virus (EBV)-encoded replication proteins that account for the basic reactions at the replication fork are thought to be the EBV Pol holoenzyme, consisting of the BALF5 Pol catalytic and the BMRF1 Pol accessory subunits, the putative helicase-primase complex, comprising the BBLF4, BSLF1, and BBLF2/3 proteins, and the BALF2 single-stranded DNA-binding protein. Immunoprecipitation analyses using anti-BSLF1 or anti-BBLF2/3 protein-specific antibody with clarified lysates of B95-8 cells in a viral productive cycle suggested that the EBV Pol holoenzyme physically interacts with the BBLF4-BSLF1-BBLF2/3 complex to form a large complex. Although the complex was stable in 500 mM NaCl and 1% NP-40, the BALF5 protein became dissociated in the presence of 0.1% sodium dodecyl sulfate. Experiments using lysates from insect cells superinfected with combinations of recombinant baculoviruses capable of expressing each of viral replication proteins showed that not the BMRF1 Pol accessory subunit but rather the BALF5 Pol catalytic subunit directly interacts with the BBLF4-BSLF1-BBLF2/3 complex. Furthermore, double infection with pairs of recombinant viruses revealed that each component of the BBLF4-BSLF1-BBLF2/3 complex makes contact with the BALF5 Pol catalytic subunit. The interactions of the EBV DNA polymerase with the EBV putative helicase-primase complex warrant particular attention because they are thought to coordinate leading- and lagging-strand DNA synthesis at the replication fork.     

Pin1 Interacts with the Epstein-Barr Virus DNA Polymerase Catalytic Subunit and Regulates Viral DNA Replication

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein is known as a regulator which recognizes phosphorylated Ser/Thr-Pro motifs and increases the rate of cis and trans amide isomer interconversion, thereby altering the conformation of its substrates. We found that Pin1 knockdown using short hairpin RNA (shRNA) technology resulted in strong suppression of productive Epstein-Barr virus (EBV) DNA replication. We further identified the EBV DNA polymerase catalytic subunit, BALF5, as a Pin1 substrate in glutathione S-transferase (GST) pulldown and immunoprecipitation assays. Lambda protein phosphatase treatment abolished the binding of BALF5 to Pin1, and mutation analysis of BALF5 revealed that replacement of the Thr178 residue by Ala (BALF5 T178A) disrupted the interaction with Pin1. To further test the effects of Pin1 in the context of virus infection, we constructed a BALF5-deficient recombinant virus. Exogenous supply of wild-type BALF5 in HEK293 cells with knockout recombinant EBV allowed efficient synthesis of viral genome DNA, but BALF5 T178A could not provide support as efficiently as wild-type BALF5. In conclusion, we found that EBV DNA polymerase BALF5 subunit interacts with Pin1 through BALF5 Thr178 in a phosphorylation-dependent manner. Pin1 might modulate EBV DNA polymerase conformation for efficient, productive viral DNA replication.     

Phosphoinositol 3 kinase inhibitor, LY294002 increases bcl-2 protein and inhibits okadaic acid-induced apoptosis in Bcl-2 expressing renal epithelial cells

Protein phosphorylation plays an indispensable role in cellular regulation of mitosis, metabolism, differentiation, and death. We previously reported that the protein phosphatase inhibitor okadaic acid (OKA) induces apoptosis in renal epithelial cells in culture. In the present study, we examined the role of phosphotidylinositol 3 (PI3) kinase signaling in okadaic acid-induced apoptosis by pre-treating normal rat kidney renal epithelial cells expressing human bcl-2 with the PI3 kinase inhibitors, LY294002 and wortmannin, followed by apoptosis-inducing concentrations of okadaic acid. Given the reported cell survival activity of PI3 kinase signaling mostly attributed to Akt kinase activation, we hypothesized that inhibition of PI3 kinase would enhance okadaic-induced apoptosis. Surprisingly, our data show that pretreatment with LY294002, but not wortmannin, attenuated okadaic acid-induced apoptosis. In contrast, to LY294002, wortmannin enhanced apoptosis. Interestingly, we also found that LY294002 treatment increased bcl-2 protein levels in normal rat kidney epithelial cells expressing bcl-2 (NRK-bcl-2). In untreated cells, bcl-2 appeared to be mainly perinuclear, coincident with the nuclear membrane, or in the cytosol. In OKA treated cells that were pre-treated with Ly294002, bcl-2 was highly co-localized with mitochondria, but in cells treated with okadaic acid alone, bcl-2 was associated with fragmented chromatin. In this model, it appears that LY294002 may exert anti-apoptotic effects by a previously unreported treatment related increase in bcl-2. Although it is widely accepted that bcl-2 protein can inhibit apoptosis, we propose that the subcellular location of bcl-2 is an important determinant in whether bcl-2 effectively inhibits apoptosis.     

Activation of Akt2 Inhibits anoikis and apoptosis induced by myogenic differentiation.

Akt2, a homolog of Akt1, encodes a serine/threonine protein kinase that is amplified in ovarian and pancreatic cancers. The antiapoptotic activities of the Akt1 proto-oncogene product have been well documented, but the role of Akt2 in cellular survival is poorly understood. Here, we demonstrate that Akt2 mRNA, protein and kinase activity are upregulated during serum deprivation-induced C2C12 cell myogenic differentiation, a process that is associated with the acquisition of an apoptosis-resistant phenotype. Transient transfection of plasmids encoding wild-type and constitutively-active Akt2 conferred resistance against apoptosis in differentiating C2C12 cells, while a kinase-negative Akt2 construct did not. Adenovirus-mediated transfer of the constitutively-active Akt2 cDNA also suppressed apoptosis during serum deprivation-induced myogenic differentiation and it protected cells from apoptosis induced by cell detachment. These data indicate that Akt2 functions as an anti-apoptotic gene during cellular differentiation, a property that may contribute to its oncogenicity.     

The MHV68 M2 protein drives IL-10 dependent B cell proliferation and differentiation

Murine gammaherpesvirus 68 (MHV68) establishes long-term latency in memory B cells similar to the human gammaherpesvirus Epstein Barr Virus (EBV). EBV encodes an interleukin-10 (IL-10) homolog and modulates cellular IL-10 expression; however, the role of IL-10 in the establishment and/or maintenance of chronic EBV infection remains unclear. Notably, MHV68 does not encode an IL-10 homolog, but virus infection has been shown to result in elevated serum IL-10 levels in wild-type mice, and IL-10 deficiency results in decreased establishment of virus latency. Here we show that a unique MHV68 latency-associated gene product, the M2 protein, is required for the elevated serum IL-10 levels observed at 2 weeks post-infection. Furthermore, M2 protein expression in primary murine B cells drives high level IL-10 expression along with increased secretion of IL-2, IL-6, and MIP-1alpha. M2 expression was also shown to significantly augment LPS driven survival and proliferation of primary murine B cells. The latter was dependent on IL-10 expression as demonstrated by the failure of IL10-/- B cells to proliferate in response to M2 protein expression and rescue of M2-associated proliferation by addition of recombinant murine IL-10. M2 protein expression in primary B cells also led to upregulated surface expression of the high affinity IL-2 receptor (CD25) and the activation marker GL7, along with down-regulated surface expression of B220, MHC II, and sIgD. The cells retained CD19 and sIgG expression, suggesting differentiation to a pre-plasma memory B cell phenotype. These observations are consistent with previous analyses of M2-null MHV68 mutants that have suggested a role for the M2 protein in expansion and differentiation of MHV68 latently infected B cells-perhaps facilitating the establishment of virus latency in memory B cells. Thus, while the M2 protein is unique to MHV68, analysis of M2 function has revealed an important role for IL-10 in MHV68 pathogenesis-identifying a strategy that appears to be conserved between at least EBV and MHV68.     

Endothelial dysfunction in the early- and late-stage type-2 diabetic Goto-Kakizaki rat aorta

Hodgkin and Reed-Sternberg (H-RS) cells of classical Hodgkin lymphoma (cHL) present an impaired expression of immunoglobulin genes, but escape apoptotic death. We investigated whether nitric oxide synthases (NOS) are expressed by H-RS cells, studied their association with EBV status and the expression of apoptotic proteins, and investigated their relationship to the clinical outcome of 171 patients. NOS1 and NOS2 were expressed in a large number of cases, whereas NOS3 expression was not detected. Positive associations were found between NOS1 and p53, bax and NOS2, bcl-2 and NOS2, bax and p53, and between bax and fasL. Inverse correlations were established between EBV and NOS2 and between EBV and bcl-2. A shorter overall survival (OS) was associated with strong expression of NOS2. In conclusion, NOS are expressed by H-RS cells of cHL.     

Expression of bcl-2 protein in bronchoalveolar lavage cell populations from patients with idiopathic pulmonary fibrosis.

OBJECTIVE: To investigate changes in the expression of the antiapoptotic protein bcl-2 in bronchoalveolar lavage fluid (BALF) cell populations in patients with idiopathic pulmonary fibrosis (IPF). STUDY DESIGN: Ten patients with IPF underwent fiberoptic bronchoscopy and bronchoalveolar lavage (BAL) in the area of maximal radiographic shadowing (based on high-resolution computed tomography findings). Results were compared with those of 10 normal people in the control group. Cellular bcl-2 expression was identified using an immunoperoxidase staining method. RESULTS: A statistically significant (P < .001) increase in the expression of bcl-2 in BALF neutrophils and eosinophils was observed in patients with IPF as compared with controls. BAL macrophages exhibited only a slight (statistically insignificant) increase in bcl-2 expression in IPF patients. No bcl-2 expression was observed in BAL lymphocytes from IPF patients in contrast to the control group. CONCLUSION: The overexpression of bcl-2 on BALF neutrophils and eosinophils, cells that characterize the special cellular profile of alveolitis in IPF, could be one of the pathophysiologic mechanisms of this disease.