Epstein-Barr virus encodes a novel homolog of the bcl-2 oncogene that inhibits apoptosis and associates with Bax and Bak

The sequenced gammaherpesviruses each contain a single viral bcl-2 homolog (v-bcl-2) which may encode a protein that functions in preventing the apoptotic death of virus-infected cells. Epstein-Barr virus (EBV), a gammaherpesvirus associated with several lymphoid and epithelial malignancies, encodes the v-Bcl-2 homolog BHRF1. In this report the previously uncharacterized BALF1 open reading frame in EBV is identified as having significant sequence similarity to other v-bcl-2 homologs and cellular bcl-2. Transfection of cells with a BALF1 cDNA conferred apoptosis resistance. Furthermore, a recombinant green fluorescent protein-BALF1 fusion protein suppressed apoptosis and associated with Bax and Bak. These results indicate that EBV encodes a second functional v-bcl-2.     

Molecular evolution and secondary structural conservation in the B-cell lymphoma leukemia 2 (bcl-2) family of proto-oncogene products

The nature of the bcl-2 family of protooncogenes was analyzed by sequence alignment, secondary structure prediction, and phylogenetic techniques. Phylogenies were inferred from both the nucleic acid and amino acid sequences of the human, murine, rat, and chicken sequences for BCL-2 and BCL-X, human MCL1, murine A1, the nematode Caenorhabditis elegans and Caenorhabditis briggsiae ced-9 proteins, and the sequences BHRF1 from Epstein-Barr and LMW5-HL from African swine fever viruses. Both sequence alignment and secondary structure prediction techniques supported the conservation of both the overall secondary structure and the carboxy-terminal transmembrane domain in all members of the family. All the treeing methods employed (distance matrix, maximum likelihood, and parsimony) supported a tree in which the proapoptotic proteins BCL-2 and BCL-X represent the most recent additions to the group. All the trees also indicated that the viral proteins BHRF1 and LMW-HL arose from a common ancestor, an ancestor they shared in common with the pro-apoptotic control protein BAX, indicating that this function of BAX evolved only recently. The most ancient branches are represented by the nematode ced-9 protein and by the control genes MCL1 and A1, which in the treeing methods employed represent separate lineages within the most ancient grouping. These results demonstrate the evolution of a highly conserved family of developmental control genes from nematode to man—genes that encode proteins essential for normal development but which are highly conserved in terms of predicted structure and possible cellular localization. The evolutionary analysis also indicates that the family may be even larger than originally predicted and that other members are waiting to be discovered. Correspondence to: D. Lloyd Evans     

BHRF1, the Epstein-Barr virus gene with homology to Bc12, is dispensable for B-lymphocyte transformation and virus replication.

The Epstein-Barr virus (EBV) BHRF1 open reading frame is abundantly expressed early in the lytic replication cycle. BHRF1 is also transiently expressed in some latently infected cell lines in the absence of expression of other lytic cycle proteins. BHRF1 shares distant, but significant, colinear primary amino acid sequence homology to Bc12, a cellular gene strongly implicated in the evolution of follicular lymphoma. The experiments reported here used a molecular genetic approach to examine the role of BHRF1 in EBV infection. Isogenic EBV recombinants having either wild-type BHRF1 or a null mutation due to a translational stop signal in place of the 24th BHRF1 codon were used to infect primary B lymphocytes. The BHRF1 mutant recombinants did not differ from the wild type in their ability to infect and transform the growth of primary B lymphocytes, to replicate in the resultant lymphoblastoid cell lines, or to initiate a second round of primary cell transformation. Deletion of the entire BHRF1 open reading frame did not destroy the ability of the mutant virus to maintain cell growth transformation. The significance of these findings with regard to the role of BHRF1 in EBV infection is discussed.     

Identification of an African swine fever virus gene with similarity to a myeloid differentiation primary response gene and a neurovirulence-associated gene of herpes simplex virus.

Here we describe an open reading frame (LMW23-NL) in the African swine fever virus genome that possesses striking similarity to a murine myeloid differentiation primary response gene (MyD116) and the neurovirulence-associated gene (ICP34.5) of herpes simplex virus. In all three proteins, a centrally located acidic region precedes a highly conserved, hydrophilic 56-amino-acid domain located at the carboxy terminus. LMW23-NL predicts a highly basic protein of 184 amino acids with an estimated molecular mass of 21.3 kDa. The similarity of LMW23-NL to genes involved in myeloid cell differentiation and viral host range suggests a role for it in African swine fever virus host range.     

BHRF1 of Epstein-Barr virus, which is homologous to human proto-oncogene bcl2, is not essential for transformation of B cells or for virus replication in vitro.

The Epstein-Barr virus (EBV) genome contains an open reading frame, BHRF1, that encodes a presumptive membrane protein with sequence similarity to the proto-oncogene bcl2, which is linked to human B-cell follicular lymphoma. Potential roles for BHRF1 in EBV's ability to growth transform human B cells and to replicate in B cells in culture were investigated by generating EBV mutants that lack most of the open reading frame. This was accomplished by recombination of plasmids carrying mutations in BHRF1 with the transformation-defective EBV strain P3HR1. Because BHRF1 resides close to the deletion in P3HR1 that renders this strain transformation defective, B-cell transformation could be used to select for recombination events in the region. B-cell clones were established by recombinants which lacked most of the BHRF1 open reading frame, although most of these initial B-cell transformants also carried nonrecombinant (BHRF1+) P3HR1 genomes, at levels ranging from a fraction of a copy to four copies per cell. Secondary B-cell transformants that lacked BHRF1+ EBV at detectable levels were found to release transforming, BHRF1-deficient EBV at levels that were within the normal range for EBV-immortalized B-cell clones. These studies demonstrate that BHRF1 is nonessential for growth transformation of B cells and for virus replication and release from these cells in culture.     

Suppression of c-Myc-Induced Apoptosis by the Epstein-Barr Virus Gene Product BHRF1

Constitutive expression of the c-myc proto-oncogene in growth factor-deprived fibroblasts promotes proliferation and induces apoptosis. In these cells, apoptosis can be inhibited by survival factors such as insulin-like growth factor I or the bcl-2 proto-oncogene product. Deregulated c-Myc expression is a common feature in Epstein-Barr virus-positive Burkitt’s lymphoma in which the c-myc gene is reciprocally translocated and placed under the control of one of the immunoglobulin loci. BHRF1 is an Epstein-Barr virus protein expressed early in the lytic cycle. BHRF1 is a member of the Bcl-2 family and has been shown to suppress apoptosis and to increase cell survival in different settings. In the present study, we report that BHRF1 inhibits c-Myc-induced apoptosis which occurs in the absence of survival factors. It does not, however, affect the capacity of c-Myc to promote cell growth. These findings demonstrate that BHRF1 has not only structural but also functional similarities to Bcl-2.     

Structural basis for apoptosis inhibition by Epstein-Barr virus BHRF1

Epstein-Barr virus (EBV) is associated with human malignancies, especially those affecting the B cell compartment such as Burkitt lymphoma. The virally encoded homolog of the mammalian pro-survival protein Bcl-2, BHRF1 contributes to viral infectivity and lymphomagenesis. In addition to the pro-apoptotic BH3-only protein Bim, its key target in lymphoid cells, BHRF1 also binds a selective sub-set of pro-apoptotic proteins (Bid, Puma, Bak) expressed by host cells. A consequence of BHRF1 expression is marked resistance to a range of cytotoxic agents and in particular, we show that its expression renders a mouse model of Burkitt lymphoma untreatable. As current small organic antagonists of Bcl-2 do not target BHRF1, the structures of it in complex with Bim or Bak shown here will be useful to guide efforts to target BHRF1 in EBV-associated malignancies, which are usually associated with poor clinical outcomes.     

Solution structure of the BHRF1 protein from Epstein-Barr virus, a homolog of human Bcl-2

The three-dimensional structure of BHRF1, the Bcl-2 homolog from Epstein-Barr virus (EBV), has been determined by NMR spectroscopy. Although the overall structure is similar to other Bcl-2 family members, there are important structural differences. Unlike some of the other Bcl-2 family members, BHRF1 does not contain the prominent hydrophobic groove that mediates binding to pro-apoptotic family members. In addition, in contrast to the anti-apoptotic Bcl-2 proteins, BHRF1 does not bind tightly to peptides derived from the pro-apoptotic proteins Bak, Bax, Bik, and Bad. The lack of an exposed, pre-formed binding groove in BHRF1 and the lack of significant binding to peptides derived from pro-apoptotic family members that bind to other anti-apoptotic family members, suggest that the mechanism of the BHRF1 anti-apoptotic activity does not parallel that of cellular Bcl-x(L) or Bcl-2.     

The cellular protein PRA1 modulates the anti-apoptotic activity of Epstein-Barr virus BHRF1, a homologue of Bcl-2, through direct interaction

The Epstein-Barr virus-encoded early protein, BHRF1, is a structural and functional homologue of the anti-apoptotic protein, Bcl-2. There is accumulating evidence that BHRF1 protects a variety of cell types from apoptosis induced by various external stimuli. To identify specific proteins from normal epithelial cells that interact with BHRF1 and that might promote or inhibit its anti-apoptotic activity, we screened a yeast two-hybrid cDNA library derived from human normal foreskin keratinocytes and identified a cellular gene encoding human prenylated rab acceptor 1 (hPRA1). The interaction of hPRA1 with BHRF1 was confirmed using glutathione S-transferase pull-down assays, confocal laser scanning microscopy, and co-immunoprecipitation. Two regions of PRA1, amino acids 30-53 and the carboxyl-terminal 21 residues, are important for BHRF1 interactions and two regions of BHRF1, amino acids 1-18 and 89-142, including the Bcl-2 homology domains BH4 and BH1, respectively, are crucial for PRA1 interactions. PRA1 expression interferes with the anti-apoptotic activity of BHRF1, although not of Bcl-2. These results indicate that the PRA1 interacts selectively with BHRF1 to reduce its anti-apoptotic activity and might play a role in the impeding completion of virus maturation.     

<Emphasis Type="Italic">Herpesvirus pan</Emphasis> encodes a functional homologue of BHRF1, the Epstein-Barr virus v-Bcl-2

Background  

Epstein-Barr virus (EBV) latently infects about 90% of the human population and is associated with benign and malignant diseases of lymphoid and epithelial origin. BHRF1, an early lytic cycle antigen, is an apoptosis suppressing member of the Bcl-2 family. In vitro studies imply that BHRF1 is dispensable for both virus replication and transformation. However, the fact that BHRF1 is highly conserved not only in all EBV isolates studied to date but also in the analogous viruses Herpesvirus papio and Herpesvirus pan that infect baboons and chimpanzees respectively, suggests BHRF1 may play an important role in vivo.     

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.     

cis-Acting effects on RNA processing and Drosha cleavage prevent Epstein-Barr virus latency III BHRF1 expression

In Epstein-Barr virus (EBV) latency III (LTIII) infection, BHRF1 encodes three microRNAs (miRNAs). Herein we report that Drosha cleavage of LTIII BHRF1 RNA and cis-acting splicing effects inhibit splicing and inhibit BHRF1 RNA and protein expression. Evidence shown here supports the view that Drosha cleavage to generate mature miRNAs and cis-acting sequences that prevent mRNA maturation are independent processes that prevent LTIII BHRF1 expression in lymphoblastoid cell lines.     

An African swine fever virus Bc1-2 homolog, 5-HL, suppresses apoptotic cell death.

Here, we show that the African swine fever virus 5-HL gene is a highly conserved viral gene and contains all known protein domains associated with Bcl-2 activity, including those involved with dimerization, mediating cell death, and protein-binding functions, and that its protein product, p21, suppresses apoptotic cell death in the mammalian lymphoid cell line FL5.12. Thus, 5-HL is a true functional viral member of the Bcl-2 gene family.     

Epstein-Barr virus BHRF1 protein protects intestine 407 epithelial cells from apoptosis induced by tumor necrosis factor alpha and anti-Fas antibody.

Tumor necrosis factor (TNF) and cytotoxic T lymphocytes, which utilize Fas to induce apoptosis in target cells, are known to play a critical role in the host defense against viral infection. In this study, the Epstein-Barr virus BHRF1 protein was stably expressed in intestine 407 cells which were susceptible to cell death mediated through both the TNF receptor and Fas. WST-1 conversion assays and acridine orange staining showed that vector-transfected control cells were killed by TNF-alpha or anti-Fas antibody in a dose-dependent manner, whereas BHRF1-expressing cells were resistant to apoptosis induced by these mediators. DNA fragmentation, a characteristic of apoptosis induced by TNF-alpha and the anti-Fas antibody, was suppressed in BHRF1-expressing cells. These results indicate that the BHRF1 protein protects cells from apoptosis mediated by the TNF receptor and Fas. The role of BHRF1 as an inhibitor of cytokine-induced apoptosis during the Epstein-Barr virus lytic cycle in vivo is discussed.     

Epstein-Barr virus BHRF1 functions downstream of Bid cleavage and upstream of mitochondrial dysfunction to inhibit TRAIL-induced apoptosis in BJAB cells

We have previously reported that TNF-related apoptosis inducing ligand (TRAIL) causes cleavage of Bid via activation of caspase-8 and the loss of mitochondrial membrane potential (DeltaPsim), resulting in apoptosis. Experiments with BJAB clones expressing Epstein-Barr virus (EBV) anti-apoptotic protein BHRF1 showed that BHRF1 drastically inhibited TRAIL-mediated apoptosis. Although Western blot analysis demonstrated that TRAIL-induced Bid cleavage was not inhibited by BHRF1, the decrease in DeltaPsim caused by TRAIL was effectively blocked by BHRF1. These findings suggest that in BJAB cells, BHRF1 acts downstream of Bid cleavage and upstream of mitochondrial damage, resulting in inhibition of TRAIL-induced apoptosis.     

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) forms complexes with a cellular anti-apoptosis protein Bcl-2 or its EBV counterpart BHRF1 through HS1-associated protein X-1

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) plays a critical role in EBV-induced transformation. An earlier report (Y. Kawaguchi et al., J. Virol. 74: 10104-10111, 2000) showed that EBNA-LP interacts with a cellular protein HS1-associated protein X-1 (HAX-1). The predicted amino acid sequence of HAX-1 exhibits similarity to that of another cellular protein Nip3 which has been shown to interact with cellular and viral anti-apoptotic proteins such as Bcl-2 and BHRF1, an EBV homolog of Bcl-2. Here we investigated whether HAX-1, like Nip3, interacts with Bcl-2 proteins and report the following. (i) A purified chimeric protein consisting of gluthathione S-transferase (GST) fused to BHRF1 (GST-BHRF1) or Bcl-2 (GST-Bcl-2) specifically pulled down HAX-1 transiently expressed in COS-7 cells. (ii) GST-BHRF1 or GST-Bcl-2 was not able to pull down EBNA-LP transiently expressed in COS-7 cells, whereas each of the GST fusion proteins formed complexes with EBNA-LP in the presence of RAX-1. These results indicated that EBNA-LP interacts with the viral and cellular Bcl-2 proteins through HAX-1, suggesting that EBNA-LP possesses a potential function in the regulation of apoptosis in EBV-infected cells.     

Functional Differences between BHRF1, the Epstein-Barr Virus-Encoded Bcl-2 Homologue, and Bcl-2 in Human Epithelial Cells

BHRF1, a component of the restricted early antigen complex of the Epstein-Barr virus lytic cycle, encodes a 17-kDa protein with both sequence and functional homology to the antiapoptotic Bcl-2 oncogene. Recent work has suggested that BHRF1 behaves like Bcl-2 in protecting cells from apoptosis induced by a range of stimuli. In this study, the effect of BHRF1 and Bcl-2 on the growth and differentiation of the SCC12F human epithelial cell line was examined. The levels of stable transfected BHRF1 expression achievable in SCC12F cells was consistently lower than that obtained with Bcl-2. While both BHRF1 and Bcl-2 inhibited epithelial differentiation, the effect of Bcl-2 was more pronounced, resulting in an almost complete blockade of differentiation in organotypic raft cultures. However, BHRF1-expressing SCC12F cells proliferated at a much higher rate than SCC12F cells expressing Bcl-2, and this effect was supported by cell cycle analysis which demonstrated that BHRF1, but not Bcl-2, promotes rapid transit through the cell cycle. These data highlight important differences between BHRF1 and Bcl-2 and suggest that BHRF1 may function to promote the survival and proliferation of lytically infected cells. The proliferative properties of BHRF1 described in this study, together with the demonstration that other oncogenic gamma herpesviruses encode Bcl-2 homologues, suggests that these proteins may serve to increase the susceptibility of virus-infected cells to oncogenic transformation, thereby contributing to the development of virus-associated tumors.