Biochemical characterization of Epstein-Barr virus nuclear antigen 2A.

The Epstein-Barr virus nuclear antigen 2A (EBNA-2A) was immunoprecipitated from latently Epstein-Barr virus-infected lymphocytes with a polyclonal serum raised against the EBNA-2A C terminus. The nucleus contained three subfractions of EBNA-2A which could be distinguished by their resistance to salt extraction: (i) a nucleoplasmatic fraction that was solubilized at 50 mM NaCl, (ii) a chromatin-associated fraction extractable at 1.5 M NaCl, and (iii) a nuclear matrix-associated fraction solubilized only by boiling with buffer containing 2% sodium dodecyl sulfate. The three subfractions were phosphorylated; it was demonstrated that the nucleoplasmatic and the chromatin-associated fractions were phosphorylated at serine and threonine residues. The half-life of the EBNA-2A protein was determined by cycloheximide treatment and by pulse-chase experiments and was found to be at least 24 h. The turnover of the phosphate residues bound to the two salt-soluble subfractions was determined to be approximately 6 to 9 h, suggesting a possible role of the phosphorylation in the regulation of the biological activity of EBNA-2A. Dephosphorylation of EBNA-2A resulted in an increased mobility of the protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and indicated the presence of differentially phosphorylated subclasses of the protein. Analysis of EBNA-2A by sucrose gradient centrifugation revealed the existence of two subclasses of complexed molecules which exhibited sedimentation coefficients of approximately 13S and 34S.     

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 nuclear antigen 2 transactivates latent membrane protein LMP1.

Several lines of evidence are compatible with the hypothesis that Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) or leader protein (EBNA-LP) affects expression of the EBV latent infection membrane protein LMP1. We now demonstrate the following. (i) Acute transfection and expression of EBNA-2 under control of simian virus 40 or Moloney murine leukemia virus promoters resulted in increased LMP1 expression in P3HR-1-infected Burkitt's lymphoma cells and the P3HR-1 or Daudi cell line. (ii) Transfection and expression of EBNA-LP alone had no effect on LMP1 expression and did not act synergistically with EBNA-2 to affect LMP1 expression. (iii) LMP1 expression in Daudi and P3HR-1-infected cells was controlled at the mRNA level, and EBNA-2 expression in Daudi cells increased LMP1 mRNA. (iv) No other EBV genes were required for EBNA-2 transactivation of LMP1 since cotransfection of recombinant EBNA-2 expression vectors and genomic LMP1 DNA fragments enhanced LMP1 expression in the EBV-negative B-lymphoma cell lines BJAB, Louckes, and BL30. (v) An EBNA-2-responsive element was found within the -512 to +40 LMP1 DNA since this DNA linked to a chloramphenicol acetyltransferase reporter gene was transactivated by cotransfection with an EBNA-2 expression vector. (vi) The EBV type 2 EBNA-2 transactivated LMP1 as well as the EBV type 1 EBNA-2. (vii) Two deletions within the EBNA-2 gene which rendered EBV transformation incompetent did not transactivate LMP1, whereas a transformation-competent EBNA-2 deletion mutant did transactivate LMP1. LMP1 is a potent effector of B-lymphocyte activation and can act synergistically with EBNA-2 to induce cellular CD23 gene expression. Thus, EBNA-2 transactivation of LMP1 amplifies the biological impact of EBNA-2 and underscores its central role in EBV-induced growth transformation.     

Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein.

Infection of Epstein-Barr virus-negative human B-lymphoma cell lines with the fully transforming B95.8 Epstein-Barr virus strain was associated with complete virus latent gene expression and a change in the cell surface and growth phenotype toward that of in vitro-transformed lymphoblastoid cell lines. In contrast, the cells infected with the P3HR1 Epstein-Barr virus strain, a deletion mutant that cannot encode Epstein-Barr nuclear antigen 2 (EBNA2) or a full-length EBNA-LP, expressed EBNAs1, 3a, 3b, and 3c but were negative for the latent membrane protein (LMP) and showed no change in cellular phenotype. This suggests that EBNA2 and/or EBNA-LP may be required for subsequent expression of LMP in Epstein-Barr virus-infected B cells. Recombinant vectors capable of expressing the B95.8 EBNA2A protein were introduced by electroporation into two P3HR1-converted B-lymphoma cell lines, BL30/P3 and BL41/P3. In both cases, stable expression of EBNA2A was accompanied by activation of LMP expression from the resident P3HR1 genome; control transfectants that did not express the EBNA2A protein never showed induction of LMP. In further experiments, a recombinant vector capable of expressing the full-length B95.8 EBNA-LP was introduced into the same target lines. Strong EBNA-LP expression was consistently observed in the transfected clones but was never accompanied by induction of LMP. The EBNA2A gene transfectants expressing EBNA2A and LMP showed a dramatic change in cell surface and growth phenotype toward a pattern like that of lymphoblastoid cell lines; some but not all of these changes could be reproduced in the absence of EBNA2A by transfection of P3HR1-converted cell lines with a recombinant vector expressing LMP. These studies suggest that EBNA2 plays an important dual role in the process of B-cell activation to the lymphoblastoid phenotype; the protein can have a direct effect upon cellular gene expression and is also involved in activating the expression of a second virus-encoded effector protein, LMP.     

SCFSkp2 complex targeted by Epstein-Barr virus essential nuclear antigen

The stability of cell cycle checkpoint and regulatory proteins is controlled by the ubiquitin-proteasome degradation machinery. A critical regulator of cell cycle molecules is the E3 ubiquitin ligase SCFSkp2, known to facilitate the polyubiquitination and degradation of p27, E2F, and c-myc. SCFSkp2 is frequently deregulated in human cancers. In this study, we have revealed a novel link between the essential Epstein-Barr virus (EBV) nuclear antigen EBNA3C and the SCFSkp2 complex, providing a mechanism for cell cycle regulation by EBV. EBNA3C associates with cyclin A/cdk2 complexes, disrupting the kinase inhibitor p27 and enhancing kinase activity. The recruitment of SCFSkp2 activity to cyclin A complexes by EBNA3C results in ubiquitination and SCFSkp2-dependent degradation of p27. This is the first report of a viral protein usurping the function of the SCFSkp2 cell cycle regulatory machinery to regulate p27 stability, establishing the foundation for a mechanism by which EBV regulates cyclin/cdk activity in human cancers.     

Mitosis-specific hyperphosphorylation of Epstein-Barr virus nuclear antigen 2 suppresses its function

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is a key gene expressed in EBV type III latent infection that can transactivate numerous promoters, including those for all the other type III viral latency genes as well as cellular genes responsible for cell proliferation. EBNA-2 is essential for EBV-mediated immortalization of primary B lymphocytes. We now report that EBNA-2, a phosphoprotein, is hyperphosphorylated specifically in mitosis. Evidence that the cyclin-dependent kinase p34(cdc2) may be involved in this hyperphosphorylation includes (i) coimmunoprecipitation of EBNA-2 and p34(cdc2), suggesting physical association; (ii) temporal correlation between hyperphosphorylation of EBNA-2 and an increase in p34(cdc2) kinase activity; and (iii) ability of purified p34(cdc2)/cyclin B1 kinase to phosphorylate EBNA-2 in vitro. Hyperphosphorylation of EBNA-2 appears to suppress its ability to transactivate the latent membrane protein 1 (LMP-1) promoter by about 50%. The association between EBNA-2 and PU.1 is also decreased by about 50% in M-phase-arrested cells, as shown by coimmunoprecipitation from cell lysates, suggesting that hyperphosphorylation of EBNA-2 impairs its affinity for PU.1. Finally, endogenous LMP-1 mRNA levels in M phase are around 55% of those in asynchronously growing cells. These results suggest that regulation of gene expression during type III latency may be regulated in a cell-cycle-related manner.     

Epstein-Barr virus nuclear antigen 2 transactivates the long terminal repeat of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1)-infected subjects show a high incidence of Epstein-Barr virus (EBV) infection. This suggests that EBV may function as a cofactor that affects HIV-1 activation and may play a major role in the progression of AIDS. To test this hypothesis, we generated two EBV-negative human B-cell lines that stably express the EBNA2 gene of EBV. These EBNA2-positive cell lines were transiently transfected with plasmids that carry either the wild type or deletion mutants of the HIV-1 long terminal repeat (LTR) fused to the chloramphenicol acetyltransferase (CAT) gene. There was a consistently higher HIV-1 LTR activation in EBNA2-expressing cells than in control cells, which suggested that EBNA2 proteins could activate the HIV-1 promoter, possibly by inducing nuclear factors binding to HIV-1 cis-regulatory sequences. To test this possibility, we used CAT-based plasmids carrying deletions of the NF-kappa B (pNFA-CAT), Sp1 (pSpA-CAT), or TAR (pTAR-CAT) region of the HIV-1 LTR and retardation assays in which nuclear proteins from EBNA2-expressing cells were challenged with oligonucleotides encompassing the NF-kappa B or Sp1 region of the HIV-1 LTR. We found that both the NF-kappa B and the Sp1 sites of the HIV-1 LTR are necessary for EBNA2 transactivation and that increased expression resulted from the induction of NF-kappa B-like factors. Moreover, experiments with the TAR-deleted pTAR-CAT and with the tat-expressing pAR-TAT plasmids indicated that endogenous Tat-like proteins could participate in EBNA2-mediated activation of the HIV-1 LTR and that EBNA2 proteins can synergize with the viral tat transactivator. Transfection experiments with plasmids expressing the EBNA1, EBNA3, and EBNALP genes did not cause a significant HIV-1 LTR activation. Thus, it appears that among the latent EBV genes tested, EBNA2 was the only EBV gene active on the HIV-1 LTR. The transactivation function of EBNA2 was also observed in the HeLa epithelial cell line, which suggests that EBV and HIV-1 infection of non-B cells may result in HIV-1 promoter activation. Therefore, a specific gene product of EBV, EBNA2, can transactivate HIV-1 and possibly contribute to the clinical progression of AIDS.     

Prediction and demonstration of a novel Epstein-Barr virus nuclear antigen.

The protein sequence predicted by the Epstein Barr virus (EBV) BERF4 open reading frame includes a tetrapeptide, Lys-Arg-Pro-Arg (KRPR), shown for other proteins to be a component of a signal for rapid nuclear localization. A subgenomic fragment of EBV DNA containing BERF4 has been incorporated into an expression vector, transfected onto primate cells and the nuclear distribution of the resulting protein established by immunofluorescence using EBV positive human sera. These sera contained high titres of antibodies to a fusion protein, produced in E. coli, consisting of beta-galactosidase and the C-terminal 167 amino acids of BERF4. Immunoaffinity purified antibodies reactive with the EBV component of the fusion show the molecular weight of this antigen in EBV immortalized B-cell lines to be about 160 kD. The demonstration that BERF4 contains an exon encoding a nuclear protein identifies a new EBNA gene (EBNA-6) and suggests that KRPR is a signal sequence common to a number of viral and cellular nuclear polypeptides which bind to nucleic acids and may therefore be of predictive value in identifying karyophilic proteins.     

High-resolution footprints of the DNA-binding domain of Epstein-Barr virus nuclear antigen 1.

The DNA-binding domain of Epstein-Barr virus nuclear antigen 1 was found by hydroxyl radical footprinting to protect backbone positions on one side of its DNA-binding site. The guanines contacted in the major groove by the DNA-binding domain of Epstein-Barr virus nuclear antigen 1 were identified by methylation protection. No difference was found in the interaction of the DNA-binding domain of Epstein-Barr virus nuclear antigen 1 with tandemly repeated and overlapping binding sites.     

Biochemical characterization of Epstein-Barr virus nuclear antigen 2A and an associated ATPase activity.

A partial purification of the Epstein-Barr-virus nuclear antigen 2A (EBNA 2A) protein from the Epstein-Barr-virus-infected lymphoblastoid cell line, Cherry, has been designed. The main purification step was immunoaffinity chromatography, based on the mAb, 115E, directed towards the carboxy terminus of EBNA 2A. This was followed by chromatography over a Blue Sepharose column. According to silver-stained SDS/PAGE, EBNA 2A was estimated to be 20% pure. The purified fractions contained an ATPase activity that was inhibited by the mAb 115E. Immunopurification of six EBNA-2A-positive cell lines and their negative counterpart showed that only fractions from EBNA-2A-positive lines contained ATPase activity. In gel-filtration experiments EBNA 2A eluted as a 75-kDa protein in conjunction with an ATPase activity. The EBNA 2A protein was covalently labeled by the ATP analog [14C]5'-[p-(fluorosulfonyl)benzoyl]adenosine. The ATPase activity was found to be optimal in the presence of 0.25 mM MgCl2 or CaCl2, whereas, in the presence of MnCl2 and ZnCl2, the activity was only about 50% of the control. High concentrations of Na2VO3 and heparin do not interfere with the activity, while 2.5 mM NaF or 0.5 M NaCl give a 50% reduction of the activity. The Km for ATP and for GTP was 13 microM and 11 microM, respectively, and the Vmax for ATP was about six-times higher than with GTP as substrate. Other low-molecular-mass non-protein phosphate esters, such as phosphoserine or phosphothreonine inhibited the ATPase activity with a Ki of 18 and 32 microM, respectively. Phosphotyrosine had a Ki of 480 microM. Serine, threonine and tyrosine had no inhibitory effect on the ATPase activity.     

Characterization of Epstein-Barr virus antigens. I. Biochemical analysis of the complement-fixing soluble antigen and relationship with Epstein-Barr virus-associated nuclear antigen.

The Epstein-Barr virus-soluble (S) antigen extracted from RAJI cells was characterized by sucrose gradient centrifugation, gel filtration, and ion-exchange chromatography. The sedimentation coefficient was estimated to be 8.5S corresponding to a molecular weight of 180,000. The S antigen binds to DEAE-A25 ion exchanger from which it can be eluted with 0.3 M NaCl in Tris buffer (pH 7.2). All fractions which contained complement-fixing S antigen also inhibited the anticomplement immunofluorescence reaction as used to detect the Epstein-Barr virus-associated nuclear antigen. These results are consistent with the hypothesis that the S and Epstein-Barr virus-associated nuclear antigens are either a single antigen or that both activities are present on the same molecule.     

Identification of an Epstein-Barr virus nuclear antigen by fluoroimmunoelectrophoresis and radioimmunoelectrophoresis.

A 65,000-dalton (65K) antigen found in Raji cells by fluoroimmunoelectrophoresis and radioimmunoelectrophoresis has been identified as an Epstein-Barr virus nuclear antigen (EBNA). This identification is based on the following evidence. The 65K antigen is detected in Raji cells but not in three Epstein-Barr virus (-) human B cell lines. It is not detected with EBNA (-) sera. The 65K antigen is found predominantly in the nucleus and co-elutes with EBNA during partial purification by DNA-Sepharose and Blue Dextran-Sepharose chromatography. Finally, the partially purified 65K antigen is an effective absorbant of EBNA antibody as measured in an anticomplement immunofluorescence assay. Antigens with molecular weights of 72, 70, and 73K have been detected in B95-8, P3HR-1, and Namalwa cells, respectively. These antigens are the likely homologues of the 65K Raji EBNA. In addition, an Epstein-Barr virus-associated, 81K DNA-binding antigen has been detected in both B95-8 and Raji cells.     

Expression of p40/Epstein-Barr virus nuclear antigen 1 binding protein 2

Nucleolar protein p40/EBP2 is a proliferation-associated antigen that interacts with Epstein-Barr virus nuclear antigen 1 (EBNA1) to maintain the Epstein-Barr virus (EBV) episomes. The yeast p40/EBP2 functions in the processing of 27S-A into 27S-B ribosomal RNA. The present study reports high evolutionary conservation of the cDNA-derived amino acid sequences of p40/EBP2 from frog, chicken, pig, rat, mouse, bovine, and human. p40/EBP2 is ubiquitously expressed in human tissues. It is highly expressed in myelogenous leukemia K-562 compared to other cell lines tested. The human p40/EBP2 gene is located in chromosome 1 with nine exons and eight introns. The minimal promoter region resides 300 nucleotides upstream of a putative ATG initiation codon preceded by a pyrimidine-rich region. These two regions contain eight Sp1 and four c-Ets-1 putative binding sites. Analysis of the p40/EBP2 gene and its promoter region will facilitate studies on the regulation of its expression in EBV-infected and noninfected cells.     

Influence of the Epstein-Barr virus nuclear antigen EBNA 2 on the growth phenotype of virus-transformed B cells.

Epstein-Barr virus (EBV) isolates show sequence divergence in the BamHI YH region of the genome which encodes the nuclear antigen EBNA 2, a protein thought to be involved in the initiation of virus-induced B-cell transformation; type A isolates (such as B95-8 EBV) encode a 82- to 87-kilodalton EBNA 2A protein, whereas type B isolates (such as AG876 EBV) encode an antigenically distinct 75-kilodalton EBNA 2B protein. In the present work 12 type A isolates and 8 type B isolates have been compared for their ability to transform resting human B cells in vitro into permanent lymphoblastoid cell lines. Although the kinetics of initial focus formation was not markedly dependent upon the EBNA 2 type of the transforming virus, on subsequent passage type A virus-transformed cells (type A transformants) yielded cell lines much more readily than did type B transformants. Direct comparison between the two types of transformant revealed clear differences in several aspects of growth phenotype. Compared with type A transformants, cell lines established with type B virus isolates consistently displayed an unusual growth pattern with poor survival of individual cells shed from lymphoblastoid clumps, a lower growth rate and a greater sensitivity to seeding at limiting dilutions, and a significantly lower saturation density that could not be corrected by supplementation of the medium with culture supernatant containing B-cell growth factors. This is the first direct evidence that, in EBV-transformed B-cell lines, the EBNA 2 protein plays a continuing role in determining the cellular growth phenotype.     

Efficient expression of an Epstein-Barr nuclear antigen in Drosophila cells transfected with Epstein-Barr virus DNA.

In a search for exogenous promoters which function in cultured Drosophila cells, we have co-transfected a D. melanogaster cell line with an Epstein-Barr virus (EBV) cosmid clone which encodes the Epstein-Barr nuclear antigen (EBNA-1). Here we report that Drosophila cells containing stably integrated copies of EBNA-1 encoding DNA synthesise a polypeptide of mol. wt. identical to that of authentic EBNA-1, which is detectable with EBNA-positive but not EBNA-negative human serum. As in EBV-transformed lymphoblastoid cells, this neo-antigen is associated with the nucleus of transfected cells suggesting that cellular localisation signals which operate in mammalian cells are also recognised in insect cells.     

Partial purification of the Epstein-Barr virus nuclear antigen(s)

The Epstein-Barr virus nuclear antigen (EBNA) is speculated to be involved in cell transformation by the virus. Studies on the molecular properties of EBNA, however, have yielded conflicting results. In this study, three Epstein-Barr virus(EBV)-induced antigens were isolated and purified from extracts prepared from Raji cells. These antigens were able to block the anticomplement immunofluorescence reaction, indicating that all three were related to EBNA. The soluble antigen was found wholly in the cytosol fraction. An EBV-induced nuclear antigen I was found both in the cytosol and the nucleus. The EBV-induced nuclear antigen II was found associated with the chromatin. The soluble antigen and the nuclear antigen I were separated and partially purified using phosphocellulose chromatography. Each was further purified 1,400-fold with respect to the whole cell state by chromatography on CL-Sepharose 6B followed by blue dextran-Sepharose. subunit molecular weights of 70,000 were determined for each of these antigens, both in the crude and purified state, by radioimmunoelectrophoresis and gel filtration. The nuclear antigen II was purified 2,500-fold using hydroxylapatite, CL-Sepharose 6B, and blue dextran-Sepharose chromatographies. This antigen displayed two subunits by radioimmunoelectrophoresis with molecular weights of 65,000 and 70,000. Although all antigens shared similar molecular weights, the extent of their homology remains to be determined.     

The DNA segregation mechanism of Epstein-Barr virus nuclear antigen 1

Latent Epstein–Barr virus (EBV) genomes are maintained in human cells as low copy number episomes that are thought to be partitioned by attachment to the cellular mitotic chromosomes through the viral EBNA1 protein. We have identified a human protein, EBP2, which interacts with the EBNA1 sequences that govern EBV partitioning. Here we show that, in mitosis, EBP2 localizes to the condensed cellular chromosomes producing a staining pattern that is indistinguishable from that of EBNA1. The localization of EBNA1 proteins with mutations in the EBP2 binding region was also examined. An EBNA1 mutant (Δ325–376) disrupted for EBP2 binding and segregation function was nuclear but failed to attach to the cellular chromosomes in mitosis. Our results indicate that amino acids 325–376 mediate the binding of EBNA1 to mitotic chromosomes and strongly suggest that EBNA1 mediates EBV segregation by attaching to EBP2 on the cellular mitotic chromosomes.