BZLF1 Protein Research Articles

The Epstein-Barr Virus Immediate-Early Protein BZLF1 Induces Expression of E2F-1 and Other Proteins Involved in Cell Cycle Progression in Primary Keratinocytes and Gastric Carcinoma Cells

The Epstein-Barr Virus Immediate-Early Protein BZLF1 Induces Expression of E2F-1 and Other Proteins Involved in Cell Cycle Progression in Primary Keratinocytes and Gastric Carcinoma Cells

Control of Epstein-Barr virus reactivation by activated CD40 and viral latent membrane protein 1.

In humans, Epstein-Barr virus (EBV) establishes a persistent latent infection in peripheral resting B lymphocytes. Virus reactivation is highly restricted. Whereas in healthy humans the infection usually is benign, immunocompromised patients show an increased risk for EBV-associated malignancies, accompanied by an increase in virus replication and in the number of virus-infected cells. To search for viral and host factors regulating virus reactivation, we used conditionally EBV-immortalized B cells. We found that CD40-CD40 ligand interaction and the viral mimic of activated CD40, EBV latent membrane protein 1, suppress virus reactivation. Both inhibit anti-IgM or phorbolester-induced transcription of the viral immediate early protein BZLF1, which controls entry into the viral lytic cycle. The finding that latent membrane protein 1 and CD40 contribute to the regulation of latency may have important implications for the balance between EBV and its host in normal as well as in immunocompromised individuals.

No Significant Association of Epstein-Barr Virus Infection with Invasive Breast Carcinoma

We studied 48 cases of invasive breast carcinoma for evidence of Epstein-Barr virus (EBV), which is associated with many human malignancies. In situ hybridization studies to detect the presence of EBV-encoded small nonpolyadenylated RNA (EBER)-1 were performed in paraffin sections. Immunohistochemical studies to detect EBV nuclear antigen (EBNA)-1, latent membrane protein (LMP)-1, and the transactivating immediate-early BZLF1 (ZEBRA) protein were also performed in paraffin sections. The presence of EBV genomic DNA was studied by polymerase chain reaction (PCR) amplification using sets of primers flanking the EBNA-4 and the EBV-LMP-1 genes in frozen tissues. Southern blot analysis using a probe flanking the EBV terminal repeat region was then attempted in cases that were PCR-positive. Five of 48 cases (10%) of breast carcinoma showed focal EBER-positive tumor cells. Twelve cases (25%) were positive for EBNA-1 by immunohistochemistry, all but one different from the EBER-positive cases. None of the cases were positive for LMP-1 or ZEBRA protein by immunohistochemistry. PCR studies for EBNA-4 and LMP-1 were each positive in five cases (including three cases in common). However, Southern blot studies successfully performed in all but one of the PCR-positive cases were completely negative. The identification of EBV by any methodology was not correlated with tumor size, grade, or lymph node status. This study demonstrated evidence of EBV infection in tissues involved by invasive breast carcinomas in a significant subset of cases. However, the lack of localization of EBV infection to a significant population of the tumor cells in any case, the negativity by Southern blot hybridization, and the lack of expression of multiple antigens in any case strongly argue against a significant role for EBV in the pathogenesis of breast carcinoma.

Epstein-barr virus immediate-early protein BZLF1 is SUMO-1 modified and disrupts promyelocytic leukemia bodies.

Although the immediate-early proteins of both herpes simplex virus (HSV) and cytomegalovirus (CMV) are known to modify promyelocytic leukemia (PML) (ND10) bodies in the nucleus of the host cell, it has been unclear whether lytic infection with gamma herpesviruses induces a similar effect. The PML protein is induced by interferon, involved in major histocompatibility complex class I presentation, and necessary for certain types of apoptosis. Therefore, it is likely that PML bodies function in an antiviral capacity. SUMO-1 modification of PML is known to be required for the formation of PML bodies. To examine whether Epstein-Barr virus (EBV) lytic replication interferes with PML bodies, we expressed the EBV immediate-early genes BZLF1 (Z) and BRLF1 (R) in EBV-positive cell lines and examined PML localization. Both Z and R expression resulted in PML dispersion in EBV-positive cells. Z but not R expression is sufficient to disrupt PML bodies in EBV-negative cell lines. We show that dispersion of PML bodies by Z requires a portion of the transcriptional activation domain of Z but not the DNA-binding function. As was previously reported for the HSV-1 ICP0 and CMV IE1 proteins, Z reduces the amount of SUMO-1-modified PML. We also found that Z itself is SUMO-1 modified (through amino acid 12) and that Z competes with PML for limiting amounts of SUMO-1. These results suggest that disruption of PML bodies is important for efficient lytic replication of EBV. Furthermore, Z may potentially alter the function of a variety of cellular proteins by inhibiting SUMO-1 modification.

EBV replication enzymes.

The Epstein-Barr virus (EBV) can choose between two alternative life styles. It infects B lymphocytes, transforming them into lymphoblastoid lines and, in contrast to neurotropic herpesviruses such as herpes simplex virus type I that establish latency in nondividing neurons, must maintain its latent genomes in cells that have the potential to divide. In B lymphoblastoid cell lines established by EBV infection, the viral genome is maintained as covalently closed circular plasmids forming nucleosomal structures with histone proteins. The number of copies is maintained at 10–50 per cell to be duplicated once during each cell division cycle by the host cellular DNA replication machinery. When production of virus is induced, the circular genome becomes a ready template for amplification, generating thousands of copies per cell during lytic infection. Replication intermediates are head-to-tail concatamers, perhaps through a rolling-circle DNA replication, which are cleaved and packaged into infectious viral particles. The lytic phase of EBV DNA replication is dependent on seven viral replication proteins: BZLF1, BALF5, BMRF1, BALF2, BBLF4, BSLF1, and BBLF2/3 gene products. The BZLF1 protein is an oriLyt-binding protein and also acts as the lytic transactivator. The BALF5 gene encodes the DNA Pol catalytic subunit and the BMRF1 gene encodes the DNA Pol accessory subunit. They form a complex to act as the Pol holoenzyme. A single-stranded (ss)DNA-binding protein is encoded by the BALF2 gene.

Epstein-Barr Virus Immediate-Early Protein BZLF1 Is SUMO-1 Modified and Disrupts Promyelocytic Leukemia Bodies

Epstein-Barr Virus Immediate-Early Protein BZLF1 Is SUMO-1 Modified and Disrupts Promyelocytic Leukemia Bodies

Lytic Epstein-Barr virus infection in the synovial tissue of patients with rheumatoid arthritis.

To evaluate the existence of Epstein-Barr virus (EBV) infection in the synovial tissue of patients with rheumatoid arthritis (RA). Synovial tissues were obtained at synovectomy or arthroplasty from 32 patients with RA and 30 control patients with osteoarthritis (OA). EBV DNA was detected by Southern blot hybridization and/or polymerase chain reaction (PCR) amplification. To localize the EBV-infected cells, tissue sections were studied by RNA in situ hybridization (ISH) for the EBV-encoded small RNA 1 (EBER-1), by DNA ISH for the Bam HI W region of EBV DNA, and by immunohistochemistry for EBV lytic proteins BZLF1 and gp350/220. EBV DNA was detected by PCR in 15 of the 32 samples from RA patients (47%), but in none of those from the 30 OA patients (P < 0.01). Of the 15 PCR-positive samples, 9 contained >1 EBV copy/1,000 cells (referred to as EBV 2+), and 6 contained 1 copy/1,000-5,000 cells (EBV 1+). Among the 9 EBV 2+ samples, 3 were also positive for EBV DNA by Southern blot hybridization, 5 were positive for EBER-1 by RNA ISH, and 3 were positive for EBV DNA by DNA ISH. Immunohistochemical analysis showed positive signals in all samples for BZLF1 and in 7 samples for gp350/ 220. In each examination, the positive signals were detected not only in lymphocytes, but also in synovial lining cells. EBV was frequently detected in the synovial tissue of RA patients. The infected cells were both lymphocytes and synovial cells, and expressed EBV proteins associated with virus replication. These findings suggest that EBV may play a role in the pathogenesis of RA.

The PKC targeting protein RACK1 interacts with the Epstein-Barr virus activator protein BZLF1.

Phorbol esters reactivate Epstein-Barr virus (EBV) from latently infected cells via transcriptional activation of the viral immediate-early gene BZLF1. BZLF1 is a member of the extended AP-1 family of transcription factors that binds to specific BZLF1-binding motifs within early EBV promoters and to consensus AP-1 sites. Regulation of BZLF1's activity is achieved at the transcriptional level as well as through post-translational modifications. Recently, we reported that the transcriptional activity of BZLF1 is augmented by TPA [Baumann, M., Mischak, H., Dammeier, S., Kolch, W., Gires, O., Pich, D., Zeidler, R., Delecluse, H. J. & Hammerschmidt, W., (1998) J. Virol. 72, 8105-8114]. The increase of BZLF1's activity depends on a single serine residue (S186) that is phosphorylated by protein kinase C (PKC) in vitro and in vivo after stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA). Here, we identified RACK1 as a binding partner of BZLF1 in a yeast interaction trap assay. RACK stands for receptor of activated C-kinase and is involved in targeting activated PKCs and other signaling proteins. In vivo, RACK1 binds directly to the transactivation domain of BZLF1. Although a functional relationship between BZLF1 and PKC could be mediated by RACKs, RACK1 did not have a detectable effect on the phosphorylation status of BZLF1 in in vitro or in vivo phosphorylation assays. We suggest that RACK1 may act as a scaffolding protein on BZLF1 independently of activated PKCs.

Epstein-Barr virus immediate-early proteins BZLF1 and BRLF1 activate the ATF2 transcription factor by increasing the levels of phosphorylated p38 and c-Jun N-terminal kinases.

Expression of either Epstein-Barr virus (EBV) immediate-early protein BZLF1 (Z) or BRLF1 (R) is sufficient to convert EBV infection from the latent to lytic form. Disruption of viral latency requires transcriptional activation of the Z and R promoters. The Z and R proteins are transcriptional activators, and each immediate-early protein activates expression of the other immediate-early protein. Z activates the R promoter through a direct binding mechanism. However, R does not bind directly to the Z promoter. In this study, we demonstrate that the ZII element (a cyclic AMP response element site) in the Z promoter is required for efficient activation by R. The ZII element has been shown to be important for induction of lytic EBV infection by tetradecanoyl phorbol acetate and surface immunoglobulin cross-linking and is activated by Z through an indirect mechanism. We demonstrate that both R and Z activate the cellular stress mitogen-activated protein (MAP) kinases, p38 and JNK, resulting in phosphorylation (and activation) of the cellular transcription factor ATF2. Furthermore, we show that the ability of R to induce lytic EBV infection in latently infected cells is significantly reduced by inhibition of either the p38 kinase or JNK pathways. In contrast, inhibition of stress MAP kinase pathways does not impair the ability of Z expression vectors to disrupt viral latency, presumably because expression of Z under the control of a strong heterologous promoter bypasses the need to activate Z transcription. Thus, both R and Z can activate the Z promoter indirectly by inducing ATF2 phosphorylation, and this activity appears to be important for R-induced disruption of viral latency.

Lack of evidence for an involvement of Epstein-Barr virus infection of synovial membranes in the pathogenesis of rheumatoid arthritis.

To test the hypothesis that Epstein-Barr virus (EBV) infection of cells within the synovial membrane contributes to the pathogenesis of rheumatoid arthritis (RA). Biopsy samples of synovial membrane from 37 patients with RA and from 51 patients with other joint diseases were studied for evidence of EBV infection using in situ hybridization specific for the EBV-encoded RNAs (EBERs). Latent membrane protein 1 (LMP1) and the lytic-cycle BZLF1 protein were detected by immunohistochemistry. Rare EBER-positive B lymphocytes were detected in 7 RA biopsy samples. EBV was not detectable in any other cells. Expression of the LMP1 and BZLF1 proteins of EBV was not observed in any of the samples. No EBV infection was detected in synovial membranes from patients with other joint diseases. Our data indicate that EBV infection is not directly involved in the pathogenesis of RA. Any contribution of EBV to the pathogenic process leading to RA is likely to be indirect.

Specificity of T cells in synovial fluid: high frequencies of CD8(+) T cells that are specific for certain viral epitopes.

CD8+ T cells dominate the lymphocyte population insynovial fluid in chronic inflammatory arthritis. It is known that theseCD8+ T cells are often clonally or oligoclonally expanded, but theirspecificity and their relevance to the pathogenesis of joint disease hasremained unclear. We found that as many as 15.5% of synovial CD8+ Tcells may be specific for a single epitope from an Epstein-Barr virus lyticcycle protein. The virus-specific T cells within the joint showed increasedexpression of markers of activation and differentiation compared with those inthe periphery, and retained their functional capacity to secreteproinflammatory cytokines on stimulation. These activated, virus-specificCD8+ T cells could therefore interact with synoviocytes, either bycell-cell contact or by a cytokine network, and play a 'bystander'role in the maintenance of inflammation in patients with arthritis.

Epstein-Barr Virus Immediate-Early Proteins BZLF1 and BRLF1 Activate the ATF2 Transcription Factor by Increasing the Levels of Phosphorylated p38 and c-Jun N-Terminal Kinases

Epstein-Barr Virus Immediate-Early Proteins BZLF1 and BRLF1 Activate the ATF2 Transcription Factor by Increasing the Levels of Phosphorylated p38 and c-Jun N-Terminal Kinases

Latency of Epstein-Barr virus is stabilized by antisense-mediated control of the viral immediate-early gene BZLF-1.

The ability of the Epstein-Barr virus (EBV) to avoid lytic replication and to establish a latent infection in B-lymphocytes is fundamental for its lifelong persistence and the pathogenesis of various EBV-associated diseases. The viral immediate-early gene BZLF-1 plays a key role for the induction of lytic replication and its activity is strictly regulated on different levels of gene expression. Recently, it was demonstrated that BZLF-1 is also controlled by a posttranscriptional mechanism. Transient synthesis of a mutated competitor RNA saturated this mechanism and caused both expression of the BZLF-1 protein and the induction of lytic viral replication. Using short overlapping fragments of the competitor, it is shown that this control acts on the unspliced primary transcript. RT-PCR demonstrated unspliced BZLF-1 RNA in latently infected B-lymphocytes in the absence of BZLF-1 protein. Due to the complementarity of the gene BZLF-1 and the latency-associated gene EBNA-1 on the opposite strand of the genome, we propose an antisense-mediated mechanism. RNase protection assays demonstrated transcripts in antisense orientation to the BZLF-1 transcript during latency, which comprise a comparable constellation to other herpesviruses. A combined RNAse protection/RT-PCR assay detected the double-stranded hybrid RNA, consisting of the unspliced BZLF-1 transcript and a noncoding intron of the EBNA-1 gene. Binding of BZLF-1 transcripts is suggested to be an important backup control mechanism in addition to transcriptional regulation, stabilizing latency and preventing inappropriate lytic viral replication in vivo.

Cellular transcription factors recruit viral replication proteins to activate the Epstein-Barr virus origin of lytic DNA replication, oriLyt.

DNA replication of Epstein-Barr virus (EBV) during the productive phase of the life cycle of this herpesvirus depends on the cis-acting element oriLyt. It consists of two essential domains, the upstream and the downstream component. Whereas the upstream component contains several DNA-binding motifs for the viral activator protein BZLF1, the downstream component is known to be the binding site of several cellular proteins. We identified cellular transcription factors that bind synergistically to a functionally relevant subsequence of the downstream component, the TD element. Two of these transcription factors, ZBP-89 and Sp1, stimulate replication as shown by protein fusions with the GAL4 DNA-binding domain and a single GAL4 DNA-binding motif inserted into the TD element. In protein binding assays, we observed an interaction of Sp1 and ZBP-89 with the viral DNA polymerase and its processivity factor. Our data indicate that cellular transcriptional activators tether viral replication proteins to the lytic origin via direct protein-protein interactions to assemble the viral replication complex at oriLyt.

Frequency of CD8+ T Lymphocytes Specific for Lytic and Latent Antigens of Epstein–Barr Virus in Healthy Virus Carriers

We investigated CD8+ T cell frequencies of five different Epstein–Barr virus-specific cytotoxic T lymphocyte epitopes located within proteins of the replicative cycle and the latent state in healthy long-term virus carriers with IFN-γ enzyme-linked immunospot assay. Frequencies of the HLA-A3-restricted epitope RVRAYTYSK (RVR) whose minimal length was mapped in this study to amino acid position 148–156 of the immediate-early protein BRLF1 were compared with those of a further known HLA-A3-restricted epitope within EBNA3A, RLRAEAQVK (RLR). Determination of frequencies of CD8+ T lymphocytes directed against lytic antigen epitope RVR revealed that only one of eight donors recognized this epitope. Frequency was calculated to be 65 RVR-specific CD8+ T lymphocytes per 106 PBMC. None of the HLA-A3-positive donors exhibited IFN-γ release after antigenic stimulation with the EBNA3A-specific peptide epitope RLR. Furthermore, we chose three known HLA-B8-restricted epitopes, RAKFKQLL (RAK), FLRGRAYGL (FLR), and QAKWRLQTL (QAK), of the lytic protein BZLF1 and the latent protein EBNA3A. Examination of eight HLA-B8-positive virus carriers revealed that the BZLF1-specific epitope RAK was recognized by all donors with a median frequency of 233 RAK-specific CD8+ T lymphocytes per 106 PBMC. Only 50% of these donors reacted against EBNA3A-specific epitope FLR and a minority (25%) reacted against EBNA3A-specific epitope QAK.

The Epstein-Barr virus BZLF1 protein interacts physically and functionally with the histone acetylase CREB-binding protein.

The Epstein-Barr virus (EBV) immediate-early protein BZLF1 (Z) is a key regulator of the EBV latent-to-lytic switch. Z is a transcriptional activator which induces EBV early gene expression. We demonstrate here that Z interacts with CREB-binding protein (CBP), a histone acetylase and transcriptional coactivator. This interaction requires the amino-terminal region of CBP as well as the transactivation and leucine zipper domains of Z. We show that CBP enhances Z-mediated transactivation of EBV early promoters, in reporter gene assays and in the context of the endogenous genome. We also demonstrate that Z decreases CREB transactivation function and that this inhibitory effect is reversed by overexpression of CBP. We show that Z also interacts directly with CREB. However, mutational analysis indicates that Z inhibition of CREB activity requires the direct interaction between Z and CBP but not the direct interaction between Z and CREB. We propose that Z interacts with CBP to enhance viral early gene transcription. In addition, the Z-CBP interaction may control host cellular transcription factor activity through competition for limiting amounts of cellular CBP.

Nitric Oxide Down-Regulates Epstein–Barr Virus Reactivation in Epithelial Cell Lines

Nitric oxide (NO), a mediator of biological functions, has an antimicrobial activity against a variety of pathogens including viruses. In this study, we found that a constitutive, low level of inducible NO synthase (iNOS) mRNA was expressed in the EBV-infected gastric tissue-derived GT38 and GT39 cell lines, by analysis with the reverse transcription–polymerase chain reaction (RT–PCR) and Southern blotting. Treatment of these cells with a specific NOS inhibitor, NG-monomethyl-l-arginine (l-NMMA), induced the immediate-early, EBV transactivator gene BZLF1 protein ZEBRA, suggesting a significant increase in EBV reactivation by l-NMMA. Northern blotting demonstrated that BZLF1 and BRLF1 transcripts were also induced by 12-O-tetradecanoylphorbol-13 acetate (TPA). Meanwhile, constitutive expression of iNOS mRNA was inhibited by TPA. l-NMMA also enhanced TPA-induced expression of the BZLF1 gene. On the other hand, a NO donor, S-nitroso-N-acetylpenicillamine (SNAP), which releases NO in an aqueous solution, inhibited the TPA-induced BZLF1 gene expression in a dose-dependent manner at both mRNA and protein levels. These results demonstrated that NO is a regulatory factor in maintaining virus latency via inhibiting EBV reactivation in the infected epithelial cells.

The Epstein-Barr Virus BZLF1 Protein Interacts Physically and Functionally with the Histone Acetylase CREB-Binding Protein

The Epstein-Barr Virus BZLF1 Protein Interacts Physically and Functionally with the Histone Acetylase CREB-Binding Protein

Productive Infection of Epstein–Barr Virus (EBV) in EBV-Genome-Positive Epithelial Cell Lines (GT38 and GT39) Derived from Gastric Tissues

We characterized the expression of Epstein–Barr virus (EBV) on two epithelial cell lines, GT38 and GT39, derived from human gastric tissues. The EBV nuclear antigen (EBNA) was detected in all cells of both cell lines. The EBV immediate-early BZLF1 protein (ZEBRA), the early antigen diffuse component (EA-D), and one of the EBV envelope proteins (gp350/220) were expressed spontaneously in small proportions in the cells. EBNA1, EBNA2, latent membrane protein 1, ZEBRA, and EA-D molecules were then observed by Western blotting in the cells. The lytic cycle was enhanced with treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) orn-butyrate. The virus particles were observed in the TPA treated GT38 cells by electron microscopy. Infectious EBV was detected with the transformation of cord blood lymphocytes and also with the induction of early antigen to Raji cells by the supernatants of both cell lines. A major single and minor multiple fused terminal fragments and a ladder of smaller fragments of the EBV genome were detected with aXhoI probe in both cell lines. These epithelial cell lines and viruses will be useful in studying their association with EBV in gastric epithelial cells.

Epstein-Barr-virus-infected human T-cell line with a unique pattern of viral-gene expression

Epstein-Barr-virus(EBV)-gene expression was analyzed in clonal sub-lines of the human T-cell line MT-2 that are persistently infected by the virus with a positive selection marker. Immunoblot analyses showed the expression of EBV proteins associated with the latent viral cycle, including the EBV nuclear antigen 1 (EBNA1) and the latent membrane protein 1 (LMP1), whereas EBNA2 was not detected. RT-PCR further demonstrated the expression of LMP2A, EBV-encoded small RNA 1 (EBER1), and complementary-strand transcripts from the BamHI-A fragment. EBNA2 mRNA was detected at a very low level. In addition to these latent viral functions, the BZLF1 protein, a transcription factor that activates a cascade of replicative-gene expression in EBV-infected B cells, was detected in these MT-2 clones by immunoblot analysis and immuno-enzymatic staining. Moreover, mRNA from other genes of the early virus-replicative cycle, such as BRLF1, BSMLF1, and BALF2, as well as BZLF1 was detected by Northern-blot analysis. Since sub-lines of the human B-cell lines BJAB and Louckes infected with similar recombinant EBV did not express these early genes, these data suggest that MT-2 cells are relatively permissive for expression of early EBV-replicative functions.