Epstein-Barr Virus Early Genes Research Articles

Abstract 5786: Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes stimulate interferon-β production by nasopharyngeal carcinoma (NPC) cells to enhance the natural killer cell-mediated cytotoxicity against NPC

Abstract Introduction: The prevailing histologic subtype of nasopharyngeal carcinoma (NPC) in the endemic regions is non-keratinizing carcinoma which shows almost consistent association with latent infection by Epstein-Barr virus (EBV). Previously our group developed a series of Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes, which can disrupt the EBV latency maintained by EBNA1, and such lytic induction could alert the immune cells. Methods: Natural killer (NK) cells of the innate immunity play a significant role in controlling EBV infection. In this study, we investigated whether our EBNA1 probes (ZRL5P4 & L2P4) could activate NK cells to eliminate NPC. Results: Studies of clinical specimens indicated that tumor infiltration of the cytotoxic NK cell subset is a favorable prognostic marker for NPC patients. Furthermore, the cytotoxicity of circulating NK cells derived from NPC patients is equally well the cells from healthy individuals. If those patients’ NK cells can be attracted to infiltrate into the tumors, the tumor burden should be relieved. Indeed, the NPC xenograft with the ZRL5P4 treatment showed heavy mouse NK cell infiltration and tumor size reduction. The essential role of those mouse NK cells was demonstrated. Next, we showed that ZRL5P4 could induce the production of a soluble factor by NPC cells to stimulate the NK cell cytotoxicity. Such NK cell activity was verified in a metastasis NPC model. Mechanistically, the EBNA1 probe treatment could induce the expression of EBV early lytic gene Rta and EBNA1 itself. Both Rta and EBNA1 are known transcription factors to drive the expression of LMP1, the NF-κB signaling was then activated, which turned on the production of the well-known NK cell activation cytokine IFN-β. Conclusion: Taken together, our results demonstrated that our EBNA1 probes can mobilize NK cells to the tumor sites and activate their anti-tumor activity via the EBV reactivation and stimulation of IFN-β production. Citation Format: Luo Chen, Xiao Han Liu, Hei Tung Yuen, William C. Cho, Ka-Leung Wong, Hong Lok Lung. Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes stimulate interferon-β production by nasopharyngeal carcinoma (NPC) cells to enhance the natural killer cell-mediated cytotoxicity against NPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5786.

Retinoblastoma Protein Is Required for Epstein-Barr Virus Replication in Differentiated Epithelia.

Coinfection of human papillomavirus (HPV) and Epstein-Barr virus (EBV) has been detected in oropharyngeal squamous cell carcinoma. Although HPV and EBV replicate in differentiated epithelial cells, we previously reported that HPV epithelial immortalization reduces EBV replication within organotypic raft culture and that the HPV16 oncoprotein E7 was sufficient to inhibit EBV replication. A well-established function of HPV E7 is the degradation of the retinoblastoma (Rb) family of pocket proteins (pRb, p107, and p130). Here, we show that pRb knockdown in differentiated epithelia and EBV-positive Burkitt lymphoma (BL) reduces EBV lytic replication following de novo infection and reactivation, respectively. In differentiated epithelia, EBV immediate early (IE) transactivators were expressed, but loss of pRb blocked expression of the early gene product, EA-D. Although no alterations were observed in markers of epithelial differentiation, DNA damage, and p16, increased markers of S-phase progression and altered p107 and p130 levels were observed in suprabasal keratinocytes after pRb knockdown. In contrast, pRb interference in Akata BX1 Burkitt lymphoma cells showed a distinct phenotype from differentiated epithelia with no significant effect on EBV IE or EA-D expression. Instead, pRb knockdown reduced the levels of the plasmablast differentiation marker PRDM1/Blimp1 and increased the abundance of c-Myc protein in reactivated Akata BL with pRb knockdown. c-Myc RNA levels also increased following the loss of pRb in epithelial rafts. These results suggest that pRb is required to suppress c-Myc for efficient EBV replication in BL cells and identifies a mechanism for how HPV immortalization, through degradation of the retinoblastoma pocket proteins, interferes with EBV replication in coinfected epithelia. IMPORTANCE Terminally differentiated epithelium is known to support EBV genome amplification and virion morphogenesis following infection. The contribution of the cell cycle in differentiated tissues to efficient EBV replication is not understood. Using organotypic epithelial raft cultures and genetic interference, we can identify factors required for EBV replication in quiescent cells. Here, we phenocopied HPV16 E7 inhibition of EBV replication through knockdown of pRb. Loss of pRb was found to reduce EBV early gene expression and viral replication. Interruption of the viral life cycle was accompanied by increased S-phase gene expression in postmitotic keratinocytes, a process also observed in E7-positive epithelia, and deregulation of other pocket proteins. Together, these findings provide evidence of a global requirement for pRb in EBV lytic replication and provide a mechanistic framework for how HPV E7 may facilitate a latent EBV infection through its mediated degradation of pRb in copositive epithelia.

EBV dUTPase: A Novel Modulator of Inflammation and the Tumor Microenvironment in EBV-Associated Malignancies.

There is increasing evidence that put into question the classical dogma that the Epstein-Barr virus (EBV) exists in cells as either a lytic virus in which new progeny is produced or in a latent state in which no progeny is produced. Notably, a third state has now been described, known as the abortive-lytic phase, which is characterized by the expression of some immediate early (IE) and early (E) genes, but no new virus progeny is produced. While the function of these IE and E gene products is not well understood, several recent studies support the concept they may contribute to tumor promotion by altering the tumor microenvironment (TME). The mechanisms by which these viral gene products may contribute to tumorigenesis remain unclear; however, it has been proposed that some of them promote cellular growth, immune evasion, and/or inhibit apoptosis. One of these EBV early gene products is the deoxyuridine triphosphate nucleotidohydrolase (dUTPase) encoded by BLLF3, which not only contributes to the establishment of latency through the production of activin A and IL-21, but it may also alter the TME, thus promoting oncogenesis.

Rta is the principal activator of Epstein-Barr virus epithelial lytic transcription.

The transition from latent Epstein-Barr virus (EBV) infection to lytic viral replication is mediated by the viral transcription factors Rta and Zta. Although both are required for virion production, dissecting the specific roles played by Rta and Zta is challenging because they induce each other’s expression. To circumvent this, we constructed an EBV mutant deleted for the genes encoding Rta and Zta (BRLF1 and BZLF1, respectively) in the Akata strain BACmid. This mutant, termed EBVΔRZ, was used to infect several epithelial cell lines, including telomerase-immortalized normal oral keratinocytes, a highly physiologic model of EBV epithelial cell infection. Using RNA-seq, we determined the gene expression induced by each viral transactivator. Surprisingly, Zta alone only induced expression of the lytic origin transcripts BHLF1 and LF3. In contrast, Rta activated the majority of EBV early gene transcripts. As expected, Zta and Rta were both required for expression of late gene transcripts. Zta also cooperated with Rta to enhance a subset of early gene transcripts (Rtasynergy transcripts) that Zta was unable to activate when expressed alone. Interestingly, Rta and Zta each cooperatively enhanced the other’s binding to EBV early gene promoters, but this effect was not restricted to promoters where synergy was observed. We demonstrate that Zta did not affect Rtasynergy transcript stability, but increased Rtasynergy gene transcription despite having no effect on their transcription when expressed alone. Our results suggest that, at least in epithelial cells, Rta is the dominant transactivator and that Zta functions primarily to support DNA replication and co-activate a subset of early promoters with Rta. This closely parallels the arrangement in KSHV where ORF50 (Rta homolog) is the principal activator of lytic transcription and K8 (Zta homolog) is required for DNA replication at oriLyt.

An Epigenetic Journey: Epstein-Barr Virus Transcribes Chromatinized and Subsequently Unchromatinized Templates during Its Lytic Cycle.

The Epstein-Barr virus (EBV) lytic phase, like those of all herpesviruses, proceeds via an orderly cascade that integrates DNA replication and gene expression. EBV early genes are expressed independently of viral DNA amplification, and several early gene products facilitate DNA amplification. On the other hand, EBV late genes are defined by their dependence on viral DNA replication for expression. Recently, a set of orthologous genes found in beta- and gammaherpesviruses have been determined to encode a viral preinitiation complex (vPIC) that mediates late gene expression. The EBV vPIC requires an origin of lytic replication in cis, implying that the vPIC mediates transcription from newly replicated DNA. In agreement with this implication, EBV late gene mRNAs localize to replication factories. Notably, these factories exclude canonical histones. In this review, we compare and contrast the mechanisms and epigenetics of EBV early and late gene expression. We summarize recent findings, propose a model explaining the dependence of EBV late gene expression on lytic DNA amplification, and suggest some directions for future study.

The Methylation Status and Expression of Epstein-Barr Virus Early Genes BARF1 and BHRF1 in Epstein-Barr Virus-Associated Gastric Carcinomas.

Epstein-Barr virus (EBV) is an important DNA virus which establishes latent infection in human malignancies. Expression of EBV-encoded genes in the associated tumors is strongly modulated by promoter CpG methylation of EBV genome. This study aimed to explore the methylation status of the promoters of EBV BamHI-A rightward frame 1 (BARF1) and BamHI-H rightward open reading frame 1 (BHRF1) and their influence on transcriptional expression, to further understand the roles of BARF1 and BHRF1 in the occurrence of EBV-associated cancer. We evaluated the methylation status of BARF1 and BHRF1 promoters in 43 EBV-associated gastric carcinoma (EBVaGC) tissues and EBV-positive cell lines. Their expressions were evaluated by real-time quantitative PCR. We found that the promoters of BARF1 and BHRF1 were methylated by varying degrees in different EBV-positive cell lines and were almost hypermethylated in all EBVaGC tissues. The methylation status of BARF1 and BHRF1 promoters were significantly reduced by 5-Aza-CdR along with the increasing gene expressions. Hypermethylation of Ap and Hp mediates the frequent silencing of BARF1 and BHRF1 in EBV-associated tumors, which could be reactivated by a demethylation agent, suggesting that promoter demethylation and activation is important for BARF1 and BHRF1 transcription and their further action.

Sequence analysis of Epstein-Barr virus (EBV) early genes BARF1 and BHRF1 in NK/T cell lymphoma from Northern China.

BackgroundNK/T cell lymphoma is an aggressive lymphoma almost always associated with EBV. BamHI-A rightward open reading frame 1 (BARF1) and BamHI-H rightward open reading frame 1 (BHRF1) are two EBV early genes, which may be involved in the oncogenicity of EBV. It has been found that V29A strains, a BARF1 mutant subtype, showed higher prevalence in NPC, which may suggest the association between this variation and nasopharyngeal carcinoma (NPC). To characterize the sequence variation patterns of the Epstein-Barr virus (EBV) early genes and to elucidate their association with NK/T cell lymphoma, we analyzed the sequences of BARF1 and BHRF1 in EBV-positive NK/T cell lymphoma samples from Northern China.MethodsIn situ hybridization (ISH) performed for EBV-encoded small RNA1 (EBER1) with specific digoxigenin-labeled probes was used to select the EBV positive lymphoma samples. Nested-polymerase chain reaction (nested-PCR) and DNA sequence analysis technique were used to obtain the sequences of BARF1 and BHRF1. The polymorphisms of these two genes were classified according to the signature changes and compared with the known corresponding EBV gene variation data.ResultsTwo major subtypes of BARF1 gene, designated as B95-8 and V29A subtype, were identified. B95-8 subtype was the dominant subtype. The V29A subtype had one consistent amino acid change at amino acid residue 29 (V → A). Compared with B95-8, AA change at 88 (L → V) of BHRF1 was found in the majority of the isolates, and AA79 (V → L) mutation in a few isolates. Functional domains of BARF1 and BHRF1 were highly conserved. The distributions of BARF1 and BHRF1 subtypes had no significant differences among different EBV-associated malignancies and healthy donors.ConclusionThe sequences of BARF1 and BHRF1 are highly conserved which may contribute to maintain the biological function of these two genes. There is no evidence that particular EBV substrains of BARF1 or BHRF1 is region-restricted or disease-specific.

Either ZEB1 or ZEB2/SIP1 Can Play a Central Role in Regulating the Epstein-Barr Virus Latent-Lytic Switch in a Cell-Type-Specific Manner

We previously reported that the cellular protein ZEB1 can repress expression of the Epstein-Barr virus (EBV) BZLF1 gene in transient transfection assays by directly binding its promoter, Zp. We also reported that EBV containing a 2-bp substitution mutation in the ZEB-binding ZV element of Zp spontaneously reactivated out of latency into lytic replication at a higher frequency than did wild-type EBV. Here, using small interfering RNA (siRNA) and short hairpin RNA (shRNA) technologies, we definitively show that ZEB1 is, indeed, a key player in maintaining EBV latency in some epithelial and B-lymphocytic cell lines. However, in other EBV-positive epithelial and B-cell lines, another zinc finger E-box-binding protein, ZEB2/SIP1, is the key player. Both ZEB1 and ZEB2 can bind Zp via the ZV element. In EBV-positive cells containing only ZEB1, knockdown of ZEB1 led to viral reactivation out of latency, with synthesis of EBV immediate-early and early lytic gene products. However, in EBV-positive cells containing both ZEBs, ZEB2, not ZEB1, was the primary ZEB family member bound to Zp. Knockdown of ZEB2, but not ZEB1, led to EBV lytic reactivation. Thus, we conclude that either ZEB1 or ZEB2 can play a central role in the maintenance of EBV latency, doing so in a cell-type-dependent manner.

Recurrent chemical reactivations of EBV promotes genome instability and enhances tumor progression of nasopharyngeal carcinoma cells

Nasopharyngeal carcinoma (NPC) is an endemic malignancy prevalent in South East Asia. Epidemiological studies have associated this disease closely with Epstein-Barr virus (EBV) infection. Previous studies also showed that EBV reactivation is implicated in the progression of NPC. Thus, we proposed that recurrent reactivations of EBV may be important for its pathogenic role. In this study, NPC cell lines latently infected with EBV, NA and HA, and the corresponding EBV-negative NPC cell lines, NPC-TW01 (TW01) and HONE-1, were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium n-butyrate (SB) for lytic cycle induction. A single treatment with TPA/SB revealed that DNA double-strand breaks and formation of micronuclei (a marker for genome instability) were associated with EBV reactivation in NA and HA cells. Examination of EBV early genes had identified several lytic proteins, particularly EBV DNase, as potent activators that induced DNA double-strand breaks and contribute to genome instability. Recurrent reactivations of EBV in NA and HA cells resulted in a marked increase of genome instability. In addition, the degree of chromosomal aberrations, as shown by chromosome structural variants and DNA copy-number alterations, is proportional to the frequency of TPA/SB-induced EBV reactivation. Whereas these DNA abnormalities were limited in EBV-negative TW01 cells with mock or TPA/SB treatment, and were few in mock-treated NA cells. The invasiveness and tumorigenesis assays also revealed a profound increase in both characteristics of the repeatedly reactivated NA cells. These results suggest that recurrent EBV reactivations may result in accumulation of genome instability and promote the tumor progression of NPC.

Inhibition of nasopharyngeal carcinoma growth by RTA‐expressing baculovirus vectors containing oriP

Nasopharyngeal carcinoma (NPC) is closely associated with latent Epstein-Barr virus (EBV) infection. Activation of latent EBV into lytic replication by introducing viral lytic gene BRLF1 (RTA) has been shown to be a potential approach to suppress the growth of EBV-associated NPC tumor. The baculovirus vectors with RTA expression cassette (BV-R), RTA and the EBV latent replication origin (OriP, BV-RO), or RTA, OriP and EBNA-1 gene (BV-ROE-CMV), were constructed and examined for their ability to mediate RTA expression, initiate lytic replication and induce cell death in EBV-positive cell lines Hone1-EBV, HK1-EBV and C666-1 in vitro. Their effect to inhibit the growth of EBV-positive NPC tumors was also evaluated in nude mice. The baculovirus vectors BV-RO and BV-ROE-CMV mediated efficient expression of RTA in EBV-positive NPC cells. Lytic EBV replication and cell death were observed in these infected cells. Both vectors also significantly inhibited the growth of EBV-positive NPC tumor in nude mice. EBV early lytic gene expression and tumor cell death were observed in these treated tumors. The presence of OriP improved the performance of the RTA-expressing baculovirus vectors to induce EBV lytic replication and cell death in vitro, and suppress the growth of EBV positive NPC tumors in vivo. By confining RTA and EBNA-1 expression to EBV-positive cells, BV-RO is expected to be a better candidate in application than BV-ROE-CMV in the long term.

Alveolar epithelial cell injury with Epstein-Barr virus upregulates TGFβ1 expression

Idiopathic pulmonary fibrosis (IPF) is a refractory and lethal interstitial lung disease characterized by alveolar epithelial cells apoptosis, fibroblast proliferation, and ECM protein deposition. Epstein-Barr virus (EBV) has previously been localized to alveolar epithelial cells of IPF patients and is associated with a poor prognosis. In this study, we utilized a microarray-based differential gene expression analysis strategy to identify molecular drivers of EBV-associated lung fibrosis. Two cell lines, primary human alveolar epithelial cells type 2 and A549 cells, were infected with EBV. EBV lytic phase induction increased active and total transforming growth factor-beta1 (TGFbeta1) transcript expression in association with reduced cell proliferation and increased caspase 3/7 activity. Exposing EBV-infected cells to ganciclovir resulted in TGFbeta1 deregulation and reduced expression of EBV early response genes, BRLF1 and BZLF1. We targeted the BRLF1 and BZLF1 gene products, Rta and Zta, by silencing RNA, and this resulted in the normalization of TGFbeta1 transcript and cell proliferation levels. Our study using a viral cell line model complements existing human and animal model data and further provides evidence to suggest that viral epithelial cell injury may play a role in IPF.

Structural Basis of Lytic Cycle Activation by the Epstein-Barr Virus ZEBRA Protein

Epstein-Barr virus (EBV) causes infectious mononucleosis and is linked to several human malignancies. EBV has a biphasic infection cycle consisting of a latent and a lytic, replicative phase. The switch from latent to lytic infection is triggered by the EBV immediate-early transcription factor ZEBRA (BZLF1, Zta, Z, EB1). We present the crystal structure of ZEBRA's DNA binding domain bound to an EBV lytic gene promoter element. ZEBRA exhibits a variant of the basic-region leucine zipper (bZIP) fold in which a C-terminal moiety stabilizes the coiled coil involved in dimer formation. The structure provides insights into ZEBRA's broad target site specificity, preferential activation of specific EBV promoters in their methylated state, ability to dimerize despite lacking a leucine zipper motif, and failure to heterodimerize with cellular bZIP proteins. The structure will allow for the design of new therapeutic agents that block activation of the EBV lytic cycle.

Characterization of the Epstein-Barr virus BALF2 promoter.

BALF2, which encodes the major DNA-binding protein of Epstein-Barr virus (EBV), is expressed during the early stage of the lytic cycle. The location of the BALF2 promoter was identified by primer extension, which indicated that the transcription start is located at nucleotide 164,782 of the EBV genome. Transfection analyses revealed that, similar to other EBV early promoters, the BALF2 promoter is activated by the EBV-encoded transcription factors Rta and Zta. The promoter is also synergistically activated if both transcription factors are present in B lymphocytes and in epithelial cells. Deletion analysis and electrophoretic mobility-shift assay revealed that the region between nucleotides -134 and -64 contains Zta-response elements and the region between nucleotides -287 and -254 contains Rta-response elements. This study demonstrates the importance of Rta and Zta in regulating the transcription of EBV early genes.

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.

Rescue of the Epstein–Barr Virus BZLF1 Mutant, Z(S186A), Early Gene Activation Defect by the BRLF1 Gene Product

Expression of the Epstein–Barr virus (EBV) immediate-early protein, BZLF1 (Z), is sufficient to disrupt viral latency. Z transcriptionally activates the EBV early genes by binding to upstream Z-responsive elements (ZREs). Recently, a serine-to-alanine mutation of Z residue 186 (within the basic DNA binding domain) was shown to inhibit the ability of Z to induce lytic infection in latently infected cells, although the Z(S186A) mutant could still bind several known ZREs and activated an early EBV promoter (BMRF1) in transient reporter gene assays (Francis, A. L., Gradoville, L., and Miller, G. (1997).J. Virol.71, 3054–3061). We now show that a specific deficiency in the ability to bind to ZRE elements in the immediate-early BRLF1 promoter may account for the inability of Z(S186A) to activate BRLF1 expression. Furthermore, we demonstrate that the ability of Z(S186A) to induce early BMRF1 and BHRF1 gene expression is rescued by cotransfection with a BRLF1 expression vector. However, the Z(S186A)/BRLF1 (R) combination cannot induce full lytic replication, suggesting that Z(S186A) may also be deficient in a replication-specific function. These results suggest that in the context of the intact viral genome, both Z and R expression are required for activation of early gene transcription in latently infected cells.

Epstein--Barr virus (EBV) EB1/Zta protein provided in trans and competent for the activation of productive cycle genes does not activate the BZLF1 gene in the EBV genome

The Epstein-Barr Virus (EBV) gene BZLF1 encodes the transcription fact or EB1 (also known as Zta) which is essential for the switch from latency to the lytic cycle: EB1 expressed from a plasmid transfected into B cell lines carrying latent EBV episomes, induces a productive viral cycle. Furthermore, EB1-specific DNA-binding sequences (ZREs) have been found in the promoters of many EBV early genes, including the BZLF1 promoter PZ and the PR promoter. At promoter PR, bicistronic mRNAs are initiated which contain, from 5' to 3', the BRLF1 and the BZLF1 open reading frames (ORFs) encoding respectively the R and EB1 proteins. The current model for the activation of the EBV lytic cycle implies that downregulation of the PZ promoter activity is a key element for latency and that a limiting step in the activation of the productive cycle is the translation of EB1. Once made, EB1 autoactivates promoter PZ, activates the PR promoter at which an mRNA coding for the EBV transcription factor R is initiated and activates the EBV early genes and the ORIlyt, due to unrestricted accessibility of the EB1-responsive elements in the viral genome. We show here that EB1 expressed from a plasmid activated most if not all of the EBV early genes in the viral genome but not its own gene, BZLF1. Moreover, transfected EB1 induced the transcription of the bicistronic mRNAs from which R is efficiently translated but not EB1. Our results demonstrate that EB1 provided in trans, although competent to activate the productive cycle genes, was not sufficient to overcome the downregulation of the PZ promoter.

Physical and functional interaction of the Epstein-Barr virus BZLF1 transactivator with the retinoic acid receptors RAR alpha and RXR alpha.

Epstein-Barr virus (EBV) reactivation, indicated by induction of EBV early antigens from latently infected lymphoid cell lines by phorbol esters, is inhibited by retinoic acid (RA). Viral reactivation, which is triggered by the immediate-early BZLF-1 (Z) viral gene product, is repressed by retinoic acid receptors (RARs) RAR alpha and RXR alpha. These proteins negatively regulate Z-mediated transactivation of the promoter for an EBV early gene product, early antigen-diffuse (EaD). Here we confirm a direct physical interaction between the AP1-like protein Z and RXR alpha and map the domains of interaction in the Z protein and RXR alpha. The domain required for homodimerization of Z is separate from that required for its interaction with RXR alpha. Z also has the effect of repressing activation of an RAR-responsive cellular promoter (BRE). Point mutants in the dimerization domain of Z unable to interact with RXR alpha do not repress RXR alpha-mediated transactivation of BRE, the promoter for RAR beta, which suggests that interaction between the two proteins is required for this repressor effect. The domain of RXR alpha required for interaction with Z has been mapped, and is again separate from that required for homodimerization. These results indicate that a 'cross-coupling' or direct interaction between Z and RAR alpha and RXR alpha can modulate the reactivation of latent EBV infection and suggest that, reciprocally, the viral protein Z may influence cellular regulatory pathways.

The Switch between Latency and Replication of Epstein-Barr Virus

Latency is characteristic behavior of many viruses that persist in their hosts. During latency an intact viral genome resides in the cell, but most viral genes are silent. Diverse inducing stimuli, by which the environment interacts with the host cell, trigger the latent viral genome into replication. Experiments are described that led to the identification of a crucial Epstein-Barr virus (EBV) gene, called ZEBRA, which acts as a switch between latency and replication of this herpes virus. During latency of EBV, ZEBRA expression is repressed. Inducing stimuli cause synthesis of ZEBRA which, in turn, activates expression of several individual EBV early genes. The ZEBRA polypeptide is a site-specific DNA binding protein that is likely to function as a transcriptional transactivator. The recognition that a single viral product may control the latency-to-replication switch offers promise of a detailed understanding of a central process in virology.

The Epstein-Barr virus early protein EB1 activates transcription from different responsive elements including AP-1 binding sites.

When expressed in Epstein-Barr virus (EBV) latently infected B cells, the EBV early protein EB1 trans-activates as many EBV early genes as does TPA. Several EB1 responsive elements (ZRE) have been identified in EBV early promoters and are located at relatively short distances from the TATA box. One of them (ZRE-M) overlaps with a consensus TPA responsive element (TRE) defined as an AP-1/c-jun/c-fos binding site and is located in an EBV promoter controlling the expression of the post-transcriptional activator EB2. Another (ZREZ) is located in the promoter controlling the expression of EB1 and does not respond to TPA. These two ZREs have no apparent sequence homology. Although EB1 activates transcription from the AP-1 enhancer sequence and from the ZREZ, the activation is severely impaired by distance, suggesting that EB1 is more likely to be a promoter factor than an enhancer factor. These properties also suggest that EB1 is not functionally related to c-jun and c-fos. However, since EB1 can activate transcription from AP-1 binding sites when properly positioned, the role of this factor in the oncogenic properties of EBV should be considered.

The Epstein-Barr virus (EBV) early promoter DR contains a cis-acting element responsive to the EBV transactivator EB1 and an enhancer with constitutive and inducible activities

The Epstein-Barr Virus (EBV) DR promoter controlled the expression of the PstI repeat region IR4. This promoter was activated by the EBV trans-acting factor EB1, mainly at the transcriptional level, and the activation was mediated by the TATA box and two cis-acting regulatory regions, one proximal to the TATA box and one distal to the TATA box. The distal region had enhancer properties. In HeLa cells, it activated transcription from the herpes simplex virus type 1 thymidine kinase promoter linked to the chloramphenicol acetyltransferase gene when located in inverted orientation upstream of the thymidine kinase promoter or downstream of the chloramphenicol acetyltransferase gene coding sequence. This enhancer also activated transcription from the simian virus 40 early upstream regulatory elements. These results indicate that the DR These results indicate that the DR enhancer can constitutively activate heterologous promoters in HeLa cells. However, the DR enhancer was not active in EBV genome-negative B cell lines, but it became active when these cells were infected by EBV and when the expression of the EBV early genes was induced by EB1. This suggests that an EBV early gene product induces the DR enhancer activity. The DR promoter TATA box-proximal cis-acting regulatory element contained EB1-responsive sequences.