Epstein-Barr virus BZLF1 protein binds to mitotic chromosomes

Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis and is associated with several types of cancers, including nasopharyngeal carcinoma and Burkitt's lymphoma. An EBV protein that plays an integral role during lytic replication is the immediate-early protein BZLF1. Our laboratory has found that BZLF1 (Z) localizes to host chromosomes during mitosis. Two Z-interacting proteins are also found localized to mitotic chromosomes in the presence of Z. The association between Z and mitotic chromosomes may lead to the sequestering of Z-interacting proteins within the cell and potentially cause an alteration of chromosome compaction or chromatin structure.     

Epstein-Barr virus immediate-early proteins BZLF1 and BRLF1 alter mitochondrial morphology during lytic replication

Epstein-Barr virus (EBV) is a human DNA virus that is responsible for the syndrome infectious mononucleosis, and is associated with several forms of cancer. During both lytic and latent viral infection, viral proteins manipulate the host's cellular components to aid in viral replication and maintenance. Here, it is demonstrated that induction of EBV lytic replication results in a dramatic reorganization of mitochondria accompanied by a significant alteration of mitochondrial membrane potential and a rapid and transient increase in the microtubular cytoskeleton. Moreover, we show that expression of the EBV immediate-early genes BZLF1 and BRLF1 contributes to the mitochondrial alteration but not the increase in the microtubule cytoskeleton, suggesting that the mechanism for the observed cytoplasmic restructuring involves a number of coordinated viral and host proteins.     

Low level of lytic replication in a recombinant Epstein-Barr virus carrying an origin of replication devoid of BZLF1-binding sites

Binding of the BZLF1 viral transactivator to Epstein-Barr virus (EBV) oriLyt has been reported to be essential for viral DNA replication. We have constructed a recombinant virus (E2-oriLyt-R) in which the oriLyt BZLF1-binding sites (ZRE) were exchanged against papilloma E2-binding sites. A fusion protein between the BZLF1 protein-transactivating domain and the E2 protein-binding domain was able to reactivate lytic replication in E2-oriLyt-R. However, BZLF1 alone could also induce E2-oriLyt-R, albeit with much lower efficiency. ZRE are therefore important but not absolutely essential cis elements for lytic replication. This shows the importance of recombinants to evaluate viral functions.     

Functional and physical interaction between p53 and BZLF1: implications for Epstein-Barr virus latency.

The p53 tumor suppressor protein, which is commonly mutated in human cancers, has been shown to interact directly with virally encoded from papillomavirus, adenovirus, and simian virus 40. The disruption of p53 function may be required for efficient replication of certain viruses and may also play a role in the development of virally induced malignancies. Infection with Epstein-Barr virus (EBV) has been associated with the development of B-cell lymphomas and nasopharyngeal carcinoma. Here we show that the EBV immediate-early protein, BZLF1 (Z), which is responsible for initiating the switch from latent to lytic infection, can interact directly in vitro and in vivo with the tumor suppressor protein, p53. This interaction requires the coiled-coil dimerization domain of the Z protein and the carboxy-terminal portion of p53. Overexpression of wild-type p53 inhibits the ability of Z to disrupt viral latency. Likewise, Z inhibits p53-dependent transactivation in lymphoid cells. The direct interaction between Z and p53 may play a role in regulating the switch from latent to lytic viral infection.     

Epstein-Barr virus immediate-early protein BZLF1 inhibits tumor necrosis factor alpha-induced signaling and apoptosis by downregulating tumor necrosis factor receptor 1

Tumor necrosis factor alpha (TNF-alpha) is a key mediator of host immune and inflammatory responses and inhibits herpesvirus replication by cytolytic and noncytolytic mechanisms. TNF-alpha effects are primarily mediated through the major TNF-alpha receptor, TNF-R1, which is constitutively expressed in most cell types. Here we show that the Epstein-Barr virus (EBV) immediate-early protein BZLF1 prevents TNF-alpha activation of target genes and TNF-alpha-induced cell death. These effects are mediated by down-regulation of the promoter for TNF-R1. Additionally, we demonstrate that expression of TNF-R1 is downregulated during the EBV lytic replication cycle. Thus, EBV has developed a novel mechanism for evading TNF-alpha antiviral effects during lytic reactivation or primary infection.     

Plasma cell-specific transcription factor XBP-1s binds to and transactivates the Epstein-Barr virus BZLF1 promoter

Epstein-Barr virus (EBV) in vivo is known to establish persistent infection in resting, circulating memory B cells and to productively replicate in plasma cells. Until now, the molecular mechanism of how EBV switches from latency to lytic replication in vivo was not known. Here, we report that the plasma cell differentiation factor, XBP-1s, activates the expression of the master regulator of EBV lytic activation, BZLF1. Using reporter assays, we observed that XBP-1s was able to transactivate the BZLF1 promoter, Zp, in a plasma cell line and other lymphoid cell lines but, interestingly, not in epithelial cell lines. We have identified an XBP-1s binding site on the ZID/ZII region of Zp, which when abolished by site-directed mutagenesis led to abrogation of XBP-1s binding and promoter activation. Using the chromatin immunoprecipitation assay, we observed direct binding of XBP-1s to endogenous Zp in an EBV-infected plasma cell line. Finally, in the same cell line, we observed that overexpression of XBP-1s resulted in increased expression of BZLF1, while knockdown of XBP-1s with short hairpin RNA drastically reduces BZLF1 expression. We suggest that EBV harnesses the B-cell terminal differentiation pathway via XBP-1s as a physiological signal to reactivate and begin viral replication. We are currently investigating other signals, such as the endoplasmic reticulum stress response proteins, which act upstream of XBP-1s, to identify other interacting factors that initiate and/or amplify the lytic switch.     

Epstein-Barr virus protein kinase BGLF4 is a virion tegument protein that dissociates from virions in a phosphorylation-dependent process and phosphorylates the viral immediate-early protein BZLF1

Epstein-Barr virus (EBV) BGLF4 is a viral protein kinase that is expressed in the lytic phase of infection and is packaged in virions. We report here that BGLF4 is a tegument protein that dissociates from the virion in a phosphorylation-dependent process. We also present evidence that BGLF4 interacts with and phosphorylates BZLF1, a key viral regulator of lytic infection. These conclusions are based on the following observations. (i) In in vitro tegument release assays, a significant fraction of BGLF4 was released from virions in the presence of physiological NaCl concentrations. (ii) Addition of physiological concentrations of ATP and MgCl(2) to virions enhanced BGLF4 release, but phosphatase treatment of virions significantly reduced BGLF4 release. (iii) A recombinant protein containing a domain of BZLF1 was specifically phosphorylated by purified recombinant BGLF4 in vitro, and BGLF4 altered BZLF1 posttranslational modification in vivo. (iv) BZLF1 was specifically coimmunoprecipitated with BGLF4 in 12-O-tetradecanoylphorbol-13-acetate-treated B95-8 cells and in COS-1 cells transiently expressing both of these viral proteins. (v) BGLF4 and BZLF1 were colocalized in intranuclear globular structures, resembling the viral replication compartment, in Akata cells treated with anti-human immunoglobulin G. Our results suggest that BGLF4 functions not only in lytically infected cells by phosphorylating viral and cellular targets but also immediately after viral penetration like other herpesvirus tegument proteins.     

Herpes simplex virus type 1 infection activates the Epstein-Barr virus replicative cycle via a CREB-dependent mechanism

The reactivation of latent Epstein-Barr virus (EBV) to lytic replication is important in pathogenesis and requires virus-host cellular interactions. However, the mechanism underlying the reactivation of EBV is not yet fully understood. In the present study, herpes simplex virus type 1 (HSV-1) was shown to induce the reactivation of latent EBV by triggering BZLF1 expression. The BZLF1 promoter (Zp) was not activated by HSV-1 essential glycoprotein-induced membrane fusion. Nevertheless, Zp was activated within 6 h post HSV-1 infection in virus entry-dependent and replication-independent manners. Using a panel of Zp deletion mutants, HSV-1 was shown to promote Zp through a cyclic adenosine monophosphate (cAMP) response element (CRE) located in ZII. The phosphorylated cAMP response element-binding (phos-CREB) protein, the cellular transactivator that binds to CRE, also increased after HSV-1 infection. By transient transfection, cAMP-dependent protein kinase A and HSV-1 US3 protein were found to be capable of activating Zp in CREB- and CRE-dependent manners. The relationship between EBV activation and HSV-1 infection revealed a possible common mechanism that stimulated latent EBV into lytic cycles in vivo.     

Epstein-Barr virus BZLF1 gene, a switch from latency to lytic infection, is expressed as an immediate-early gene after primary infection of B lymphocytes

We demonstrate here that the Epstein-Barr virus (EBV) BZLF1 gene, a switch from latent infection to lytic infection, is expressed as early as 1.5 h after EBV infection in Burkitt's lymphoma-derived, EBV-negative Akata and Daudi cells and primary B lymphocytes. Since BZLF1 mRNA is expressed even when the cells are infected with EBV in the presence of anisomycin, an inhibitor of protein synthesis, its expression does not require prerequisite protein synthesis, indicating that BZLF1 is expressed as an immediate-early gene following primary EBV infection of B lymphocytes.     

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 (EBV) immediate-early protein BZLF1 mediates the switch between the latent and lytic forms of EBV infection and has been previously shown to induce a G(1)/S block in cell cycle progression in some cell types. To examine the effect of BZLF1 on cellular gene expression, we performed microarray analysis on telomerase-immortalized human keratinocytes that were mock infected or infected with a control adenovirus vector (AdLacZ) or a vector expressing the EBV BZLF1 protein (AdBZLF1). Cellular genes activated by BZLF1 expression included E2F-1, cyclin E, Cdc25A, and a number of other genes involved in cell cycle progression. Immunoblot analysis confirmed that BZLF1 induced expression of E2F-1, cyclin E, Cdc25A, and stem loop binding protein (a protein known to be primarily expressed during S phase) in telomerase-immortalized keratinocytes. Similarly, BZLF1 increased expression of E2F-1, cyclin E, and stem loop binding protein (SLBP) in primary tonsil keratinocytes. In contrast, BZLF1 did not induce E2F-1 expression in normal human fibroblasts. Cell cycle analysis revealed that while BZLF1 dramatically blocked G(1)/S progression in normal human fibroblasts, it did not significantly affect cell cycle progression in primary human tonsil keratinocytes. Furthermore, in EBV-infected gastric carcinoma cells, the BZLF1-positive cells had an increased number of cells in S phase compared to the BZLF1-negative cells. Thus, in certain cell types (but not others), BZLF1 enhances expression of cellular proteins associated with cell cycle progression, which suggests that an S-phase-like environment may be advantageous for efficient lytic EBV replication in some cell types.     

Reactivation of lytic replication from B cells latently infected with Epstein-Barr virus occurs with high S-phase cyclin-dependent kinase activity while inhibiting cellular DNA replication

Productive infection and replication of herpesviruses usually occurs in growth-arrested cells, but there has been no direct evidence in the case of Epstein-Barr virus (EBV), since an efficient lytic replication system without external stimuli does not exist for the virus. Expression of the EBV lytic-switch transactivator BZLF1 protein in EBV-negative epithelial tumor cell lines, however, is known to arrest the cell cycle in G(0)/G(1) by induction of the tumor suppressor protein p53 and the cyclin-dependent kinase (CDK) inhibitors p21(WAF-1/CIP-1) and p27(KIP-1), followed by the accumulation of a hypophosphorylated form of the Rb protein. In order to determine the effect of the onset of lytic viral replication on cellular events in latently EBV-infected B LCLs, a tightly controlled induction system of the EBV lytic-replication program by inducible BZLF1 protein expression was established in B95-8 cells. The induction of lytic replication completely arrested cell cycle progression and cellular DNA replication. Surprisingly, the levels of p53, p21(WAF-1/CIP-1), and p27(KIP-1) were constant before and after induction of the lytic program, indicating that the cell cycle arrest induced by the lytic program is not mediated through p53 and the CDK inhibitors. Furthermore, although cellular DNA replication was blocked, elevation of cyclin E/A expression and accumulation of hyperphosphorylated forms of Rb protein were observed, a post-G(1)/S phase characteristic of cells. Thus, while the EBV lytic program promoted specific cell cycle-associated activities involved in the progression from G(1) to S phase, it inhibited cellular DNA synthesis. Such cellular conditions appear to especially favor viral lytic replication.