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 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.     

Analysis of the BZLF1 promoter of Epstein-Barr virus: identification of an anti-immunoglobulin response sequence.

The induction of the viral lytic cycle in latently Epstein-Barr virus (EBV)-infected B cells is initiated by activation of the BZLF1 gene, whose expression is sufficient to disrupt EBV latency, suggesting that BZLF1 acts as the switch to change from a latent to a lytic replicative cycle. In the present studies, a series of deletion plasmids encompassing positions bp -552 to +12 of the BZLF1 promoter were constructed and tested for their response to anti-immunoglobulin (anti-Ig), an inducer of the viral lytic cycle, upon transfection into EBV-negative and -positive lymphoid cells. The promoter consisted of three functionally distinct regions. Region I (bp -552 to -221) had a negative influence on promoter activity; its deletion made the promoter highly responsive to anti-Ig. Region II (bp -203 to -177) was important for conferring responsiveness to anti-Ig. The response to anti-Ig did not require the presence of the EBV genome or EBV gene products. This sequence also enhanced expression of the chloramphenicol acetyltransferase (cat) gene from the simian virus 40 promoter in response to anti-Ig, even when inserted downstream of the cat gene. Region III (-134 to -116) was a positive element that was transactivated by the BZLF1 gene product.     

The Epstein-Barr virus BMLF1 promoter contains an enhancer element that is responsive to the BZLF1 and BRLF1 transactivators.

We have previously shown that the Epstein-Barr virus (EBV) immediate-early gene product, BZLF1, can activate expression of the EBV BMLF1 immediate-early promoter in EBV-positive, but not EBV-negative, B cells, suggesting that the BZLF1 effect may be mediated through another EBV gene product (S. Kenney, J. Kamine, E. Holley-Guthrie, J.-C. Lin, E.-C. Mar, and J. S. Pagano, J. Virol. 63:1729-1736, 1989). Here, we show that the EBV BRLF1 immediate-early gene product transactivates the BMLF1 promoter in either EBV-positive or EBV-negative B cells. Deletional analysis revealed that both the BZLF1-responsive region and the BRLF1-responsive region of the BMLF1 promoter are contained within the same 140-base-pair FokI-PvuII fragment located 300 base pairs upstream of the mRNA start site. This FokI-PvuII fragment functions as an enhancer element in the presence of the BRLF1 transactivator and contains the sequence CCGTGGAGA ATGTC, which is strikingly similar to the BRLF1-responsive region of the EBV DR/DL enhancer (A. Chevallier-Greco, H. Gruffat, E. Manet, A. Calender, and A. Sergeant, J. Virol. 63:615-623, 1989). The effect of BZLF1 on the BMLF1 promoter is likely to be indirect and mediated through the BRLF1 transactivator.     

The Epstein-Barr virus (EBV) BZLF1 immediate-early gene product differentially affects latent versus productive EBV promoters.

The Epstein-Barr virus (EBV) BZLF1 gene product is thought to mediate the disruption of latent EBV infection. We have examined the regulatory effects of BZLF1 by studying its transactivating effects on seven different EBV promoters. We find that whereas the BZLF1 gene product increases the activity of the two early promoters, BMLF1 and BMRF1, it decreases the activity of three latent promoters (the BamHI-C and BamHI-W Epstein-Barr nuclear antigen promoters and the latent membrane protein promoter). The BZLF1-induced changes in promoter-directed chloramphenicol acetyltransferase activity occur in EBV-negative as well as EBV-positive cell lines and are accompanied by a similar change in chloramphenicol acetyltransferase mRNA. Deletion analysis of the BamHI Z fragment indicates that in a portion of the amino-terminal half of the BZLF1 gene product (amino acids 24 to 86) is not essential for positive transactivating effects but is required for down-regulating effects. Thus, different domains of the same EBV immediate-early gene product can either increase the function of EBV promoters active in productive infection or decrease the function of key promoters active in latent infection.     

The spliced BZLF1 gene of Epstein-Barr virus (EBV) transactivates an early EBV promoter and induces the virus productive cycle.

A spliced cDNA spanning the Epstein-Barr virus BZLF1 gene expresses the BZLF1 protein and is active in inducing the virus productive cycle. A deletion mutant which lacks the N-terminal half of the protein is inactive. Cotransfection experiments in EBV-negative B-lymphocyte cell lines demonstrated that the BZLF1 gene activates the promoter for the BSLF2 + BMLF1 gene in the absence of any other EBV gene product. These results confirmed that the spliced BZLF1 gene is the transactivating gene structure in BamHI-Z.     

Terminal differentiation into plasma cells initiates the replicative cycle of Epstein-Barr virus in vivo

In this paper we demonstrate that the cells which initiate replication of Epstein-Barr virus (EBV) in the tonsils of healthy carriers are plasma cells (CD38hi, CD10-, CD19+, CD20lo, surface immunoglobulin negative, and cytoplasmic immunoglobulin positive). We further conclude that differentiation into plasma cells, and not the signals that induce differentiation, initiates viral replication. This was confirmed by in vitro studies showing that the promoter for BZLF1, the gene that begins viral replication, becomes active only after memory cells differentiate into plasma cells and is also active in plasma cell lines. This differs from the reactivation of BZLF1 in vitro, which occurs acutely and is associated with apoptosis and not with differentiation. We suggest that differentiation and acute stress represent two distinct pathways of EBV reactivation in vivo. The fraction of cells replicating the virus decreases as the cells progress through the lytic cycle such that only a tiny fraction actually release infectious virus. This may reflect abortive replication or elimination of cells by the cellular immune response. Consistent with the later conclusion, the cells did not down regulate major histocompatibility complex class I molecules, suggesting that this is not an immune evasion tactic used by EBV and that the cells remain vulnerable to cytotoxic-T-lymphocyte attack.     

The Cellular Ataxia Telangiectasia-Mutated Kinase Promotes Epstein-Barr Virus Lytic Reactivation in Response to Multiple Different Types of Lytic Reactivation-Inducing Stimuli

The Epstein-Barr virus (EBV) latent-to-lytic switch is mediated by the viral proteins BZLF1 (Z), BRLF1 (R), and BRRF1 (Na). Since we previously showed that DNA-damaging agents (including chemotherapy and irradiation) can induce EBV lytic reactivation and recently demonstrated that wild-type p53 contributes to lytic reactivation, we investigated the role of the ATM kinase during EBV reactivation. ATM phosphorylates and activates p53, as well as numerous other substrates involved in the cellular DNA damage response. Using an ATM inhibitor (KU55933), we found that ATM activity is required for efficient induction of EBV lytic gene expression by a variety of different stimuli, including a histone deacetylase (HDAC) inhibitor, the transforming growth factor β (TGF-β) cytokine, a demethylating agent (5-azacytidine), B cell receptor engagement with anti-IgG antibody, hydrogen peroxide, and the proteosome inhibitor bortezomib. In EBV-infected AGS (gastric) cells, knockdown of ATM, or p53, expression inhibits EBV reactivation. Conversely, treatment of these cells with nutlin-3 (which activates p53 and ATM) robustly induces lytic reactivation in a p53- and ATM-dependent manner. The ability of the EBV R and Na proteins to induce lytic reactivation in EBV-infected AGS cells is ATM dependent. However, overexpression of Z induces lytic gene expression in the presence or absence of ATM activity. Our results suggest that ATM enhances Z promoter activity in the context of the intact EBV genome and that p53 contributes to the ATM effect. Nevertheless, since we found that ATM inhibitors also reduce lytic reactivation in Burkitt lymphoma cells that have no p53, additional ATM substrates must also contribute to the ATM effect.