scholarly journals 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

2010 ◽  
Vol 84 (12) ◽  
pp. 6139-6152 ◽  
Author(s):  
Amy L. Ellis ◽  
Zhenxun Wang ◽  
Xianming Yu ◽  
Janet E. Mertz
Keyword(s):  
Cell Lines ◽  
Epstein Barr Virus ◽  
Cellular Protein ◽  
Lytic Replication ◽  
Viral Reactivation ◽  
Dependent Manner ◽  
Cell Type ◽  
Barr Virus ◽  
A Cell ◽  
Epstein Barr

ABSTRACT 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.

scholarly journals Cell Cycle Association of the Retinoblastoma Protein Rb and the Histone Demethylase LSD1 with the Epstein-Barr Virus Latency Promoter Cp

2008 ◽  
Vol 82 (7) ◽  
pp. 3428-3437 ◽  
Author(s):  
Charles M. Chau ◽  
Zhong Deng ◽  
Hyojueng Kang ◽  
Paul M. Lieberman
Keyword(s):  
Cell Cycle ◽  
Epstein Barr Virus ◽  
Affinity Purification ◽  
Histone H3 ◽  
Histone Demethylase ◽  
Stable Complex ◽  
Dependent Manner ◽  
Barr Virus ◽  
A Cell ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus C promoter (Cp) regulates the major multicistronic transcript encoding the EBNA-LP, 1, 2, and 3 genes required for B-cell proliferation during latency. The growth-transforming potential of these viral genes suggests that they must be tightly regulated with the host cell cycle and differentiation process. To better understand Cp regulation, we used DNA affinity purification to identify cellular and viral proteins that bind to Cp in latently infected cells. Several previously unknown factors were identified, including the cell cycle regulatory proteins E2F1 and Rb. E2F1 bound to a specific site in Cp located in the core Cp region 3′ of the known EBNA2-responsive RBP-Jk (CSL, CBF1) binding site. The histone H3 K4 demethylase LSD1 (BCC110) was also identified by DNA affinity and was shown to form a stable complex with Rb. Coimmunoprecipitation assays demonstrated that E2F1, Rb, and LSD1 bind to Cp in a cell cycle-dependent manner. Rb and LSD1 binding to Cp increased after the S phase, corresponding to a decrease in histone H3 K4 methylation and Cp transcription. Coimmunoprecipitation and immunofluorescence assays reveal that LSD1 interacts with Rb. Surprisingly, LSD1 did not coimmunoprecipitate with E2F1, suggesting that it associates with Rb independently of E2F1. Depletion of LSD1 by small interfering RNAs inhibited Cp basal transcription levels, and overexpression of LSD1 altered the cell cycle profile in p53-positive (p53+), but not p53-negative (p53−), HCT cells. These findings indicate that Cp is a cell cycle-regulated promoter that is under the control of Rb and the histone demethylase LSD1 in multiple latency types.

scholarly journals The BFRF1 Gene of Epstein-Barr Virus Encodes a Novel Protein

2000 ◽  
Vol 74 (7) ◽  
pp. 3235-3244 ◽  
Author(s):  
Antonella Farina ◽  
Roberta Santarelli ◽  
Roberta Gonnella ◽  
Roberto Bei ◽  
Raffaella Muraro ◽  
...  
Keyword(s):  
Cell Lines ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Particle Identification ◽  
Early Gene ◽  
Cell Fractionation ◽  
Barr Virus ◽  
F Region ◽  
Epstein Barr

ABSTRACT Computer analysis of the Epstein-Barr virus (EBV) genome indicates there are ∼100 open reading frames (ORFs). Thus far about 30 EBV genes divided into the categories latent and lytic have been identified. The BamHI F region of EBV is abundantly transcribed during lytic replication. This region is highly conserved among herpesviruses, thus suggesting that some common function could be retained in the ORFs encompassed within this viral fragment. To identify putative novel proteins and possible new markers for viral replication, we focused our attention on the first rightward ORF in theBamHI F region (BFRF1). Histidine and glutathione S-transferase-tagged BFRF1 fusion proteins were synthesized to produce a mouse monoclonal antibody (MAb). Analysis of human sera revealed a high seroprevalence of antibodies to BFRF1 in patients affected by nasopharyngeal carcinoma or Burkitt's lymphoma, whereas no humoral response to BFRF1 could be detected among healthy donors. An anti-BFRF1 MAb recognizes a doublet migrating at 37 to 38 kDa in cells extracts from EBV-infected cell lines following lytic cycle activation and in an EBV-negative cell line (DG75) transfected with a plasmid expressing the BFRF1 gene. Northern blot analysis allowed the detection of a major transcript of 3.7 kb highly expressed in EBV-positive lytic cycle-induced cell lines. Treatment with inhibitors of viral DNA polymerase, such as phosphonoacetic acid and acyclovir, reduced but did not abolish the transcription ofBFRF1, thus indicating that BFRF1 can be classified as an early gene. Cell fractionation experiments, as well as immunolocalization by immunofluorescence microscopy, immunohistochemistry, and immunoelectron microscopy, showed that BFRF1 is localized on the plasma membrane and nuclear compartments of the cells and is a structural component of the viral particle. Identification of BFRF1 provides a new marker with which to monitor EBV infection and might help us better understand the biology of the virus.

scholarly journals Sendai virus trailer RNA simultaneously blocks two apoptosis-inducing mechanisms in a cell type-dependent manner

2005 ◽  
Vol 86 (8) ◽  
pp. 2305-2314 ◽  
Author(s):  
Marian Wiegand ◽  
Sascha Bossow ◽  
Wolfgang J. Neubert
Keyword(s):  
Cell Lines ◽  
Cytopathic Effect ◽  
Sendai Virus ◽  
Cellular Protein ◽  
Dependent Manner ◽  
Cell Type ◽  
Initiator Caspases ◽  
A Cell ◽  
Cell Type Specific ◽  
Intracellular Antigen

Induction of apoptosis during Sendai virus (SeV) infection has previously been documented to be triggered by initiator caspases (for strain F) or by a contribution of the cellular protein TIAR (T-cell-activated intracellular antigen-related) (for strain Z). Here, evidence was provided that both TIAR and caspases are simultaneously involved in apoptosis induction as a result of infection with SeV strain F. SeV F infection induced death in all tested cell lines, which could only be partially prevented through the pan-caspase inhibitor z-VAD-fmk. However, infection of seven different cell lines with the SeV mutant Fctr48z overexpressing a TIAR-sequestering RNA from the modified leader resulted in a cell type-dependent reduced cytopathic effect (CPE); in an earlier study a similar mutant derived from SeV Z was shown to prevent the induction of any CPE. Finally, blocking of caspases through z-VAD-fmk combined with Fctr48z infection led to complete abrogation of CPE, clearly demonstrating the existence of two separate mechanisms inducing cell death during SeV F infections. Interestingly, a cell type-specific interference between these two mechanisms could be detected during infection with the mutant virus Fctr48z: RNA transcribed from the mutated leader was able to trans-dominantly inhibit caspase-mediated apoptosis. Thus, virus-expressed factors enabling a well-balanced ratio of suppression and triggering of apoptosis seem to be essential for optimal virus replication.

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

2001 ◽  
Vol 75 (5) ◽  
pp. 2388-2399 ◽  
Author(s):  
Amy L. Adamson ◽  
Shannon Kenney
Keyword(s):  
Cell Lines ◽  
Transcriptional Activation ◽  
Epstein Barr Virus ◽  
Promyelocytic Leukemia ◽  
Lytic Replication ◽  
Immediate Early ◽  
Barr Virus ◽  
Early Protein ◽  
Pml Bodies ◽  
Epstein Barr

ABSTRACT 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.

scholarly journals Plasma Cell-Specific Transcription Factor XBP-1s Binds to and Transactivates the Epstein-Barr Virus BZLF1 Promoter

2007 ◽  
Vol 81 (24) ◽  
pp. 13566-13577 ◽  
Author(s):  
Chia Chi Sun ◽  
David A. Thorley-Lawson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Plasma Cell ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Endoplasmic Reticulum Stress Response ◽  
Barr Virus ◽  
Epstein Barr ◽  
Bzlf1 Promoter

ABSTRACT 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.

scholarly journals Cellular Differentiation Regulator BLIMP1 Induces Epstein-Barr Virus Lytic Reactivation in Epithelial and B Cells by Activating Transcription from both the R and Z Promoters

2014 ◽  
Vol 89 (3) ◽  
pp. 1731-1743 ◽  
Author(s):  
Jessica A. Reusch ◽  
Dhananjay M. Nawandar ◽  
Kenneth L. Wright ◽  
Shannon C. Kenney ◽  
Janet E. Mertz
Keyword(s):  
Transcription Factors ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Cell Types ◽  
Lytic Replication ◽  
Barr Virus ◽  
Lytic Reactivation ◽  
Epstein Barr ◽  
Cellular Transcription

ABSTRACTEpstein-Barr virus (EBV) maintains a lifelong latent infection within a subset of its host's memory B cells, while lytic EBV replication takes place in plasma cells and differentiated epithelial cells. Therefore, cellular transcription factors, such as BLIMP1, that are key mediators of differentiation likely contribute to the EBV latent-to-lytic switch. Previous reports showed that ectopic BLIMP1 expression induces reactivation in some EBV-positive (EBV+) B-cell lines and transcription from Zp, with all Z+cells in oral hairy leukoplakia being BLIMP1+. Here, we examined BLIMP1's role in inducing EBV lytic gene expression in numerous EBV+epithelial and B-cell lines and activating transcription from Rp. BLIMP1 addition was sufficient to induce reactivation in latently infected epithelial cells derived from gastric cancers, nasopharyngeal carcinomas, and normal oral keratinocytes (NOK) as well as some, but not all B-cell lines. BLIMP1 strongly induced transcription from Rp as well as Zp, with there being three or more synergistically acting BLIMP1-responsive elements (BRE) within Rp. BLIMP1's DNA-binding domain was required for reactivation, but BLIMP1 did not directly bind the nucleotide (nt) −660 Rp BRE. siRNA knockdown of BLIMP1 inhibited 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced lytic reactivation in NOK-Akata cells, cells that can be reactivated by R, but not Z. Thus, we conclude that BLIMP1 expression is both necessary and sufficient to induce EBV lytic replication in many (possibly all) EBV+epithelial-cell types, but in only a subset of EBV+B-cell types; it does so, at least in part, by strongly activating expression of both EBV immediately early genes,BZLF1andBRLF1.IMPORTANCEThis study is the first one to show that the cellular transcription factor BLIMP1, a key player in both epithelial and B-cell differentiation, induces reactivation of the oncogenic herpesvirus Epstein-Barr virus (EBV) out of latency into lytic replication in a variety of cancerous epithelial cell types as well as in some, but not all, B-cell types that contain this virus in a dormant state. The mechanism by which BLIMP1 does so involves strongly turning on expression of both of the immediate early genes of the virus, probably by directly acting upon the promoters as part of protein complexes or indirectly by altering the expression or activities of some cellular transcription factors and signaling pathways. The fact that EBV+cancers usually contain mostly undifferentiated cells may be due in part to these cells dying from lytic EBV infection when they differentiate and express wild-type BLIMP1.

scholarly journals Cellular MicroRNAs 200b and 429 Regulate the Epstein-Barr Virus Switch between Latency and Lytic Replication

2010 ◽  
Vol 84 (19) ◽  
pp. 10329-10343 ◽  
Author(s):  
Amy L. Ellis-Connell ◽  
Tawin Iempridee ◽  
Iris Xu ◽  
Janet E. Mertz
Keyword(s):  
Gene Expression ◽  
Epstein Barr Virus ◽  
Family Members ◽  
Lytic Replication ◽  
Dependent Manner ◽  
Lytic Gene ◽  
Barr Virus ◽  
Ebv Infection ◽  
Lytic Gene Expression ◽  
Epstein Barr

ABSTRACT We previously showed that the cellular proteins ZEB1 and ZEB2/SIP1 both play key roles in regulating the latent-lytic switch of Epstein-Barr Virus (EBV) by repressing BZLF1 gene expression. We investigated here the effects of cellular microRNA (miRNA) 200 (miR200) family members on the EBV infection status of cells. We show that miR200b and miR429, but not miR200a, can induce EBV-positive cells into lytic replication by downregulating expression of ZEB1 and ZEB2, leading to production of infectious virus. The levels of miR200 family members in EBV-infected cells strongly negatively correlated with the levels of the ZEBs (e.g., −0.89 [P < 0.001] for miR429 versus ZEB1) and positively correlated with the degree of EBV lytic gene expression (e.g., 0.73 [P < 0.01] for miR429 versus BZLF1). The addition of either miR200b or miR429 to EBV-positive cells led to EBV lytic reactivation in a ZEB-dependent manner; inhibition of these miRNAs led to decreased EBV lytic gene expression. The degree of latent infection by an EBV mutant defective in the primary ZEB-binding site of the EBV BZLF1 promoter was not affected by the addition of these miRNAs. Furthermore, EBV infection of primary blood B cells led to downregulation of these miRNAs and upregulation of ZEB levels. Thus, we conclude that miRNAs 200b and 429 are key regulators via their effects on expression of ZEB1 and ZEB2 of the switch between latent and lytic infection by EBV and, therefore, potential targets for development of new lytic induction therapeutics with which to treat patients with EBV-associated malignancies.

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