scholarly journals Identification of functional targets of the Zta transcriptional activator by formation of stable preinitiation complex intermediates.

1994 ◽  
Vol 14 (12) ◽  
pp. 8365-8375 ◽  
Author(s):  
P Lieberman
Keyword(s):  
Transcriptional Activation ◽  
Associated Factors ◽  
Transcriptional Activator ◽  
Preinitiation Complex ◽  
Mobility Shift ◽  
Response Element ◽  
Barr Virus ◽  
Epstein Barr ◽  
Promoter Dna ◽  
High Level

Transcriptional activator proteins stimulate the formation of a preinitiation complex that may be distinct from a basal-level transcription complex in its composition and stability. Components of the general transcription factors that form activator-dependent stable intermediates were determined by the use of Sarkosyl and oligonucleotide challenge experiments. High-level transcriptional activation by the Epstein-Barr virus-encoded Zta protein required an activity in the TFIID fraction that is distinct from the TATA-binding protein (TBP) and the TBP-associated factors. This additional activity copurifies with and is likely to be identical to the previously defined coactivator, USA (M. Meisterernst, A. L. Roy, H. M. Lieu, and R. G. Roeder, Cell 66:981-994, 1991). The formation of a stable preinitiation complex intermediate resistant to Sarkosyl required the preincubation of the promoter DNA with Zta, holo-TFIID (TBP and TBP-associated factors), TFIIB, TFIIA, and the coactivator USA. The formation of a Zta response element-resistant preinitiation complex required the preincubation of promoter DNA with Zta, holo-TFIID, TFIIB, and TFIIA. Agarose gel electrophoretic mobility shift showed that a preformed Zta-holo-TFIID-TFIIA complex was resistant to Sarkosyl and to Zta response element oligonucleotide challenge. DNase I footprinting suggests that only Zta, holo-TFIID, and TFIIA make significant contacts with the promoter DNA. These results provide functional and physical evidence that the Zta transcriptional activator influences at least two distinct steps in preinitiation complex assembly, the formation of the stable holo-TFIID-TFIIA-promoter complex and the subsequent binding of TFIIB and a USA-like coactivator.

Identification of functional targets of the Zta transcriptional activator by formation of stable preinitiation complex intermediates

1994 ◽  
Vol 14 (12) ◽  
pp. 8365-8375
Author(s):  
P Lieberman
Keyword(s):  
Transcriptional Activation ◽  
Associated Factors ◽  
Transcriptional Activator ◽  
Preinitiation Complex ◽  
Mobility Shift ◽  
Response Element ◽  
Barr Virus ◽  
Epstein Barr ◽  
Promoter Dna ◽  
High Level

Transcriptional activator proteins stimulate the formation of a preinitiation complex that may be distinct from a basal-level transcription complex in its composition and stability. Components of the general transcription factors that form activator-dependent stable intermediates were determined by the use of Sarkosyl and oligonucleotide challenge experiments. High-level transcriptional activation by the Epstein-Barr virus-encoded Zta protein required an activity in the TFIID fraction that is distinct from the TATA-binding protein (TBP) and the TBP-associated factors. This additional activity copurifies with and is likely to be identical to the previously defined coactivator, USA (M. Meisterernst, A. L. Roy, H. M. Lieu, and R. G. Roeder, Cell 66:981-994, 1991). The formation of a stable preinitiation complex intermediate resistant to Sarkosyl required the preincubation of the promoter DNA with Zta, holo-TFIID (TBP and TBP-associated factors), TFIIB, TFIIA, and the coactivator USA. The formation of a Zta response element-resistant preinitiation complex required the preincubation of promoter DNA with Zta, holo-TFIID, TFIIB, and TFIIA. Agarose gel electrophoretic mobility shift showed that a preformed Zta-holo-TFIID-TFIIA complex was resistant to Sarkosyl and to Zta response element oligonucleotide challenge. DNase I footprinting suggests that only Zta, holo-TFIID, and TFIIA make significant contacts with the promoter DNA. These results provide functional and physical evidence that the Zta transcriptional activator influences at least two distinct steps in preinitiation complex assembly, the formation of the stable holo-TFIID-TFIIA-promoter complex and the subsequent binding of TFIIB and a USA-like coactivator.

scholarly journals Transcriptional Activation Signals Found in the Epstein-Barr Virus (EBV) Latency C Promoter Are Conserved in the Latency C Promoter Sequences from Baboon and Rhesus Monkey EBV-Like Lymphocryptoviruses (Cercopithicine Herpesviruses 12 and 15)

1999 ◽  
Vol 73 (1) ◽  
pp. 826-833 ◽  
Author(s):  
Ezequiel M. Fuentes-Pananá ◽  
Sankar Swaminathan ◽  
Paul D. Ling
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Epstein Barr Virus ◽  
Rhesus Macaques ◽  
Regulatory Elements ◽  
Mobility Shift ◽  
Barr Virus ◽  
Epstein Barr ◽  
Transformed Cell Lines

ABSTRACT The Epstein-Barr virus (EBV) EBNA2 protein is a transcriptional activator that controls viral latent gene expression and is essential for EBV-driven B-cell immortalization. EBNA2 is expressed from the viral C promoter (Cp) and regulates its own expression by activating Cp through interaction with the cellular DNA binding protein CBF1. Through regulation of Cp and EBNA2 expression, EBV controls the pattern of latent protein expression and the type of latency established. To gain further insight into the important regulatory elements that modulate Cp usage, we isolated and sequenced the Cp regions corresponding to nucleotides 10251 to 11479 of the EBV genome (−1079 to +144 relative to the transcription initiation site) from the EBV-like lymphocryptoviruses found in baboons (herpesvirus papio; HVP) and Rhesus macaques (RhEBV). Sequence comparison of the approximately 1,230-bp Cp regions from these primate viruses revealed that EBV and HVP Cp sequences are 64% conserved, EBV and RhEBV Cp sequences are 66% conserved, and HVP and RhEBV Cp sequences are 65% conserved relative to each other. Approximately 50% of the residues are conserved among all three sequences, yet all three viruses have retained response elements for glucocorticoids, two positionally conserved CCAAT boxes, and positionally conserved TATA boxes. The putative EBNA2 100-bp enhancers within these promoters contain 54 conserved residues, and the binding sites for CBF1 and CBF2 are well conserved. Cp usage in the HVP- and RhEBV-transformed cell lines was detected by S1 nuclease protection analysis. Transient-transfection analysis showed that promoters of both HVP and RhEBV are responsive to EBNA2 and that they bind CBF1 and CBF2 in gel mobility shift assays. These results suggest that similar mechanisms for regulation of latent gene expression are conserved among the EBV-related lymphocryptoviruses found in nonhuman primates.

scholarly journals Requirement for transcription factor IIA (TFIIA)-TFIID recruitment by an activator depends on promoter structure and template competition.

1997 ◽  
Vol 17 (11) ◽  
pp. 6624-6632 ◽  
Author(s):  
P M Lieberman ◽  
J Ozer ◽  
D B Gürsel
Keyword(s):  
Transcriptional Activation ◽  
Core Promoter ◽  
Initiation Site ◽  
Transcriptional Initiation ◽  
Barr Virus ◽  
Tata Element ◽  
Epstein Barr ◽  
Substitution Mutations ◽  
Promoter Dna ◽  
Tata Box Binding Protein

Different mechanisms of transcriptional activation may be required for distinct classes of promoters and cellular conditions. The Epstein-Barr virus (EBV)-encoded transcriptional activator Zta recruits the general transcription factors IID (TFIID) and IIA (TFIIA) to promoter DNA and induces a TATA box-binding protein (TBP)-associated factor-dependent footprint downstream of the transcriptional initiation site. In this study, we investigated the functional significance of TFIID-TFIIA (D-A complex) recruitment by Zta. Alanine substitution mutations in the Zta activation domain which eliminate the ability of Zta to stimulate the D-A complex were examined. These Zta mutants were defective in the ability to activate transcription from an EBV-derived promoter (BHLF1) but activated a highly responsive synthetic promoter (Z7E4T). Both the number of activator binding sites and the core promoter region contribute to the requirement for D-A complex recruitment. These functionally distinct core promoters had significant differences in affinity for TBP and TFIID binding. The D-A complex-recruiting activity of Zta was found to be important for promoter selection in the presence of a competitor template. Conditions which limit TFIID binding to the TATA element or compromise the ability of TFIIA to bind TBP required activator stimulation of the D-A complex. These results indicate that D-A complex recruitment is one of at least two activation pathways utilized by Zta and is the essential pathway for a subset of promoters and conditions which limit TFIID binding to the TATA element.

scholarly journals The Epstein-Barr Virus EBNA-LP Protein Preferentially Coactivates EBNA2-Mediated Stimulation of Latent Membrane Proteins Expressed from the Viral Divergent Promoter

2005 ◽  
Vol 79 (7) ◽  
pp. 4492-4505 ◽  
Author(s):  
RongSheng Peng ◽  
Stephanie C. Moses ◽  
Jie Tan ◽  
Elisabeth Kremmer ◽  
Paul D. Ling
Keyword(s):  
B Cell ◽  
Transcriptional Activation ◽  
Epstein Barr Virus ◽  
Type I ◽  
Barr Virus ◽  
Cell Immortalization ◽  
Cellular Cofactor ◽  
Epstein Barr ◽  
Transformed Cell Lines ◽  
Stimulation Of

ABSTRACT The mechanistic contribution of the Epstein-Barr virus (EBV) EBNA-LP protein to B-cell immortalization remains an enigma. However, previous studies have indicated that EBNA-LP may contribute to immortalization by enhancing EBNA2-mediated transcriptional activation of the LMP-1 gene. To gain further insight into the potential role EBNA-LP has in EBV-mediated B-cell immortalization, we asked whether it is a global or gene-specific coactivator of EBNA2 and whether coactivation requires interaction between these proteins. In type I Burkitt's lymphoma cells, we found that EBNA-LP strongly coactivated EBNA2 stimulation of LMP-1 and LMP2B RNAs, which are expressed from the viral divergent promoter. Surprisingly, the viral LMP2A gene and cellular CD21 and Hes-1 genes were induced by EBNA2 but showed no further induction after EBNA-LP coexpression. We also found that EBNA-LP did not stably interact with EBNA2 in coimmunoprecipitation assays, even though the conditions were adequate to observe specific interactions between EBNA2 and its cellular cofactor, CBF1. Colocalization between EBNA2 and EBNA-LP was not detectable in EBV-transformed cell lines or transfected type I Burkitt's cells. Finally, no significant interactions between EBNA2 and EBNA-LP were found with mammalian two-hybrid assays. From this data, we conclude that EBNA-LP is not a global coactivator of EBNA2 targets, but it preferentially coactivates EBNA2 stimulation of the viral divergent promoter. While this may require specific transient interactions between these proteins that only occur in the context of the divergent promoter, our data strongly suggest that EBNA-LP also cooperates with EBNA2 through mechanisms that do not require direct or indirect complex formation between these proteins.

scholarly journals Impaired recovery of Epstein-Barr virus (EBV)—specific CD8+ T lymphocytes after partially T-depleted allogeneic stem cell transplantation may identify patients at very high risk for progressive EBV reactivation and lymphoproliferative disease

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4290-4297 ◽  
Author(s):  
Pauline Meij ◽  
Joost W. J. van Esser ◽  
Hubert G. M. Niesters ◽  
Debbie van Baarle ◽  
Frank Miedema ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
High Risk ◽  
Cd8 T Cells ◽  
Lymphoproliferative Disease ◽  
Barr Virus ◽  
Ebv Reactivation ◽  
Epstein Barr ◽  
High Level ◽  
Very High

Abstract Epstein-Barr virus (EBV)—specific cytotoxic T lymphocytes are considered pivotal to prevent lymphoproliferative disease (LPD) in allogeneic stem cell transplantation (SCT) recipients. We evaluated the recovery of EBV-specific CD8+ T cells after partially T-cell—depleted SCT and studied the interaction between EBV-specific CD8+ T cells, EBV reactivation, and EBV-LPD. EBV-specific CD8+ T cells were enumerated using 12 class I HLA tetramers presenting peptides derived from 7 EBV proteins. Blood samples were taken at regular intervals after SCT in 61 patients, and EBV DNA levels were assessed by real-time polymerase chain reaction. Forty-five patients showed EBV reactivation, including 25 with high-level reactivation (ie, more than 1000 genome equivalents [geq] per milliliter). Nine of these 25 patients progressed to EBV-LPD. CD8+ T cells specific for latent or lytic EBV epitopes repopulated the peripheral blood at largely similar rates. In most patients, EBV-specific CD8+ T-cell counts had returned to normal levels within 6 months after SCT. Concurrently, the incidence of EBV reactivations clearly decreased. Patients with insufficient EBV-specific CD8+ T-cell recovery were at high risk for EBV reactivation in the first 6 months after SCT. Failure to detect EBV-specific CD8+ T cells in patients with high-level reactivation was associated with the subsequent development of EBV-LPD (P = .048). Consequently, the earlier defined positive predictive value of approximately 40%, based on high-level EBV reactivation only, increased to 100% in patients without detectable EBV-specific CD8+ T cells. Thus, impaired recovery of EBV-specific CD8+ T cells in patients with high-level EBV reactivation may identify a subgroup at very high risk for EBV-LPD and supports that EBV-specific CD8+ T cells protect SCT recipients from progressive EBV reactivation and EBV-LPD.

scholarly journals The Epstein-Barr Virus BMRF1 Protein Activates Transcription and Inhibits the DNA Damage Response by Binding NuRD

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Samuel G. Salamun ◽  
Justine Sitz ◽  
Carlos F. De La Cruz-Herrera ◽  
Jaime Yockteng-Melgar ◽  
Edyta Marcon ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Transcriptional Activation ◽  
Epstein Barr Virus ◽  
Nurd Complex ◽  
Dna Breaks ◽  
Nucleosome Remodeling ◽  
Barr Virus ◽  
Damage Response ◽  
Epstein Barr

ABSTRACT The BMRF1 protein of Epstein-Barr virus (EBV) has multiple roles in viral lytic infection, including serving as the DNA polymerase processivity factor, activating transcription from several EBV promoters and inhibiting the host DNA damage response to double-stranded DNA breaks (DSBs). Using affinity purification coupled to mass spectrometry, we identified the nucleosome remodeling and deacetylation (NuRD) complex as the top interactor of BMRF1. We further found that NuRD components localize with BMRF1 at viral replication compartments and that this interaction occurs through the BMRF1 C-terminal region previously shown to mediate transcriptional activation. We identified an RBBP4 binding motif within this region that can interact with both RBBP4 and MTA2 components of the NuRD complex and showed that point mutation of this motif abrogates NuRD binding as well as the ability of BMRF1 to activate transcription from the BDLF3 and BLLF1 EBV promoters. In addition to its role in transcriptional regulation, NuRD has been shown to contribute to DSB signaling in enabling recruitment of RNF168 ubiquitin ligase and subsequent ubiquitylation at the break. We showed that BMRF1 inhibited RNF168 recruitment and ubiquitylation at DSBs and that this inhibition was at least partly relieved by loss of the NuRD interaction. The results reveal a mechanism by which BMRF1 activates transcription and inhibits DSB signaling and a novel role for NuRD in transcriptional activation in EBV. IMPORTANCE The Epstein-Barr virus (EBV) BMRF1 protein is critical for EBV infection, playing key roles in viral genome replication, activation of EBV genes, and inhibition of host DNA damage responses (DDRs). Here we show that BMRF1 targets the cellular nucleosome remodeling and deacetylation (NuRD) complex, using a motif in the BMRF1 transcriptional activation sequence. Mutation of this motif disrupts the ability of BMRF1 to activate transcription and interfere with DDRs, showing the importance of the NuRD interaction for BMRF1 functions. BMRF1 was shown to act at the same step in the DDR as NuRD, suggesting that it interferes with NuRD function.

LYDMA-antigens and Immunity against EBV-infected Cells Epstein-Barr Virus as a Model for the Study of Host-infection Interaction

1987 ◽  
Vol 2 (2) ◽  
pp. 125-132 ◽  
Author(s):  
Maria L. Villa ◽  
Emilio Bombardieri
Keyword(s):  
Transcriptional Activation ◽  
Epstein Barr Virus ◽  
Dna Viruses ◽  
Barr Virus ◽  
Good Potential ◽  
Infected Cells ◽  
Membrane Antigens ◽  
Epstein Barr ◽  
Host Infection

Molecular biology has shown that DNA viruses carry their own transforming genes, unlike RNA viruses (retrovirus), which use cellular “oncogenes”. Some of the products of transforming viral genes are very good potential targets for immune defence. Epstein-Barr virus (EBV) immortalization is linked to the transcriptional activation of some latently transcribed regions; the lymphocyte-determined membrane antigens (LYDMA), the product of one of these regions, are the T-cell's chosen target. EBV-induced immortalization may therefore be free from any malignant consequence as long as immortalized clones are suppressed by immunosurveillance. In vivo, LYDMA-positive clones may be susceptible to immune control; LYDMA-negative clones can transform to neoplastic cells

Inhibition of NF-κB induces apoptosis of KSHV-infected primary effusion lymphoma cells

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2537-2542 ◽  
Author(s):  
Shannon A. Keller ◽  
Elaine J. Schattner ◽  
Ethel Cesarman
Keyword(s):  
Primary Effusion Lymphoma ◽  
Leukemia Virus ◽  
Human Lymphocytes ◽  
Kaposi Sarcoma ◽  
Irreversible Inhibitor ◽  
Mobility Shift ◽  
Lymphoma Cells ◽  
Barr Virus ◽  
Human T Cell ◽  
Epstein Barr

Abstract Kaposi sarcoma–associated herpesvirus (KSHV), or human herpervirus 8 (HHV-8), is a γ-herpesvirus that infects human lymphocytes and is associated with primary effusion lymphoma (PEL). Currently, the role of viral infection in the transformation of PEL cells is unknown. One possibility is that KSHV, like the lymphotropic viruses Epstein-Barr virus (EBV) and human T-cell leukemia virus I (HTLV-I), activates the transcription factor NF-κB to promote survival and proliferation of infected lymphocytes. To examine this possibility, we assessed NF-κB activity in KSHV-infected PEL cell lines and primary tumor specimens by electrophoretic mobility shift assay (EMSA). We observed that NF-κB is constitutively activated in all KSHV-infected lymphomas, and consists of 2 predominant complexes, p65/p50 heterodimers and p50/p50 homodimers. Inhibition experiments demonstrated that Bay 11-7082, an irreversible inhibitor of IκBα phosphorylation, completely and specifically abrogated the NF-κB/DNA binding in PEL cells. PEL cells treated with Bay 11 demonstrated down-regulation of the NF-κB inducible cytokine interleukin 6 (IL-6), and apoptosis. These results suggest that NF-κB activity is necessary for survival of KSHV-infected lymphoma cells, and that pharmacologic inhibition of NF-κB may be an effective treatment for PEL.

scholarly journals Epstein-Barr Virus Immediate-Early Protein BRLF1 Induces the Lytic Form of Viral Replication through a Mechanism Involving Phosphatidylinositol-3 Kinase Activation

2001 ◽  
Vol 75 (13) ◽  
pp. 6135-6142 ◽  
Author(s):  
Catherine Dayle Darr ◽  
Amy Mauser ◽  
Shannon Kenney
Keyword(s):  
Viral Replication ◽  
Transcriptional Activation ◽  
Epstein Barr Virus ◽  
Adenovirus Vector ◽  
Pi3 Kinase ◽  
Immediate Early ◽  
Kinase Activation ◽  
Barr Virus ◽  
Epstein Barr ◽  
Phosphatidylinositol 3

ABSTRACT Expression of the Epstein-Barr virus (EBV) immediate-early (IE) protein BRLF1 induces the lytic form of viral replication in most EBV-positive cell lines. BRLF1 is a transcriptional activator that binds directly to a GC-rich motif present in some EBV lytic gene promoters. However, BRLF1 activates transcription of the other IE protein, BZLF1, through an indirect mechanism which we previously showed to require activation of the stress mitogen-activated protein kinases. Here we demonstrate that BRLF1 activates phosphatidylinositol-3 (PI3) kinase signaling in host cells. We show that the specific PI3 kinase inhibitor, LY294002, completely abrogates the ability of a BRLF1 adenovirus vector to induce the lytic form of EBV infection, while not affecting lytic infection induced by a BZLF1 adenovirus vector. Furthermore, we demonstrate that the requirement for PI3 kinase activation in BRLF1-induced transcriptional activation is promoter dependent. BRLF1 activation of the SM early promoter (which occurs through a direct binding mechanism) does not require PI3 kinase activation, whereas activation of the IE BZLF1 and early BMRF1 promoters requires PI3 kinase activation. Thus, there are clearly two separate mechanisms by which BRLF1 induces transcriptional activation.

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