CTCF Prevents the Epigenetic Drift of EBV Latency Promoter Qp

The establishment and maintenance of Epstein-Barr Virus (EBV) latent infection requires distinct viral gene expression programs. These gene expression programs, termed latency types, are determined largely by promoter selection, and controlled through the interplay between cell-type specific transcription factors, chromatin structure, and epigenetic modifications. We used a genome-wide chromatin-immunoprecipitation (ChIP) assay to identify epigenetic modifications that correlate with different latency types. We found that the chromatin insulator protein CTCF binds at several key regulatory nodes in the EBV genome and may compartmentalize epigenetic modifications across the viral genome. Highly enriched CTCF binding sites were identified at the promoter regions upstream of Cp, Wp, EBERs, and Qp. Since Qp is essential for long-term maintenance of viral genomes in type I latency and epithelial cell infections, we focused on the role of CTCF in regulating Qp. Purified CTCF bound ∼40 bp upstream of the EBNA1 binding sites located at +10 bp relative to the transcriptional initiation site at Qp. Mutagenesis of the CTCF binding site in EBV bacmids resulted in a decrease in the recovery of stable hygromycin-resistant episomes in 293 cells. EBV lacking the Qp CTCF site showed a decrease in Qp transcription initiation and a corresponding increase in Cp and Fp promoter utilization at 8 weeks post-transfection. However, by 16 weeks post-transfection, bacmids lacking CTCF sites had no detectable Qp transcription and showed high levels of histone H3 K9 methylation and CpG DNA methylation at the Qp initiation site. These findings provide direct genetic evidence that CTCF functions as a chromatin insulator that prevents the promiscuous transcription of surrounding genes and blocks the epigenetic silencing of an essential promoter, Qp, during EBV latent infection

A Gammaherpesvirus Cooperates with Interferon-alpha/beta-Induced IRF2 to Halt Viral Replication, Control Reactivation, and Minimize Host Lethality

The gammaherpesviruses, including Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), establish latency in memory B lymphocytes and promote lymphoproliferative disease in immunocompromised individuals. The precise immune mechanisms that prevent gammaherpesvirus reactivation and tumorigenesis are poorly defined. Murine gammaherpesvirus 68 (MHV68) is closely related to EBV and KSHV, and type I (alpha/beta) interferons (IFNαβ) regulate MHV68 reactivation from both B cells and macrophages by unknown mechanisms. Here we demonstrate that IFNβ is highly upregulated during latent infection, in the absence of detectable MHV68 replication. We identify an interferon-stimulated response element (ISRE) in the MHV68 M2 gene promoter that is bound by the IFNαβ-induced transcriptional repressor IRF2 during latency in vivo. The M2 protein regulates B cell signaling to promote establishment of latency and reactivation. Virus lacking the M2 ISRE (ISREΔ) overexpresses M2 mRNA and displays uncontrolled acute replication in vivo, higher latent viral load, and aberrantly high reactivation from latency. These phenotypes of the ISREΔ mutant are B-cell-specific, require IRF2, and correlate with a significant increase in virulence in a model of acute viral pneumonia. We therefore identify a mechanism by which a gammaherpesvirus subverts host IFNαβ signaling in a surprisingly cooperative manner, to directly repress viral replication and reactivation and enforce latency, thereby minimizing acute host disease. Since we find ISREs 5′ to the major lymphocyte latency genes of multiple rodent, primate, and human gammaherpesviruses, we propose that cooperative subversion of IFNαβ-induced IRFs to promote latent infection is an ancient strategy that ensures a stable, minimally-pathogenic virus-host relationship

An Epstein-Barr Virus Anti-Apoptotic Protein Constitutively Expressed in Transformed Cells and Implicated in Burkitt Lymphomagenesis: The Wp/BHRF1 Link

Two factors contribute to Burkitt lymphoma (BL) pathogenesis, a chromosomal translocation leading to c-myc oncogene deregulation and infection with Epstein-Barr virus (EBV). Although the virus has B cell growth–transforming ability, this may not relate to its role in BL since many of the transforming proteins are not expressed in the tumor. Mounting evidence supports an alternative role, whereby EBV counteracts the high apoptotic sensitivity inherent to the c-myc–driven growth program. In that regard, a subset of BLs carry virus mutants in a novel form of latent infection that provides unusually strong resistance to apoptosis. Uniquely, these virus mutants use Wp (a viral promoter normally activated early in B cell transformation) and express a broader-than-usual range of latent antigens. Here, using an inducible system to express the candidate antigens, we show that this marked apoptosis resistance is mediated not by one of the extended range of EBNAs seen in Wp-restricted latency but by Wp-driven expression of the viral bcl2 homologue, BHRF1, a protein usually associated with the virus lytic cycle. Interestingly, this Wp/BHRF1 connection is not confined to Wp-restricted BLs but appears integral to normal B cell transformation by EBV. We find that the BHRF1 gene expression recently reported in newly infected B cells is temporally linked to Wp activation and the presence of W/BHRF1-spliced transcripts. Furthermore, just as Wp activity is never completely eclipsed in in vitro–transformed lines, low-level BHRF1 transcripts remain detectable in these cells long-term. Most importantly, recognition by BHRF1-specific T cells confirms that such lines continue to express the protein independently of any lytic cycle entry. This work therefore provides the first evidence that BHRF1, the EBV bcl2 homologue, is constitutively expressed as a latent protein in growth-transformed cells in vitro and, in the context of Wp-restricted BL, may contribute to virus-associated lymphomagenesis in vivo

The Epstein-Barr virus EBNA-1 promoter Qp requires an initiator-like element.

Expression of the Epstein-Barr virus (EBV) EBNA-1 protein within EBV-positive tumor cells and subpopulations of latently infected B lymphocytes in vivo is mediated by the promoter Qp. Previous studies have established that Qp is a TATA-less promoter whose activation requires only proximal regulatory elements and that it is negatively autoregulated through two EBNA-1 binding sites downstream of the transcription initiation sites. The objective of this study was to better define the properties of an essential positive regulatory element (QRE-2) adjacent to a major transcription start site of Qp and to evaluate the contributions of other potential regulatory elements proximal to the Qp start site. Using DNA affinity purification and UV cross-linking, we have identified the QRE-2-binding protein as a single polypeptide of approximately 40 kDa. The DNA-binding properties of this protein are clearly distinct from those of the TATA-binding protein, suggesting that in the absence of a TATA box, QRE-2 may function as an initiator element to direct assembly of TFIID near the transcription start site. Mutational analysis of potential regulatory elements, furthermore, indicated that the putative E2F binding sites within the EBNA-1 binding domain can exert a positive influence on Qp that is EBNA-1 independent, suggesting that these regulatory elements play an additional if not different role in Qp regulation than previously proposed. A model for the regulation of Qp consistent with the current and previous findings which provides for a simple but efficient mechanism of ensuring the EBNA-1 expression necessary to sustain long-term latency is presented

Transcription start sites downstream of the Epstein-Barr virus (EBV) Fp promoter in early-passage Burkitt lymphoma cells define a fourth promoter for expression of the EBV EBNA-1 protein

In Epstein-Barr virus (EBV)-transformed B lymphoblastoid and many Burkitt lymphoma cell lines, the EBV EBNA-1 protein is one of six viral nuclear antigens expressed from a common transcription unit under the control of one of two promoters, Wp or Cp. In contrast, EBNA-1 is the only EBV nuclear antigen expressed in Burkitt and other EBV-positive tumors. We previously identified a promoter of EBNA-1 transcription, designated Fp, in early-passage Mutu Burkitt tumor cells, and this promoter is also active in long-term Mutu and Akata Burkitt cell lines which maintain the exclusive expression of EBNA-1 characteristic of the tumor. However, transcription initiation within Fp reporter gene plasmids in EBV-negative cells occurs at positions 100 to 200 bases downstream of the Fp start site in the BamHI-Q restriction fragment. Here we demonstrate that transcription initiation within newly established Burkitt lymphoma cell lines is consistent with the transcription initiation we observed in reporter plasmids. Furthermore, previous observations of transcription from Fp to generate EBNA-1 transcripts can be attributed to lytic-cycle gene expression. These data, in conjunction with our previous characterization of promoter regulatory elements, define a fourth EBNA-1 promoter, Qp, that is active in latently infected Burkitt tumor cells