Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye

<p>Abstract</p> <p>Background</p> <p>Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantitative polymerase chain reaction (QPCR) assays using SYBR Green I dye and a single quantification standard containing two EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed to detect and measure absolute EBV DNA load in patients with various EBV-associated diseases. EBV DNA loads and viral capsid antigen (VCA) IgG antibody titres were also quantified on a population sample.</p> <p>Results</p> <p>EBV DNA was measurable in ethylenediaminetetraacetic acid (EDTA) whole blood, peripheral blood mononuclear cells (PBMCs), plasma and cerebrospinal fluid (CSF) samples. EBV DNA loads were detectable from 8.0 × 10²to 1.3 × 10⁸copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 10³to 2.0 × 10⁵copies/ml in infectious mononucleosis (n = 7), 7.5 × 10⁴to 1.1 × 10⁵copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 10²to 5.6 × 10³copies/ml in HIV-infected patients (n = 12), and 2.0 × 10²to 9.1 × 10⁴copies/ml in the population sample (n = 218). EBNA-1 and BHRF-1 DNA were detected in 11.0% and 21.6% of the population sample respectively. There was a modest correlation between VCA IgG antibody titre and BHRF-1 DNA load (rho = 0.13, p = 0.05) but not EBNA-1 DNA load (rho = 0.11, p = 0.11).</p> <p>Conclusion</p> <p>Two sensitive and specific real-time PCR assays using SYBR Green I dye and a single quantification standard containing two EBV DNA targets, were developed for the detection and measurement of EBV DNA load in a variety of clinical samples. These assays have application in the investigation of EBV-related illnesses in immunocompromised individuals.</p

Murine Gamma-herpesvirus Immortalization of Fetal Liver-Derived B Cells Requires both the Viral Cyclin D Homolog and Latency-Associated Nuclear Antigen

Human gammaherpesviruses are associated with the development of lymphoproliferative diseases and B cell lymphomas, particularly in immunosuppressed hosts. Understanding the molecular mechanisms by which human gammaherpesviruses cause disease is hampered by the lack of convenient small animal models to study them. However, infection of laboratory strains of mice with the rodent virus murine gammaherpesvirus 68 (MHV68) has been useful in gaining insights into how gammaherpesviruses contribute to the genesis and progression of lymphoproliferative lesions. In this report we make the novel observation that MHV68 infection of murine day 15 fetal liver cells results in their immortalization and differentiation into B plasmablasts that can be propagated indefinitely in vitro, and can establish metastasizing lymphomas in mice lacking normal immune competence. The phenotype of the MHV68 immortalized B cell lines is similar to that observed in lymphomas caused by KSHV and resembles the favored phenotype observed during MHV68 infection in vivo. All established cell lines maintained the MHV68 genome, with limited viral gene expression and little or no detectable virus production - although virus reactivation could be induced upon crosslinking surface Ig. Notably, transcription of the genes encoding the MHV68 viral cyclin D homolog (v-cyclin) and the homolog of the KSHV latency-associated nuclear antigen (LANA), both of which are conserved among characterized γ2-herpesviruses, could consistently be detected in the established B cell lines. Furthermore, we show that the v-cyclin and LANA homologs are required for MHV68 immortalization of murine B cells. In contrast the M2 gene, which is unique to MHV68 and plays a role in latency and virus reactivation in vivo, was dispensable for B cell immortalization. This new model of gammaherpesvirus-driven B cell immortalization and differentiation in a small animal model establishes an experimental system for detailed investigation of the role of gammaherpesvirus gene products and host responses in the genesis and progression of gammaherpesvirus-associated lymphomas, and presents a convenient system to evaluate therapeutic modalities

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

E2F1 Mediated Apoptosis Induced by the DNA Damage Response Is Blocked by EBV Nuclear Antigen 3C in Lymphoblastoid Cells

EBV latent antigen EBNA3C is indispensible for in vitro B-cell immortalization resulting in continuously proliferating lymphoblastoid cell lines (LCLs). EBNA3C was previously shown to target pRb for ubiquitin-proteasome mediated degradation, which facilitates G1 to S transition controlled by the major transcriptional activator E2F1. E2F1 also plays a pivotal role in regulating DNA damage induced apoptosis through both p53-dependent and -independent pathways. In this study, we demonstrate that in response to DNA damage LCLs knocked down for EBNA3C undergo a drastic induction of apoptosis, as a possible consequence of both p53- and E2F1-mediated activities. Importantly, EBNA3C was previously shown to suppress p53-induced apoptosis. Now, we also show that EBNA3C efficiently blocks E2F1-mediated apoptosis, as well as its anti-proliferative effects in a p53-independent manner, in response to DNA damage. The N- and C-terminal domains of EBNA3C form a stable pRb independent complex with the N-terminal DNA-binding region of E2F1 responsible for inducing apoptosis. Mechanistically, we show that EBNA3C represses E2F1 transcriptional activity via blocking its DNA-binding activity at the responsive promoters of p73 and Apaf-1 apoptosis induced genes, and also facilitates E2F1 degradation in an ubiquitin-proteasome dependent fashion. Moreover, in response to DNA damage, E2F1 knockdown LCLs exhibited a significant reduction in apoptosis with higher cell-viability. In the presence of normal mitogenic stimuli the growth rate of LCLs knockdown for E2F1 was markedly impaired; indicating that E2F1 plays a dual role in EBV positive cells and that active engagement of the EBNA3C-E2F1 complex is crucial for inhibition of DNA damage induced E2F1-mediated apoptosis. This study offers novel insights into our current understanding of EBV biology and enhances the potential for development of effective therapies against EBV associated B-cell lymphomas

Zinc Coordination Is Required for and Regulates Transcription Activation by Epstein-Barr Nuclear Antigen 1

Epstein-Barr Nuclear Antigen 1 (EBNA1) is essential for Epstein-Barr virus to immortalize naïve B-cells. Upon binding a cluster of 20 cognate binding-sites termed the family of repeats, EBNA1 transactivates promoters for EBV genes that are required for immortalization. A small domain, termed UR1, that is 25 amino-acids in length, has been identified previously as essential for EBNA1 to activate transcription. In this study, we have elucidated how UR1 contributes to EBNA1's ability to transactivate. We show that zinc is necessary for EBNA1 to activate transcription, and that UR1 coordinates zinc through a pair of essential cysteines contained within it. UR1 dimerizes upon coordinating zinc, indicating that EBNA1 contains a second dimerization interface in its amino-terminus. There is a strong correlation between UR1-mediated dimerization and EBNA1's ability to transactivate cooperatively. Point mutants of EBNA1 that disrupt zinc coordination also prevent self-association, and do not activate transcription cooperatively. Further, we demonstrate that UR1 acts as a molecular sensor that regulates the ability of EBNA1 to activate transcription in response to changes in redox and oxygen partial pressure (pO2). Mild oxidative stress mimicking such environmental changes decreases EBNA1-dependent transcription in a lymphoblastoid cell-line. Coincident with a reduction in EBNA1-dependent transcription, reductions are observed in EBNA2 and LMP1 protein levels. Although these changes do not affect LCL survival, treated cells accumulate in G0/G1. These findings are discussed in the context of EBV latency in body compartments that differ strikingly in their pO2 and redox potential