Abstract
Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.
Highlights
Infection with Epstein-Barr virus (EBV), the first tumor virus described in humans, is associated with B-cell lymphoproliferative syndromes, such as Hodgkin and endemic Burkitt lymphoma and with diseases of epithelial cell origin such as oral hairy leukoplakia, nasopharyngeal carcinoma, and gastric carcinoma [1,2,3,4]
We show here that activation of DNA damage response signaling proteins can be elicited in the early phase of the EBV lytic cycle and in the absence of EBV DNA replication
Our findings raised two questions: 1) which viral factors are responsible for induction of DNA damage signaling during the early phase of the EBV lytic cycle? 2) What is the function of DNA damage signaling activation associated with the early phase of the EBV lytic cycle? We addressed these questions by investigating the role of early lytic proteins, ZEBRA, BGLF4, BGLF5, and BALF2, in inducing markers of DNA damage signaling and by exploring how the EBV lytic cycle is affected by DNA damage signaling
Summary
Infection with Epstein-Barr virus (EBV), the first tumor virus described in humans, is associated with B-cell lymphoproliferative syndromes, such as Hodgkin and endemic Burkitt lymphoma and with diseases of epithelial cell origin such as oral hairy leukoplakia, nasopharyngeal carcinoma, and gastric carcinoma [1,2,3,4]. DNA damage signaling pathways are induced during EBV infection and lytic reactivation in both lymphoid and epithelial cells [5,6,7,8,9]. Our study investigates the activation of DNA damage responses (DDR) as a consequence of EBV lytic cycle reactivation and expression of EBV lytic genes in cells of lymphoid and epithelial origin. Phosphorylation of Ataxia telangiectasia mutated (ATM), a transducer protein in the homologous recombination (HR) pathway of DDR, is a classic marker of DNA damage signaling activation. Several viral transcription activators, including HSV-1 ICP0, HIV-1 Tat protein, and HHV6 U19 protein, modulate DNA damage signaling responses and functionally interact with proteins involved in chromatin remodeling [14,15,16,17]. An emerging view is that chromatin remodeling may be a common mechanism for ATM kinase activation by viral transcription factors [18]
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