DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA

PLoS One. 2015 May 7;10(5):e0126088. doi: 10.1371/journal.pone.0126088. eCollection 2015.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ataxia Telangiectasia Mutated Proteins / analysis
  • Ataxia Telangiectasia Mutated Proteins / metabolism
  • Cell Line
  • Chromobox Protein Homolog 5
  • DNA Damage*
  • DNA Replication
  • DNA, Viral / genetics*
  • Deoxyribonucleases / genetics
  • Deoxyribonucleases / metabolism
  • Epstein-Barr Virus Infections / genetics*
  • Epstein-Barr Virus Infections / metabolism
  • Epstein-Barr Virus Infections / pathology*
  • Epstein-Barr Virus Infections / virology
  • Gene Expression Regulation, Neoplastic
  • Herpesvirus 4, Human / genetics*
  • Herpesvirus 4, Human / physiology
  • Histones / analysis
  • Histones / metabolism
  • Humans
  • Immediate-Early Proteins / genetics
  • Immediate-Early Proteins / metabolism
  • Intracellular Signaling Peptides and Proteins / analysis
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Phosphorylation
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism
  • Signal Transduction
  • Trans-Activators / genetics*
  • Trans-Activators / metabolism
  • Tumor Suppressor p53-Binding Protein 1
  • Viral Proteins / genetics
  • Viral Proteins / metabolism

Substances

  • BRLF1 protein, Human herpesvirus 4
  • BZLF1 protein, Herpesvirus 4, Human
  • CBX1 protein, human
  • DNA, Viral
  • H2AX protein, human
  • Histones
  • Immediate-Early Proteins
  • Intracellular Signaling Peptides and Proteins
  • TP53BP1 protein, human
  • Trans-Activators
  • Tumor Suppressor p53-Binding Protein 1
  • Viral Proteins
  • Chromobox Protein Homolog 5
  • BGLF4 protein, Epstein-Barr virus
  • Ataxia Telangiectasia Mutated Proteins
  • Protein Serine-Threonine Kinases
  • Deoxyribonucleases
  • deoxyribonuclease, Epstein-Barr virus