Abstract
Epstein-Barr virus (EBV) is an oncogenic herpesvirus that is ubiquitous in the human population. Early after EBV infection in vitro, primary human B cells undergo a transient period of hyper-proliferation, which results in replicative stress and DNA damage, activation of the DNA damage response (DDR) pathway and, ultimately, senescence. In this study, we investigated DDR-mediated senescence in early arrested EBV-infected B cells and characterized the establishment of persistent DNA damage foci. We found that arrested EBV-infected B cells exhibited an increase in promyelocytic leukemia nuclear bodies (PML NBs), which predominantly localized to markers of DNA damage, as well as telomeric DNA. Furthermore, arrested EBV-infected B cells exhibited an increase in the presence of telomere dysfunction-induced foci. Importantly, we found that increasing human telomerase reverse transcriptase (hTERT) expression with danazol, a drug used to treat telomere diseases, permitted early EBV-infected B cells to overcome cellular senescence and enhanced transformation. Finally, we report that EBV-infected B cells undergoing hyper-proliferation are more sensitive than lymphoblastoid cell lines (LCLs) to inhibition of Bloom syndrome-associated helicase, which facilitates telomere replication. Together, our results describe the composition of persistent DNA damage foci in the early stages of EBV infection and define key regulators of this barrier to long-term outgrowth.
Highlights
Epstein-Barr virus (EBV), an oncogenic γ-herpesvirus was the first human tumor virus to be discovered [1]
In this study, we characterize the formation of persistent DNA damage response (DDR) foci in senescent EBV-infected B cells and the regulators of this response impinging on B-cell transformation
We found that arrested infected B cells exhibited an increase in TIF positive localization of DDR markers to telomeric DNA, known as telomere dysfunction-induced foci (TIF)
Summary
Epstein-Barr virus (EBV), an oncogenic γ-herpesvirus was the first human tumor virus to be discovered [1]. Activation of the DDR leads to DNA repair coupled with a transient cell cycle arrest, or if the damage is too great or irreparable, apoptosis or cellular senescence. Infected cells that arrest after undergoing rapid proliferation exhibit an increase in markers of OIS, including H3K9me senescence-associated heterochromatic foci, upregulation of p16 and p21, and enhancement of p53 target gene expression [15,24]. Depending on the severity or location of DNA damage, lesions may either induce transient growth arrest to allow time for damage to be repaired, or lesions may establish persistent DNA damage foci, constitutive DDR signaling and chronic p53 activation, which leads to permanent senescence [26,27,28,29,30,31]. In this study, we characterize the formation of persistent DDR foci in senescent EBV-infected B cells and the regulators of this response impinging on B-cell transformation
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