Identification of epstein-barr virus proteins that inhibit the DNA damage response

Abstract

Epstein-Barr virus (EBV) is a common herpesvirus that is the causative agent of mononucleosis and can also induce a variety of B cell lymphomas and carcinomas. To promote viral replication and cell survival, EBV is known to manipulate the cellular DNA damage response (DDR). To date there have only been a few studies on the interplay between EBV and the DDR and the functions of many EBV proteins remain uncharacterized. In this study, we screened a library of 47 EBV proteins for the ability to interfere with the formation of 53BP1 foci, a late step in the DDR, in response to etoposide-induced DNA damage. An uncharacterized tegument protein, BKRF4, was identified as an inhibitor of the DNA damage response. By studying the effect of BKRF4 on the steps of the DDR, it was shown that BKRF4 had no effect on the induction of γ-H2AX, but blocked the subsequent step at which histones H2A/H2AX become polyubiquitylated, which in turn inhibited the recruitment of 53BP1. To identify the host interactions of BKRF4, an affinity purification-mass spectrometry experiment was performed and identified histones as the most prevalent interacting partner. In keeping with this finding, an in vitro binding assay performed with purified BKRF4 and purified histone octamers showed the direct interaction between BKRF4 and histones. This interaction predicts that BKRF4 will localize to cellular chromatin, which was confirmed by both fluorescent microscopy and biochemical fractionation. BKRF4 contains a highly acidic region and this fragment of the protein was shown to be responsible for the chromatin interaction and DDR inhibition. Therefore, our results indicate that BKRF4 is targeted to cellular chromatin through a direct interaction with histones, where it can modulate at least some histone functions including the DDR.