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A Screen for Epstein-Barr Virus Proteins That Inhibit the DNA Damage Response Reveals a Novel Histone Binding Protein

Epstein-Barr virus (EBV) infects most people worldwide and is causatively associated with several types of cancer, including ∼10% of gastric carcinomas. EBV encodes ∼80 proteins, many of which are believed to manipulate cellular regulatory pathways but are poorly characterized. The DNA damage response (DDR) is one such pathway that is critical for maintaining genome integrity and preventing cancer-associated mutations. In this study, a screen for EBV proteins that inhibit the DDR identified BKRF4 as a DDR inhibitor that binds histones and blocks their ubiquitylation at the DNA damage sites. We also present evidence that BKRF4 is expressed in both latent and lytic forms of EBV infection, where it downregulates the DDR, as well as in EBV-positive gastric tumors. The results suggest that BKRF4 could contribute to the development of gastric carcinoma through its ability to inhibit the DDR. ABSTRACT To replicate and persist in human cells, linear double-stranded DNA (dsDNA) viruses, such as Epstein-Barr virus (EBV), must overcome the host DNA damage response (DDR) that is triggered by the viral genomes. Since this response is necessary to maintain cellular genome integrity, its inhibition by EBV is likely an important factor in the development of cancers associated with EBV infection, including gastric carcinoma. Here we present the first extensive screen of EBV proteins that inhibit dsDNA break signaling. We identify the BKRF4 tegument protein as a DDR inhibitor that interferes with histone ubiquitylation at dsDNA breaks and recruitment of the RNF168 histone ubiquitin ligase. We further show that BKRF4 binds directly to histones through an acidic domain that targets BKRF4 to cellular chromatin and is sufficient to inhibit dsDNA break signaling. BKRF4 transcripts were detected in EBV-positive gastric carcinoma cells (AGS-EBV), and these increased in lytic infection. Silencing of BKRF4 in both latent and lytic AGS-EBV cells (but not in EBV-negative AGS cells) resulted in increased dsDNA break signaling, confirming a role for BKRF4 in DDR inhibition in the context of EBV infection and suggesting that BKRF4 is expressed in latent cells. BKRF4 was also found to be consistently expressed in EBV-positive gastric tumors in the absence of a full lytic infection. The results suggest that BKRF4 plays a role in inhibiting the cellular DDR in latent and lytic EBV infection and that the resulting accumulation of DNA damage might contribute to development of gastric carcinoma. IMPORTANCE Epstein-Barr virus (EBV) infects most people worldwide and is causatively associated with several types of cancer, including ∼10% of gastric carcinomas. EBV encodes ∼80 proteins, many of which are believed to manipulate cellular regulatory pathways but are poorly characterized. The DNA damage response (DDR) is one such pathway that is critical for maintaining genome integrity and preventing cancer-associated mutations. In this study, a screen for EBV proteins that inhibit the DDR identified BKRF4 as a DDR inhibitor that binds histones and blocks their ubiquitylation at the DNA damage sites. We also present evidence that BKRF4 is expressed in both latent and lytic forms of EBV infection, where it downregulates the DDR, as well as in EBV-positive gastric tumors. The results suggest that BKRF4 could contribute to the development of gastric carcinoma through its ability to inhibit the DDR.