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

Abstract

To replicate and persist in human cells, linear double-stranded (ds) DNA 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 tumours 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. Epstein-Barr virus (EBV) infects most people worldwide and is causatively associated with several types of cancer, including ∼10% of gastric carcinoma. 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. Here a screen for EBV proteins that inhibit the DDR identified BKRF4 as a DDR inhibitor that binds histones and block 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 down-regulates the DDR, as well as in EBV-positive gastric tumours. The results suggest that BKRF4 could contribute to the development of gastric carcinoma through its ability to inhibit the DDR.

Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response

“…Both the Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KHSV), which cause Burkitt's and Hodgkin's lymphoma, nasopharyngeal carcinoma, stomach adenocarcinoma, and Kaposi's sarcoma (2), inhibit the recruitment of RNF168 at DSBs. These effects are attributed to the EBV tegument protein BKRF4 and the KHSV LANA protein, which bind to chromatin and physically block the interaction of RNF168 with the nucleosome (59,60). The consequences of RNF168 inhibition on viral replication and cancer progression induced by EBV and KHSV remain to be determined.…”

A genome-wide screen of Epstein-Barr virus proteins that modulate host SUMOylation identifies a SUMO E3 ligase conserved in herpesviruses

“…Additional SUMO-regulated processes controlled by herpesviruses include interference with cell cycle progression, resulting in G1/S arrest, and inhibition of DNA damage responses [ 58 – 64 ]. Our previous screens have identified BGLF2 and BMRF1 as contributing to G1/S arrest [ 28 ] and BMRF1 as an inhibitor of DNA damage response [ 65 ]. We do not yet know the mechanism of these effects, but the fact that both proteins were found to upregulate SUMOylation suggests that they may be affecting cell cycle progression and the DDR by increasing SUMOylation of some cellular proteins.…”

“…BMRF1 belongs to the family of DNA polymerase processivity factors that are conserved in all herpesviruses [ 74 ]. However, BMRF1 also has additional roles in transcriptional activation [ 75 – 77 ], cell cycle progression [ 28 ] and in inhibiting the DNA damage response to double stranded DNA breaks [ 34 ]. It will be interesting to determine how the multiple roles of BGLF2 and BMRF1 relate to their ability to upregulate SUMOylation.…”

“…To investigate how EBV proteins impact cellular pathways, we previously generated an expression library for most of the EBV lytic proteins, and used the C-terminal FLAG tag on each protein to determine their subcellular localization [ 33 ]. These EBV proteins were then screened for the ability to disrupt or alter PML nuclear bodies [ 33 ], contribute to cell cycle arrest at the G1/S interface [ 28 ] and inhibit the cellular DNA damage response [ 34 ]; characteristics typical of EBV lytic infection. These screens have led to new functions for several EBV proteins demonstrating the utility of the approach.…”

Identification of ARKL1 as a Negative Regulator of Epstein-Barr Virus Reactivation

“…The ARKL1ΔS mutant lacking amino acids 202 to 226 was generated by gene synthesis, described in Cao et al (44), and inserted into the same restriction sites of the pCMV3FC plasmid as above. The plasmids expressing FLAG-DDX24, FLAG-FBXO38, and FLAG-BKRF4 are described previously (84)(85)(86). The BZLF1 promoter firefly luciferase (pZp-luc) and renilla luciferase plasmids (pRL-TK) were kindly provided by Takayuki Murata (53).…”