Locus-specific proteomics identifies novel regulators of Epstein-Barr virus lytic reactivation

The Epstein-Barr virus (EBV) is a human gamma-herpesvirus which infects over 90% of the global population and is associated with lymphoid and epithelioid cancers. After infection, EBV enters a latent state in B cells, whereby the viral genome persists as a nuclear episome maintained by expression of a small number of latency-associated viral proteins. The lytic viral proteins, required for DNA replication and virion production, are silenced by cellular epigenetic mechanisms. The immediate-early lytic gene BZLF1 is the most important target for transcriptional repression, as its expression triggers the lytic cascade. To gain insight into the factors restricting BZLF1 expression, we used the PICh method of locus-specific proteomics (proteomics of isolated chromatin segments) to identify proteins which occupy BZLF1 promoter DNA. We identified more than 30 proteins associated with the BZLF1 promoter, including the nucleosome remodeler CHD4 and components of the Polycomb PRC1 complex. We show that CHD4 and PRC1 components are novel repressors of BZLF1 gene expression and that both are required to prevent spontaneous lytic reactivation in Burkitt lymphoma cells. We also reveal a marked, cell-wide loss of the PRC1 histone mark (H2AK119Ub) during the lytic cycle, which is dependent on immediate-early and early lytic gene expression. A proteomic analysis of Burkitt lymphoma cells containing lytic EBV identified upregulation of USP17, a de-ubiquitinase capable of H2AK119Ub removal. Taken together, our study demonstrates the power of proteomic approaches to identify repressors of EBV reactivation and provides new insight into how EBV manipulates epigenetic mechanisms during the lytic cycle.IMPORTANCEFollowing infection, Epstein-Barr virus persists in a latent state where the viral genome resides in the cell nucleus as an episome. Cellular epigenetic proteins occupy the episomes to restrict viral gene expression and prevent lytic reactivation. In this study, we use mass spectrometry to characterize cellular proteins occupying a key viral lytic gene promoter (pBZLF1). We identify the nucleosome remodeler CHD4 and histone modifier complex PRC1 as novel repressors of pBZLF1 and show that both are required to prevent spontaneous EBV lytic reactivation in B cells. We also report that PRC1-mediated histone modification is erased during EBV lytic reactivation from both cellular and viral genomes. The human de-ubiquitinase enzyme USP17 is likely to be responsible for this effect, as upregulation of USP17 is induced by EBV lytic proteins. This study provides new insight into how EBV manipulates epigenetic mechanisms to regulate latency and lytic reactivation and reveals novel potential therapeutic targets.