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

The Epstein Barr virus (EBV) is a human gammaherpesvirus 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