University of Cambridge
Locus-specific proteomics identifies novel repressors of Epstein-Barr virus reactivation
Abstract
dc:description.abstractThe Epstein Barr virus (EBV) is a human gammaherpesvirus which infects the majority of the global population and is associated with the development of cancer and autoimmune disease. Infection with EBV is lifelong, as the virus enters a latent state in B-cells which persists until an appropriate stimulus, such as B-cell activation, triggers viral reactivation to a lytic state. During latent infection, the viral genome resides in the cell nucleus as an episome tethered to host chromosomes and expression of lytic genes is silenced by epigenetic mechanisms. The most important target for transcriptional repression is the immediate-early lytic gene BZLF1, whose expression can activate the entire lytic cascade. Our understanding of the factors which restrict BZLF1 expression is incomplete and the aim of my project was to use PICh (Proteomics of Isolated Chromatin segments), a novel locus- specific proteomic method, to systematically characterise the cellular proteins which bind the BZLF1 promoter DNA. My PICh experiment identified two new EBV repressors: the polycomb complex PRC1 and the nucleosome remodeler CHD4. These proteins were found to independently bind the latent EBV genome and CRISPR-Cas9-mediated depletion of either of these components resulted in spontaneous lytic reactivation, confirming their role as repressors. My further experiments investigated how PRC1-mediated repression was lost during lytic reactivation and unexpectedly found that the PRC1 histone mark, H2AK119Ub, was removed from both viral and human genomes during the early lytic cycle. This suggested an active process of deubiquitination, and subsequent analysis of whole-cell proteomic changes in lytic B-cells identified massive upregulation of the human deubiquitinase USP17. USP17 upregulation was driven by EBV lytic proteins and overexpression of USP17 was associated with deubiquitination of H2AK119Ub. The complexity of the USP17 gene locus, comprising at least 32 copies, made its depletion very challenging. Further experiments are therefore required to definitively show that USP17 is responsible for H2AK119Ub deubiquitination during the EBV lytic cycle and, most importantly, to ascertain whether removal of H2AK119Ub is necessary to complete lytic reactivation. In summary, my project was the first use of locus specific proteomics to characterise novel EBV repressors and identified USP17 as a target of viral manipulation during lytic EBV reactivation.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2023
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Greaves, Daniel
- Advisor dc:contributor.advisor
-
- Lehner, paul
Subjects
dc:subject × 3Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.108424
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/368087