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Exploring interactions between Epstein-Barr virus transcription factor Zta and the human genome
Epstein-Barr virus is a gamma herpesvirus that is present in human adult’s B-lymphocytes infecting 90% of the global population. EBV causes many types of lymphoma and carcinoma. The virus life cycle can be divided in two stages, latency and lytic cycle. Viral gene BZLF1 codes for the viral transcription and replication factor Zta (also known as BZLF1, ZEBRA, EB1, and Z) which is part of the signalling required to switch from latency to the lytic cycle. Zta is part of the bZIP family of proteins, it forms homodimers and can bind to specific sequences termed Zta Response Elements (ZREs). It binds to the EBV lytic origin of replication as well as to specific targeted promoters in the viral genome and regulates its expression. Recent research found and mapped interactions between the key viral transcription factor Zta and the B-cell genome, this showed interactions of Zta proximal (closer than 2Kb) and distal (farther than 2 Kb) to the transcription start site of several genes.
In this work, I asked the questions: Can enhancer properties be found in the sequences where Zta binds to? Is Zta distally regulating expression by looping of DNA? This was approached first by identifying potential sequences that could be conferring enhancing activity, then inserting them into vectors and transfecting them into two different cell lines. In this way, through luciferase reporter assays, any enhancing capabilities of the sequences were tested when placed in a proximal and distal manner to promoters known to be regulated by Zta, as well as mutated promoters not regulated by Zta. This resulted in finding discreet enhancer activity in the sequences analysed, with some being specific to the cell type that was used in the experiment. To answer the second question, chromosome conformation capture (3C) was used to test the possibility of a spatial rearrangement bringing together distal Zta binding regions and promotor regions of selected genes (looping). However, I did not find evidence of looping between Zta binding sites and the neighbouring promoters analysed, in the cell context employed
Mechanism of activation of the BNLF2a immune evasion gene of Epstein-Barr virus by Zta
The human gamma herpes virus Epstein–Barr virus (EBV) exploits multiple routes to evade the cellular immune response. During the EBV lytic replication cycle, viral proteins are expressed that provide excellent targets for recognition by cytotoxic T cells. This is countered by the viral BNLF2a gene. In B cells during latency, where BNLF2a is not expressed, we show that its regulatory region is embedded in repressive chromatin. The expression of BNLF2a mirrors the expression of a viral lytic cycle transcriptional regulator, Zta (BZLF1, EB1, ZEBRA), in B cells and we propose that Zta plays a role in up-regulating BNLF2a. In cells undergoing EBV lytic replication, we identified two distinct regions of interaction of Zta with the chromatin-associated BNLF2a promoter. We identify five potential Zta-response elements (ZREs) in the promoter that are highly conserved between virus isolates. Zta binds to these elements in vitro and activates the expression of the BNLF2a promoter in both epithelial and B cells. We also found redundancy amongst the ZREs. The EBV genome undergoes a biphasic DNA methylation cycle during its infection cycle. One of the ZREs contains an integral CpG motif. We show that this can be DNA methylated during EBV latency and that both Zta binding and promoter activation are enhanced by its methylation. In summary, we find that the BNLF2a promoter is directly targeted by Zta and that DNA methylation within the proximal ZRE aids activation. The implications for regulation of this key viral gene during the reactivation of EBV from latency are discussed