University of Sussex
Traylen, Christopher.pdf (6.2 MB)

To elucidate the Epstein-Barr virus replisome

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posted on 2023-06-09, 00:07 authored by Christopher Traylen
Epstein - Barr virus (EBV) is a member of the ?-herpesvirus subfamily of Herpesviridae. EBV is a double stranded DNA virus infecting humans causing a variety of disease from asymptomatic infection to association with certain tumours including Burkitts lymphoma, Hodgkin’s disease and nasopharyngeal carcinoma. EBV encodes an immediate-early protein called Zta (BZLF1, EB1, ZEBRA), which is an important transcription factor and replication factor direct in disrupting latency. EBV encodes viral proteins that assemble as a replisome at the viral lytic origin recognition site (Ori-Lyt). Zta binds Ori-Lyt and it is unclear how Zta interacts and recruits the complex to the site of DNA replication, while coordinating and recruiting host factors. After a mutation to three alanines (ZtaAAA) data implicates that the extreme C-terminus of Zta is essential for replication. The question posed is how does Zta assemble the replisome? Identification of the lytic changes that contribute to lytic replication, including cellular components that may contribute to EBV replication is attempted. Transfected control, Zta and ZtaAAA in HEK293-BZLF1-KO cells was compared. Size exclusion chromatography identified a higher molecular weight complex containing Zta during viral replication. SILAC (Stable isotope labelling by amino acids in cell culture) coupled to proteomics analysis identified the elution fraction composition. An interpretation of these cellular components in the context of lytic replication is explored. Identification of interactions of Zta with cellular proteins was attempted by SILAC histidine tagged Zta with pull down assay. Quantitative data was returned and a confirmation of interactions was attempted. A global proteomics approach was also performed. An enrichment method to isolate SILAC labeled Burkitts Lymphoma cells undergoing EBV lytic replication was coupled to mass spectrometry analysis to identify changes in host and viral proteins. Overall, cellular targets that may interact with Zta are to be confirmed. The global proteomics study recognized for the first time by proteomic analysis the identification of three EBV lytic replication cycle protein


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University of Sussex

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