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Utilising Omics Approaches to Understand Kaposi's Sarcoma-associated Herpesvirus

Owen, Christopher Bradley (2015) Utilising Omics Approaches to Understand Kaposi's Sarcoma-associated Herpesvirus. PhD thesis, University of Leeds.

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Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic human virus associated with a number of malignancies, including Kaposi’s sarcoma. Similar to all herpesviruses, KSHV establishes either latent or lytic infections in host cells. The latent stage involves minimal viral gene expression, enabling the virus to remain dormant, maintaining genome integrity and enabling viral persistence. Conversely, lytic replication is characterised by the expression of a highly regulated and coordinated cascade of viral gene expression, ultimately resulting in the production of new mature, infectious virions. Importantly, lytic replication is necessary for the development and spread of Kaposi’s sarcoma. State of the art high throughput, high resolution “omics” approaches, such as mass spectrometry-based quantitative proteomics and next generation sequencing-based transcriptomics, are rapidly becoming the techniques of choice for discovering novel biological phenomena due, in part, to their sensitivity and the ability to repeatedly ask new questions of an existing dataset. Herein, three high throughput, high resolution approaches, namely SILAC-based quantitative proteomics, miRNA sequencing and mRNA sequencing are employed in an attempt to identify and characterise novel interactions between KSHV and the host cell. Utilising quantitative proteomics, the essential host cell splicing factor Prp19 is identified as a novel interacting partner of the lytic KSHV ORF57 protein in subnuclear bodies. This interaction, surprisingly, does not contribute to viral mRNA maturation, but instead has implications for the cellular DNA damage response, appearing to limit the effectiveness of this important pathway, possibly to reduce any negative effects on viral replication. Proteomic analyses also highlighted a link between KSHV lytic replication and host miRNA biogenesis pathways. Through the application of miRNA sequencing, two host miRNAs, namely miR-151a-5p and miR-365a-3p, were found to be dysregulated during KSHV infection. Importantly, neither of these miRNAs appear to represent a host antiviral response. Instead, cellular target mRNAs are identified for miR-365a-3p, through the use of mRNA sequencing. These targets, termed DOCK5 and PRUNE2 are rapidly degraded during KSHV lytic replication via the viral-mediated upregulation of miR-365a-3p expression. Subsequent analysis of DOCK5 function during lytic replication suggests this interaction may promote viral egress. The data presented herein sheds light on previously unidentified mechanisms employed by KSHV to hijack the host cell, and may aid in the development of novel therapeutics against this important pathogen.

Item Type: Thesis (PhD)
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute for Molecular and Cellular Biology (Leeds)
Identification Number/EthosID: uk.bl.ethos.679805
Depositing User: Dr Christopher B Owen
Date Deposited: 17 Feb 2016 10:46
Last Modified: 26 Apr 2016 15:44
URI: http://etheses.whiterose.ac.uk/id/eprint/11782

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