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Structural and Biochemical Studies of Proteins Implicated in Kaposi's

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Structural and Biochemical Studies of Proteins Implicated in Kaposi's Structural and biochemical studies of proteins implicated in Kaposi’s Sarcoma-associated Herpesvirus pathobiology Hyunah Lee Thesis submitted to the University College London for the degree of Doctor of Philosophy 2019 Division of Structural and Molecular Biology UCL 1 Declaration I, Hyunah Lee, confirm the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract The Kaposi’s sarcoma-associated Herpes virus (KSHV) infects hundreds of millions of people world-wide contributing to the development of Kaposi’s sarcoma (the most common HIV-related cancer), Multicentric Castleman’s disease and Primary effusion lymphoma. The project focused on two key proteins: KSHV SOX and KSHV- vFLIP that operate during the lytic and latent phases of the KSHV life-cycle respectively. KSHV SOX is a virally encoded highly conserved alkaline exonuclease which plays a key role in the global and rapid degradation of host mRNA in a process termed host shutoff as well as the packaging of viral genomes into capsids following replication. Although published (Bagneries et al., 2011) and unpublished (Lee, 2015, Master’s thesis, UCL) crystal structures of SOX bound to DNA has given key insights into the mechanism of DNA recognition and cleavage, it was unclear how RNA could be recognised owing to the lack of canonical RNA binding motifs and therefore uncertain whether the mechanism of cleavage proposed for the exonucleolytic cleavage of DNA substrates would apply. A combination of structural and biochemical approaches were therefore used to address these key outstanding questions which are presented in this thesis. They include the first crystal structure of SOX-E244S mutant bound to the KSHV pre-miRNA K2-31 which was determined at 3.3 Å by molecular replacement. Analysis of this structure has revealed a distinct binding mode for RNA binding relative to DNA in which the “bridge” motif, spanning the C- and N-terminal lobes of the SOX molecule, has an essential role in substrate 3 recognition. Despite these differences, endonucleolytic processing of RNA transcripts could still be achieved by the SN2 (biomolecular nucleophilic substitution) mechanism originally proposed for the exonucleolytic cleavage of DNA. The results of biochemical and biophysical assays performed on wild-type and mutant proteins defective in host shut off further revealed that the degradation of RNA is largely sequence non-specific, but requires structured elements such as stem loop or bulge motifs. They also revealed that impaired host shut off activity could be explained by impaired RNase activity against structured substrates for a subset of the mutants. The ability of compare SOX-DNA and SOX-RNA structures afforded by these studies lead to the identification of phytic acid as a potential inhibitor of both its DNase and RNase activities confirmed by the crystal structure of SOX-phytic acid complex refined to 2.3 Å. Here it is shown that phytic acid not only has the potential to physically block the association of DNA/RNA substrates but also inhibit nucleolytic cleavage by directly co-ordinating to a catalytically important magnesium ion. During latency, KSHV produces a limited number of proteins essential for its survival. One of these is the viral FLICE inhibitory proteins (vFLIPs) that directly activates the canonical and alternative NF-κB pathways resulting in increased cell proliferation, transformation, cytokine secretion, and protection against growth factor withdrawal-induced apoptosis. Previous studies have shown that KSHV-vFLIP forms a ternary complex with the transcription factor p100 and the kinase IKKα for persistent activation of the alternative pathway shown to have an important role in cellular transformation. Although the mechanism underlying this process is unclear, a physical interaction between KSHV-vFLIP and p100 has been implicated. Having 4 successfully produced all three proteins in E. Coli and baculovirus expression systems, the nature of this complex was investigated using pull down assays and site- directed mutagenesis. From these results, it has been possible to deduce that KSHV- vFLIP interacts with residues 860-900 located at the C-terminal end of p100 and does not interact with the death domain of p100. 5 Impact Statement The findings of this thesis provide the first novel atomic structure of a viral RNase bound to RNA and has been recently published in Nucleic acids journal (Lee. H., Patschull. A., Bagneris. C., Ryan. H., Sanderson. C., Ebrahimi. B., Nobeli. I., and Barrett.T. KSHV SOX mediated host shutoff: the molecular mechanism underlying mRNA transcript processing (2017). Nucleic Acids Research). These studies have provided a platform for the identification of potential inhibitors which is further analysed in Results chapter 5 and a manuscript will be prepared for publication. Moreover, this work will be expanded by other members of the Barrett group for the identification of more potent inhibitors or compounds for KSHV anti-cancer therapy. This thesis also describes the involvement of KSHV-vFLIP in the alternative NF-κB pathway which sheds light on another defence mechanism utilised by KSHV to counteract human immune surveillance systems. Together these findings could aid in the development of novel drugs or anti-viral therapies that specifically target KSHV. 6 Acknowledgements First and foremost, I am deeply thankful to Dr Tracey Barrett for supervising this PhD. Throughout my PhD, she provided me tremendous support, advice, invaluable discussion about ideas and results which I found extremely helpful. I would like to thank Dr Claire Bagneris for the support and help in the Franklin lab and her contribution to the work presented in the thesis; Dr Ambrose Cole for advice and discussion on collecting X-ray data. I would like to also thank my PhD committee members: Prof John Christodoulou and Prof Chris Kay for advice and invaluable discussions. I would like to thank Prof Snezana Djordjevic and Miss Manu Davies for advice and support at UCL. I am especially grateful to all our Franklin lab members for their help, technical support, sharing equipment and reagents and being wonderful friends. I am grateful to all our collaborators for the opportunity to be involved in interesting projects and their support. And finally I would like to thank my family; especially my mum, dad and my brother who have always supported me and pushed me to achieve my goals. I have to give special thanks to my closest friends for supporting me throughout my PhD and being always there for me. 7 Table of Contents Abstract .................................................................................................................................................. 3 Impact Statement .............................................................................................................................. 6 Acknowledgements .......................................................................................................................... 7 List of Figures .................................................................................................................................... 14 List of Tables ...................................................................................................................................... 17 Abbreviations .................................................................................................................................... 18 Chapter 1: Introduction ............................................................................................................... 26 1.1 Human Herpesviruses .......................................................................................................... 26 1.2 Kaposi’s Sarcoma-associated Herpesvirus (KSHV) .................................................. 29 1.2.1 Infection and lifecycle of KSHV ................................................................................. 31 1.2.2 KSHV miRNAs .................................................................................................................. 34 1.3 Mechanisms of Viral host shutoff .................................................................................. 36 1.3.1 HSO in γ-herpesviruses ............................................................................................. 37 1.3.2 The mechanics of DNA and RNA turnover in BGLF5 and KSHV SOX ....... 39 1.4 The DNase activity of KSHV-SOX and its involvement in viral DNA packaging .............................................................................................................................................................. 42 1.5 The structural basis of DNA processing by SOX ......................................................... 43 1.6 The RNase Activity of KSHV-SOX ..................................................................................... 45 1.7 Evidence of structured RNA recognition by SOX ....................................................... 47 1.8 KSHV-associated antiviral therapy ................................................................................. 51 8 1.9 Structure-based design of KSHV-SOX inhibitors ....................................................... 53 1.9.1 Cancer preventive and therapeutic properties of Phytic acid ...................... 53 1.10 Viral latency and the NF-κB pathway .......................................................................... 57 1.10.1 NF-κB ..............................................................................................................................
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