Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/113005
Type: Theses
Title: The novel interacting partners of viperin and their role in establishing a host antiviral state
Author: Khantisitthiporn, Onruedee
Issue Date: 2017
School/Discipline: School of Biological Sciences
Abstract: Viral infection results in activation of the innate immune response that culminates in the production of the interferons and the establishment of an antiviral state through the expression of hundreds of interferon stimulated genes (ISGs). One such ISG is Viperin (RSAD2) that has broad antiviral activity against a range of RNA and DNA viruses. Previous work has established that viperin is antiviral against the hepatitis C virus, however the molecular mechanism(s) that underpin this antiviral activity are not well understood. It is thought that viperin interacts with the HCV NS5A protein and the host pro-viral host factor VAP-A within the HCV replication complex, although this has not formally been proven. Thus the main objective of this thesis was to investigate the localisation of viperin to the HCV replication complex at the cellular level and to identify additional viperin cellular interacting partners in the hope that we can further understand the biology of this enigmatic protein. We therefore used a cell biology approach to visualise viperin at or within the HCV replication complex. Expression of viperin tagged to the electron microscopy (EM) tag (APEX2) allowed us to precisely determine the localisation of viperin at the ultrastructural level by EM. We identified that in the presence of exogenous HCV non-structural protein expression (and hence RC formation), viperin alters the formation of convoluted membranes that in-turn could have an impact on the establishment of replication complex formation that is crucial for HCV replication. In addition to viperin binding the HCV NS5A protein, it can also bind to host proteins (FPPS, VAP-A, TRAF6, and IRAK1) to modulate cellular function such as lipid metabolism and innate immune signalling. We hypothesised that viperin exerts such a diverse range of functions through interaction with as yet unidentified cellular proteins and we employed a yeast two-hybrid screen to identify novel interacting partners. The peroxisomal biogenesis factor 19 (PEX19) was identified as a genuine viperin interaction partner and furthermore showed that expression of viperin not only co-localised with peroxisomes but redirected them to a perinuclear localisation and association with lipid droplets. The reason for this is not immediately clear, however peroxisomes have recently been shown to be a scaffold for the innate immune adaptor molecule MAVS and are now recognised as important organelles in activation of the host cellular response to viral infection. Using a combination of deconvolution and super-resolution microscopy and EM using APEX2 tagged viperin we show that viperin localises to the LD in association with peroxisomes and the mitochondria. It is, therefore, possible that viperin may direct MAVS+ve peroxisomes to sites of innate immune signalling. Consistent with this hypothesis we have shown that viperin augments IFN-β production for MAVS localised selectively on both mitochondria and the peroxisome, but not for each organelle individually. This implies that viperin may act as linker molecule for MAVS signalling from both organelles. In conclusion, we propose that viperin localises within the HCV replication complex to impart its antiviral effect. Furthermore, viperin interacts with PEX19 and recruits peroxisomes to an “innate immune synapse” to augment innate immune signalling to restrict viral infection. The work of this thesis contributes our understanding of the host-virus relationship in control of HCV and the possibility of the development of innate immune agonists as therapeutics for viral disease.
Advisor: Beard, Michael Robert
Eyre, Nicholas Stratford
Helbig, Karla
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2018
Keywords: Viperin
PEX19
peroxisomes
innate immune signalling
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at http://www.adelaide.edu.au/legals
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