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Title: The role of the deubiquitylase UCHL3 during alphavirus infection
Author: King, S. C.
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Alphaviruses are positive-sense RNA viruses from the family Togaviridae. These viruses rely on arthropod vectors, such as mosquitos, to replicate before transmission to vertebrates. There are several medically important viruses in this group, including Chikungunya virus (CHIKV) which, since 2004, has caused large number of epidemics that have infected millions of people in Asia, the Indian subcontinent, Europe, the Americas and the Pacific Islands. Consequences of CHIKV infection can include chronic and debilitating arthritis and, in some instances, life-threatening neurologic and cardiac manifestations, which are a major public health concern. Possible future outbreaks and emerging new strains underscore the need to understand this complex virus group, its pathogenicity and host-cell interactions. Currently there are no licensed vaccines or antivirals for CHIKV infection. Ubiquitination and its reversible process, deubiquitination make up the ubiquitin proteasome system which regulates the activity of many cellular pathways and processes, including cell signalling, membrane traffic, protein quality control, endocytosis, DNA repair, cell proliferation, and apoptosis. Thus, is a popular target for viruses, which can manipulate and subvert certain elements of the host ubiquitin proteasome system to enhance their own replication, either by targeting of cellular proteins or encoding viral homologues of key pathway proteins. Deubiquitylases or DUBs are a large family of enzymes shown to play a crucial role, not only in virus replication but also in regulating the antiviral innate immune response, including autophagy and apoptosis, making them attractive drug targets. To generate a better understanding of CHIKV pathogenesis and the role of host cellular factors a siRNA screen was performed using SFV (a close relative of CHIKV) as a model to identify specific host DUBs that play an antiviral role in alphavirus infection, i.e. those that play a role in defending against virus. In an experiment preceding this thesis a DUB siRNA pool library screen identified 21 antiviral candidate DUBs (CYLD, UCHL3, OTUD7A, DUB4, OTUD6B, UCHL5, USP10, USP29, OTUB2, USP31, MPND, USP16, OTUB1, DUB3, USP36, EIF3F, USP7, USP8, USP15, JOSD1, and USP9Y). In the present study, 14 of these 21 DUBs were followed up in a confirmatory deconvolution screen by depletion in SFV-infected HeLa cells. Following validation, two strong antiviral candidate DUBs were identified, USP10 and UCHL3. Since other studies have already established a role for USP10 in SFV and CHIKV infection, this work was expanded to focus on UCHL3 and its role during alphavirus infection in different cell types. The data revealed that while the capacity for SFV and CHIKV to replicate was enhanced in the absence of UCHL3 in HeLa cells, in fibroblast cells this was the reverse highlighting a potential fundamental difference between cell types used, or between siRNA knockdown vs genetic KO of UCHL3. SFV-infected HeLa cells also showed a propensity for raised apoptotic markers, including caspase 3 and 7, and PUMA in the absence of UCHL3 during infection suggesting UCHL3 is pro-survival. UCHL3 reportedly plays an important role in regulating several different cellular processes contributing to cell survival, including cellular proliferation and DNA repair. Based on the data in this thesis and indications from the literature it is now hypothesised to play an important role in the regulation of autophagy and apoptosis. This study may help open the door to further investigations into the role of UCHL3 as a potential antiviral mechanism against alphaviruses.
Supervisor: Blake, Neil ; Coulson, Judy Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral