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Title: Investigation of the role of protein kinase D in human rhinovirus replication
Author: Guedan Paredes, Ana Isabel
ISNI:       0000 0004 8499 3854
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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Viral respiratory tract infections are the major cause of asthma and COPD exacerbations and represent a significant healthcare burden. Although several viruses are responsible, the most frequent pathogen involved in exacerbations is rhinovirus (RV). Currently there are no vaccines or chemotherapy treatments available and efforts to develop anti-virals that target viral proteins have not succeeded due partly to the high ability of RV to mutate. In the last few years, anti-viral drug development strategies have focused on host proteins involved in viral replication. However, so far, this approach has also failed to succeed due to toxicity issues. Thus, the aim of this thesis was to identify novel drug targets for the treatment of RV infections, which might potentially be used to treat asthma and COPD exacerbations. RV replicates on the cytoplasmic face of the endoplasmic reticulum (ER) and Golgi membranes and phosphatidylinositol 4-kinase III β (PI4KB) and oxysterol-binding protein (OSBP) are two host proteins needed by the virus early in the replication cycle. These host targets have been the focus of anti-viral drug development in recent years. The DAG-activated kinase, protein kinase D (PKD), among many other functions in the cell, is involved in Golgi vesicle fission and transport at the trans-Golgi network (TGN) where it also regulates the activity of PI4KB, OSBP and ceramide transfer protein (CERT). Therefore, I hypothesised that PKD might be involved in viral replication due to its known interactions with previously identified host factors required by the virus. To investigate the role of PKD, I have used overexpression of PKD1 mutants, PKD knockout cells and PKD chemical inhibitors in in vitro epithelial cell models of viral infection. Furthermore, novel and more potent PKD inhibitors were made and tested in vitro. As host lipid metabolism appears to be key for viral replication, a lipidomics study through a time course of RV infection was performed and pointed to lipid enzymes involved in DAG-related pathways as novel anti-viral targets. The anti-viral effect of inhibiting these targets was tested in an in vitro model of human bronchial epithelial cell infection. I have also identified an unexpected role for PKD in the type I interferon (IFN) signalling pathway, which was studied in the context of viral infection and following recombinant IFN-β stimulation. This novel observation has important implications for the potential to use PKD inhibitors therapeutically. These findings support the involvement of PKD in RV replication and its potential as an anti-viral target, and on the other hand, a role for PKD in the type I IFN signalling pathway. Furthermore, a number of new host anti-viral targets have been discovered by the lipidomics study.
Supervisor: Solari, Roberto ; Johnston, Sebastian Sponsor: National Heart and Lung Institute
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral