Investigations into the significance of Nrf2 signalling and ubiquitination of proteins in Respiratory Syncytial Virus infection

Chudek, Dorota Investigations into the significance of Nrf2 signalling and ubiquitination of proteins in Respiratory Syncytial Virus infection. Master of Philosophy thesis, University of Liverpool.

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Abstract

Introduction and Objectives: Respiratory Syncytial Virus (RSV) is the commonest cause of bronchiolitis in infants. This negative strand RNA virus is known to infect and replicate in the airway epithelium. RSV infection induces elevated levels of reactive oxidative species and subsequent oxidative stress injury in the lungs. Nrf2, a transcription factor that regulates antioxidant protein expression, has an important role in preventing pulmonary oxidative damage. Sulforaphane is a potent, naturally occurring inducer of NRF2 found in vegetables such as broccoli. In this study we sought to determine whether Nrf2 induction by sulforaphane might reduce RSV replication in airway epithelial cells. We also selected six proteins including MAVS, DDX21, RPS10, prohibitin, annexin A1, HMGB1 from proteomics defining changes in their level of ubiquitination following RSV infection. Our aim was to determine which proteins change their level of polyubiquitination following the infection. This could help identify new biochemical pathways involved in the host defence or viral replication and new targets for potential therapeutic intervention. Method: BEAS2B cells were infected with RSV at MOI of 1 following pre-treatment with sulforaphane. Samples were harvested at time points 24 and 48 hours and analysed by Western Blotting for NrF2 and RSV. In addition, RT-qPCR was carried out for RSV quantification using an RSV N primer. A549 cells were infected with various concentrations of RSV (1:4-4:1). Samples were harvested at time point of 4 and 24 hours and analysed by Western Blotting using antibodies to ubiquitin and target proteins selected from ubiquitination proteomics data . Immunoprecipitation was used to confirm ubiquitination of these proteins and immunohistology to confirm their cellular localisation. Proteasome activity was inhibited using MG132 a specific, potent, reversible, and cell-permeable proteasome inhibitor. Results: Sulforaphane induced Nrf2 production in BEAS2Bs in a dose dependent manner. Results do not show that RSV replication is reduced in airway epithelial cells pretreated with Sulforaphane. However, preliminary data suggests that virus might have degrading effect on Nrf2. Western blots demonstrate changes in expression of target proteins and their ubiquitination following RSV infection and proteasome inhibition. Breakdown products of DDX21 and MAVS were detected in RSV infected samples. Conclusions: RSV infection changes expression of target proteins in A549 cells and might have influence on their ubiquitination, however, most probably it does not affect expression of Sulforaphane induced Nrf2. DDX21 and Nrf2 are likely to be degraded by the virus but results have to be confirmed in further experiments.

Item Type:	Thesis (Master of Philosophy)
Additional Information:	Date: 2015-08 (completed)
Depositing User:	Symplectic Admin
Date Deposited:	28 Jan 2016 10:07
Last Modified:	17 Dec 2022 01:38
DOI:	10.17638/02030240
URI:	https://livrepository.liverpool.ac.uk/id/eprint/2030240