Studies of RNF168 and CXCL5 expression by epithelial cell lines during RSV infection

Jeyatheswaran, Jonathan Studies of RNF168 and CXCL5 expression by epithelial cell lines during RSV infection. Master of Philosophy thesis, University of Liverpool.

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Abstract

Background: During herpes simplex virus type 1 (HSV-1) infection, the protein ICP0 causes degradation of RNF168 during infection, preventing the ubiquitination of histones proteins and a subsequent DNA damage response to double strand breaks. Our laboratory has shown that during RSV infection in vitro, RSV replication as measured by RSV N gene expression increased considerably when SiRNA were used to knock down RNF168 gene. This suggests that RNF168 activity may be affected by RSV expression or act to limit RSV replication normally during infection as part of a host cell intrinsic antiviral defence. Identifying and understanding the mechanism by which this occurs may be useful in the development of future drug therapies for RSV. Aims: To investigate the effect of RSV infection, interferon beta stimulation and polyI:C on RNF168 expression. To investigate how the duration of RSV infection affects the concentration levels of CXCL5. Methods: A549 cells were infected with RSV in a 24-well plate. Cells were also stimulated with IFN-β and the TLR3 ligand polyI:C. Real-time PCR for RSV N gene expression and Sandwich ELISAs for IL-6 and IL-8 were carried out to provide evidence that samples have been infected. Western Blotting was carried out to measure RNF168 expression. A Sandwich ELISA was applied to measure the concentration levels of CXCL5. Results: At a MOI of 1:1, 4 hours RSV infection suggested that there was an increase in RNF168 expression. This increase was not statistically significant (p=0.13). At 24 hours after RSV infection, MOI 1:1 the level of RNF168 expression decreased in comparison to control but this decrease was not statistically significant (p=0.14). However, at a MOI of 0.025:1, 24 hours after infection, RNF168 expression increased in comparison to the control. This change was not significant (p=0.29). IFN-β stimulation for 4 hours suggested that higher concentrations of IFN-β increased RNF168 expression. This was not a significant change (p=0.17). IFN-β stimulation for 24 hours suggested that higher concentrations caused a decrease in RNF168 expression in comparison to the control (p=0.6 for 100ng/ml). An increase in CXCL5 concentration in infected samples was seen at 6 hours in comparison to control, which supports previous proteonomic data results.

Item Type:	Thesis (Master of Philosophy)
Additional Information:	Date: 2013-08 (completed)
Subjects:	?? QR355 ??
Divisions:	Faculty of Health and Life Sciences
Depositing User:	Symplectic Admin
Date Deposited:	07 Aug 2014 10:13
Last Modified:	16 Dec 2022 04:41
DOI:	10.17638/00015615
Supervisors:	McNamara, Paul Flanagan, Brian
URI:	https://livrepository.liverpool.ac.uk/id/eprint/15615