Investigating the roles of the cellular DEAD-box protein DDX3 in the hepatitis C virus lifecycle

Stack, Seamus (2013) Investigating the roles of the cellular DEAD-box protein DDX3 in the hepatitis C virus lifecycle. PhD thesis, University of Glasgow.

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Hepatitis C Virus (HCV) is a major cause of chronic hepatitis, with present estimates predicting that approximately 170 million people are currently infected with the virus worldwide. The majority of infections progress to chronicity, ultimately leading to fibrosis, cirrhosis and hepatocellular carcinoma. HCV, which belongs to the Flaviviridae virus family, has a single-stranded RNA genome of positive polarity that encodes a unique polyprotein approximately 3000 amino acids in length.

DDX3 is a member of the DEAD-box family of RNA helicases. It is a ubiquitous cellular protein possessing ATPase and helicase activities. The exact cellular function of DDX3 is as yet undefined, but there is evidence for its involvement in biological processes as diverse as splicing, translation initiation and repression, cell cycle regulation, nucleo-cytoplasmic RNA shuttling, RNA transport, interferon induction and apoptosis.

Recent studies have shown that DDX3 may be one of the primary host targets for manipulation by a number of different viruses, including HCV. The HCV core protein has been found to directly interact with DDX3. Previous studies conducted by our research group have shown that this interaction is dispensable for viral replication. Intriguingly, our group and others have shown that knockdown of endogenous DDX3 severely impairs HCV replication. However, the exact stage of the HCV lifecycle at which DDX3 functions is unknown. To investigate this, we used a number of in vitro cell systems available to us, comprising HCV psuedoparticles (HCVpp), subgenomic replicons (SGR) and the HCV cell culture system (HCVcc), to analyse how DDX3 depletion affected numerous cellular and molecular events vital to an efficient viral lifecycle.

The effect of DDX3 knockdown on these systems was determined by transducing Huh-7 cells with lentivirus encoding short hairpin RNA against the N-terminus of DDX3. Towards this end, we confirmed the important role DDX3 plays in the HCV lifecycle and deduced that DDX3 acts at a post-translation stage of the HCV lifecycle in Huh-7 cells, inhibiting an as yet undetermined aspect of RNA replication. Results obtained also suggested that DDX3 is required for efficient HCV replication complex (RCs) function, as DDX3-deficient Huh-7 cells transiently transfected with SGR RNA exhibited diminished viral replication compared to control Huh-7 cells, yet no such reduction was observed in DDX3-depleted Huh-7 cells stably replicating SGR RNA. Although I did not observe the presence of DDX3 in RC during IF analysis, it is as yet unclear whether DDX3 is directly incorporated into these structures or influences their function in an indirect manner.

To determine if DDX3 knockdown had any specific effects on HCV assembly, we enlisted a subclone of Huh-7 cells with depleted CD81 levels called Huh7L-#4 cells for use in single-cycle infectious virus production experiments. DDX3 knockdown greatly altered infectious HCV production in these cells, to a far greater extent than that observed in Huh-7 cells, but HCV RNA replication was unperturbed in these cells. Subsequent analysis of infectious virus production and HCV RNA replication in DDX3-deficient shCD81 cells was performed to investigate if the phenotype observed in Huh7L-#4 cells was related to the absence of CD81. Our results confirmed that this was not the case.

In its totality, this study has uncovered important details regarding the importance of DDX3 in the HCV lifecycle, with data obtained suggesting that DDX3 plays a pleiotropic role in this lifecycle, with distinct responsibilities in undetermined aspects of HCV RNA replication and infectious virus production.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Virology, Hepatitis C biology, DDX3
Subjects: Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
Supervisor's Name: Patel, Dr. Arvind
Date of Award: 2013
Depositing User: Dr Seamus Stack
Unique ID: glathesis:2013-4571
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 Sep 2013 08:37
Last Modified: 01 Aug 2022 08:39

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