Investigating the interaction between the Hepatitis C virus core protein and the cellular DEAD-box protein DDX3 and its importance in virus replication

Angus, Allan G.N. (2010) Investigating the interaction between the Hepatitis C virus core protein and the cellular DEAD-box protein DDX3 and its importance in virus replication. PhD thesis, University of Glasgow.

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Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Present estimates predict that approximately 130 million people are infected with HCV with the majority of all infections progressing to chronicity, ultimately leading to fibrosis, cirrhosis and hepatocellular carcinoma. The virus, which belongs to the family Flaviviridae, has a single-stranded RNA genome of positive polarity that codes for a unique polyprotein of
approximately 3000 amino acids. Core protein forms the viral nucleocapsid and is the most highly conserved of all the viral proteins. As well as playing a central role in virion assembly, core has been shown to modulate several cellular processes mainly due to its interaction with several host factors. One such cellular interacting partner of core protein is the DEAD-box RNA helicase DDX3. Although, the binding of these two proteins has been shown by yeast-two hybrid screening and co-immunoprecipitation (co-IP) assays, an authentic interaction is yet to be
demonstrated in cells replicating the HCV JFH1 infectious clone. Nevertheless, IF analysis of JFH1-infected cells has shown a proportion of DDX3 is redistributed to distinct
cytoplasmic sites where it colocalises with core, implying that these two proteins interact during virus replication. Six JFH1 core alanine mutants (F24A, G27A, I30A, G33A, V34A
and Y35A) are known to disrupt this core-DDX3 colocalistion without altering viral genome replication following direct electroporation of viral RNA into cells. All mutants
release infectious virus particles, except JFH1G33A. However, substantial reductions in RNA replication levels were observed at early time points following infection of naïve cells with the infectious mutant virus particles. Furthermore, the G33A mutation appears to allow the viral genome some degree of elasticity as during the serial passaging of G33Aelectroporated cells, a progressive increase in infectious virus particles was observed.
Based on the diverse phenotypes of these mutants it is difficult to conclude as to which effects (if any) are directly related to the core-DDX3 interaction. In addition, DDX3 has become a topical host factor in HCV research following recent studies demonstrating that its knockdown from target cells causes an overwhelming reduction to HCV replication. However, these studies failed to demonstrate if the core-DDX3 interaction had any functional relevance to this effect. The aim of this study was to clearly define the role of the core-DDX3 interaction in HCV replication. Using highly specific antibodies, a co-IP assay was developed that was sensitive enough to detect an interaction between these two proteins from JFH1 RNAelectroporated cell lysates. Co-IP analysis of the 6 JFH1 core mutants revealed that only one of these (JFH1Y35A) appeared to completely abolish DDX3 binding. Detailed analysis of this mutant revealed its replication properties resembled those of the WT virus following both electroporation of the viral RNA into cells and infection of naïve cells with the virus particles, demonstrating that the core-DDX3 interaction is dispensable for virus
replication in cell culture. In addition, DDX3 knockdown experiments revealed that as for JFH1WT, the replication of JFH1Y35A was equally sensitive to the depletion of endogenous DDX3 levels from target cells, indicating that the requirement of DDX3 for HCV replication is unrelated to its interaction with the viral core protein. Interestingly, sucrose gradient studies showed that DDX3 protein sedimented with the extracellular core protein from JFH1WT virus particles. Analysis of the JFH1Y35A virus particles found them to be less dense with a lower content of DDX3, alluding to the exciting possibility of DDX3 being a
novel component of the virion. The results of this study indicate that the core-DDX3 interaction is not the mechanism through which HCV utilises this host factor for its replication. However, DDX3 may be incorporated into virus particles through its association with the nucleocapsid. Investigating the latter possibility is the subject of our future work. In a separate study, the replication properties of JFH1G33A and JFH1F24A were fully
characterised. These two mutants were chosen for further analysis as they possessed unique properties relating to the virus proliferation in cell culture. Of the original 6 JFH1 core mutants, only JFH1G33A had a defect in secreting infectious virus particles. Upon passaging of cells electroporated with this mutant infectious particle production increased and eventually achieved peak titers similar to WT. The increase in infectivity consistently
coincided with the appearance of a second mutation in close proximity to the originally mutated residue 33. Although, similar results were reported previously for this mutant by
Dalrymple, (2007), the present study extended this work by confirming that these second site changes were compensatory mutations, which rescued the infectivity of JFH1G33A. In
contrast to the assembly defect of JFH1G33A, JFH1F24A was efficient at secreting infectious virus following electroporation, however the infection kinetics of these particles was considerably lower than those derived from JFH1WT. Further analysis, suggested the delayed infection of JFH1F24A virus was likely due to an early event in the HCV lifecycle following entry into the cell and prior to the release of the infectious genome. Identifying the importance of glycine 33 in infectious virus assembly and phenylalanine 24 in virus spread provides the basis for further studies into the role that core plays in virus
proliferation in cell culture. In its totality, this study has uncovered important details regarding the role of core, DDX3 and the core-DDX3 interaction in the HCV lifecycle.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: HCV, DDX3, Core protein, Virus Replication
Subjects: Q Science > QR Microbiology > QR355 Virology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
Supervisor's Name: Patel, Dr. Arvind
Date of Award: 2010
Depositing User: Dr Allan G.N Angus
Unique ID: glathesis:2010-3551
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 18 Mar 2013 15:43
Last Modified: 18 Mar 2013 15:45

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