Dalrymple, David A.
Identification of hepatitis C virus core protein residues critical for the interaction with the cellular DEAD-Box Helicase DDX3 and their functional relevance.
PhD thesis, University of Glasgow.
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Hepatitis C virus (HCV) is a single-stranded RNA virus belonging to the Flaviviridae and infects approximately 170 million people worldwide. Unlike other known RNA viruses, HCV causes a persistent infection in the majority of infected people and can lead to cirrhosis of the liver and hepatocellular carcinoma. For these reasons, HCV is rightly classified as a major human pathogen.
HCV core protein is believed to form, by analogy with other members of the Flaviviridae family, the nucleocapsid of the virus. As well as this, core has been shown to modulate many cellular processes via interactions with numerous host-cell proteins. One such protein shown to interact with HCV core is the DEAD-box RNA helicase DDX3. In cells expressing either HCV core alone, or as part of the full length HCV polyprotein, DDX3 is redistributed from its normal diffuse cytoplasmic localisation to lipid droplets where it colocalises with core. The cellular function of DDX3 is still unknown although it has been suggested to be involved in processes such as splicing, translation and RNA transport.
The aim of this study was to investigate the role of DDX3 in the life cycle of HCV. This was aided by the recent discovery of a fully infectious HCV genotype 2a clone (strain JFH-1), allowing previously inaccessible aspects of the virus life cycle to be studied such as particle assembly and release. A library of HCV core mutants (residues 1-59 only) was produced by error-prone PCR and subsequently expressed in bacteria and analysed for their ability to bind bacterially expressed DDX3 using a rapid, high throughput ELISA screen. Six HCV core residues, conserved throughout all genotypes, were identified as being critical for interaction with DDX3. These residues were confirmed as being critical for the interaction by transfection of mutant core (together with E1 and E2 to ensure correct processing of core) into Huh7 cells. None of the 6 mutant core proteins were able to redistribute cellular DDX3.
In order to study the effects of abolishing the core-DDX3 interaction in terms of a fully infectious HCV life cycle, the 6 critical residues were individually mutated to alanine in the cell culture infectious strain JFH-1 genome. All 6 mutant JFH-1 RNAs were capable of replication and being translated. Further investigation however, suggested that replication rate of mutant JFH-1 RNA was >50-fold lower than that of wild type JFH-1 RNA replication. Mutant core proteins colocalised with the lipid droplet marker ADRP, indicating correct subcellular localisation of the viral protein. Western-immunoblot analysis of mutant cores also confirmed that core proteins of same molecular weight to that of wild type core were produced, suggesting mutant cores were correctly processed. Of the 6 mutant JFH-1 clones analysed, 5 of them were capable of secreting infectious HCV particles that could subsequently infect naïve Huh7 cells, as detected by immunofluorescence and RT-PCR. However, one mutant, in which residue 33 of core had been changed from glycine to alanine, was initially unable to produce infectious particles. Upon passaging of cells electroporated with this mutant, infectious particles were eventually produced. The production of infectious particles consistently coincided with the presence of a second mutation in the surrounding area of the originally mutated residue 33. However, JFH-1 RNA containing both the mutation at residue 33 and the second identified mutation nearby, was unable to produce infectious particles upon electroporation, suggesting another lesion elsewhere in the HCV genome may also be required in order to overcome the effect of mutating residue 33.
A recent report has indicated that DDX3 may be a nucleo-cytoplasmic shuttling protein, utilising the CRM1 export pathway. To confirm this, DDX3 localisation was analysed in the presence of the CRM1 inhibitor leptomycin B (LMB). In the absence of LMB, DDX3 was seen to have a diffuse cytoplasmic localisation while a small proportion was also seen in the nucleus. In the presence of LMB however, a build-up of DDX3 was seen in the nucleus, confirming that DDX3 uses the CRM1 pathway to shuttle from the nucleus to the cytoplasm.
The results of this study indicate that the interaction of the cellular DEAD-box helicase DDX3 with core protein is not essential for the life cycle of HCV. It has been shown here however, that the replication rates of mutant HCV RNA are lower than that of wild type, suggesting that DDX3 may enhance either replication itself, or translation (which in turn provides the machinery required for viral RNA replication). Investigating this possibility is the subject of our future work. The identification of glycine 33 of core protein as being essential for production of infectious virus particles (without abolishing replication) will provide the basis for further studies on the production of infectious particles and the role that core protein plays in this process. The panel of JFH-1 core mutants will also be useful in studying the core-DDX3 interaction in a much wider context involving the role of DDX3 in normal cells.
This study has uncovered important details regarding the interaction between core and DDX3 and, together with the reagents produced throughout this investigation, should enable further successful study into the role of DDX3 in the life cycle of HCV.
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