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Title: Elucidation of the mechanism of action of a mutation in the dengue virus NS4B protein that confers a persistent phenotype in cell culture
Author: Ismail, Rosmani
ISNI:       0000 0004 5922 368X
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2015
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Dengue viruses (DENVs) are mosquito-borne flaviviruses, which are responsible for a wide range of illnesses, from a mild febrile illness to the more serious dengue haemorrhagic fever and dengue shock syndromes. The DENV NS4B is a highly hydrophobic integral ER membrane protein that has been reported to perturb type I interferon (IFN) signalling and has emerged as a major target for anti-viral strategies. The objective of this study was to determine the mechanism of action of two consecutive nucleotide mutations in the NS4B gene (nt 7020 and 7021) that result in the substitution of threonine 66 with alanine, which allow the virus to replicate persistently in mammalian and mosquito cells. Initially, a transient gene expression system was developed to analyse the effect of the mutations on the localisation of NS4B and its ability to perturb the IFN response and the unfolded protein response. The mutations were not found to significantly affect any of these processes. Novel cell lines were then produced that stably expressed the NS4B gene and its mutant counterpart in an inducible fashion. The cell lines were used to identify cellular proteins (DNAJA2, PFN2, FAF2, BAX and PMSG1) that potentially interact with the NS4B proteins using a high-throughput proteomic approach. Finally, HEK293T cell lines that were either stably persistently infected with a virus containing the NS4B mutations or contained a wild type DENV replicon or a replicon containing the NS4B mutations were established. Genome sequencing demonstrated that the NS4B mutations were more stable in a replicon than in the full viral genome. The effect of the NS4B mutations on host cell processes was investigated by transcriptomic analysis using RNAseq. The transcriptomic analysis revealed that canonical cellular pathways were affected by viral replication including IFN signaling, virus recognition, ubiquitination, apoptosis, and ER stress. Further analysis showed that the NS4B mutations affected the ability of the virus and a DENV replicon to effectively suppress IFN signaling and IFN sensitive gene expression. This study provides further insight into the role of the NS4B protein in the viral-host interaction and provides a mechanism to explain how a persistent DENV infection may occur.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available