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Title: High-throughput quantitative proteomic analysis of host proteins interacting with dengue virus replication complex
Author: Yousuf, Amjad
ISNI:       0000 0004 6057 7389
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2016
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Dengue virus (DENV) is a global health problem with approximately 390 million infections annually. Currently, no completely effective vaccines or medications are available to prevent DENV infection. This study used high-throughput quantitative proteomic analysis to identify cellular proteins that modulate DENV replication and are potential targets for the development of antiviral strategies. DENV is known to replicate and package its genome in association with perinuclear ER membranes. Initially, experimental conditions were optimised to infect human Huh-7 liver cells at high efficiency, with DENV serotypes -2 and -4 and to isolate a subcellular heavy membrane fraction (16K) enriched in the DENV replication complex. Then, stable isotope labelling by amino acids in cell culture (SILAC) coupled with high-throughput mass spectrometry (MS) was used to identify changes in the Huh-7 cell proteome and the subcellular 16K fraction in response to DENV-2 and DENV-4 infection. The analysis led to the identification and quantification of 3650 and 4026 and 3461 and 3668 proteins in the 16K and total cell proteomes from DENV-2 and DENV-4 infected cells respectively. For comparison, the total cell proteomes were also analysed by tandem mass tagging combined with MS. Proteomic bioinformatics was done on the datasets which showed that DENV modulated various cellular pathways including; protein biosynthesis, the secretory pathway, lipid metabolism and the cell cycle. A comparison of changes in the total and 16K proteomes from DENV infected cells identified a number of cellular proteins that selectively increased in the replicase containing fraction. which was validated by Western blotting and immunofluorescence analysis. Seven proteins (APOB, ARFRP1, BAG2, GOLGA1, GOLGB1, GOSRI and TMED9) were further investigated for their role in DENV -2 replication by examining either viral or replicon replication in cells depleted of the proteins by siRNA knockdown which revealed proteins that both positively and negatively modulated DENV replication.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available