Use this URL to cite or link to this record in EThOS:
Title: High-throughput quantitative proteomic analysis of dengue virus infected cells
Author: Chiu, Han-Chen
ISNI:       0000 0004 5921 8071
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2015
Availability of Full Text:
Access from EThOS:
The four serotypes of dengue virus (DENV 1-4) cause the most important arthropod-borne viral disease of humans. Dengue is a global health concern with up to 390 million human infections estimated to occur annually. Despite intensive research, the pathogenesis of dengue is not well understood. The use of high-throughput transcriptomic and small interfering RNA approaches has led to the identification of cellular proteins and pathways that are required for, and modulated by, the DENV lifecycle. In comparison to other high-throughput approaches, high-throughput proteomic analysis has not been applied to the investigation of the DENV -host protein interaction. In this study, for the first time, ~table isotope labelling by ~mino acids in fell culture (SILAC) combined with high throughput mass spectrometry (MS) has been used to examine the host cell response to DENV infection. Initially, SILAC-MS analysis was used to investigate the changes that occurred in the proteome of human alveolar epithelial A549 cells in response to DENV infection. Nuclear and cytoplasmic fractions prepared from mock and DENV -2 infected A549 cells were analysed. 2115 and 3098 proteins in nuclear and cytoplasmic fractions respectively were identified and quantified in both the mock and DENV -2 infected A549 cells. The SILAC-MS results were subjected to bioinformatics analysis and validated for eight selected proteins by Western blot and immunofluorescence analysis. Two of the selected proteins, ELKS/Rab6-interactinglCAST family member 1 (ERC 1) and PRA 1 family protein 2 (PRAF2), significantly decreased during infection and were investigated in more detail to determine their relevance to the DENV lifecycle. Knockdown of ERCI but not PRAF2 inhibited DENV -2 replication whereas overexpression of PRAF2 but not ERCI inhibited DENV-2 infection. Co-immunoprecipitation analysis combined with SILAC-MS revealed that ERCI and PRAF2 interacted with the DENV NS5 and NS 1 respectively and cellular proteins involved in trafficking, suggesting that DENV may modulate intracellular transport processes during its release. Comparative analysis of DENV -2 and DENV -4 infected human hepatocyte Huh-7 cells was then done by SILAC-MS. The SILAC-MS results identified common and cell specific pathways that were modulated by DENV infection and identified cellular proteins that were differentially effected by DENV -2 and DENV -4 infection. Furthermore, the analysis of cell specific pathways that were modulated by DENV infection was done by comparison ofDENV-2 infected A549 and DENV-2 infected Huh- 7 cells. Overall the work described in this thesis demonstrated that SILAC-MS is a powerful approach for the analysis of changes in the host proteome upon DENV infection. A number of novel protein changes were identified that can now be furher examined to increase our understanding of DENV replication and identify targets against which antiviral strategies can be developed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available