High-throughput proteomic analysis of the Dengue virus replication complex.

Abstract

Dengue virus (DENV) is the causative agent of Dengue, a subtropical disease affecting up to 390 million people per year. Dengue can progress to a severe form, and there are no licensed treatments. DENV-2 infected Huh-7 cells, Huh-7 cells stably maintaining a self-replicating DENV-2 sub-genomic RNA (termed the replicon) and mock infected cells were used to prepare cellular lysates from which a heavy membrane fraction (16K) containing the replication complex was isolated. Proteins in the fraction were analysed by Tandem Mass Tagging (TMT) combined with mass spectrometry. Bioinformatic analysis of the data revealed a strong enrichment of proteins associated with mitochondrial terms in DENV-2 infected cells, but more secretory pathway proteins in the replicon containing cells compared to mock infected cells. Using the data, potential host factors (HFs) were selected and a silencing RNA (siRNA) screen was attempted to validate their involvement in DENV-2 infection, but ultimately this failed as there was not time to optimise the conditions fully. Overall, the mass spectrometry and bioinformatic techniques used showed greater power than previous studies making them a good way to identify potential HFs.

Date of Award	5 Dec 2023
Original language	English
Awarding Institution	University of Bristol
Supervisor	Andrew D Davidson (Supervisor)