Comparative proteomic analysis of dengue virus type 2 infected and replicon cell lines

: unveiling host responses to dengue virus infection and pharmacological inhibition of lysosomal degradation

Abstract

Dengue, the most prevalent arboviral disease affecting over 300 million individuals yearly, is caused by infection with dengue virus (DENV). To understand the proteomic modulations occurring at cellular level as a result of DENV infection, this study utilised quantitative tandem liquid chromatography-mass spectrometry (LC- MS/MS) to analyse the cellular proteomes of DENV-2 infected and replicon cell lines.

Initially, bioinformatic analysis was conducted on a proteomic dataset produced from mock, DENV-2 infected HEK-293T cells, and replicon HEK-293T cells to compare their cellular proteomes. Results indicated that the replicon may be a good model to study DENV replication, and that proteins only dysregulated during DENV infection were associated with ER-related processes, as well as “ubiquitin-dependent ERAD pathway”, and “oxidative phosphorylation”. Western blotting was carried out using the respective cells grown in serum-free or serum-containing medium to establish the effects of serum and serum depletion on the cellular proteomes. Whilst the abundances of ALG8, SEC11C, and FAHD1 were similar in the two conditions, others such as CDK6 may need further validation.

This study also investigated the host cellular proteins degraded by lysosomal- dependent processes during DENV infection. Bioinformatic analysis was conducted on a proteomic dataset generated from mock and DENV-2 infected Huh-7 cells treated with lysosomal inhibitor bafilomycin A1 or vehicle control DMSO. The results showed that several proteins involved in “complement and coagulation cascades” (FGA/B/G, C3, F2, CLU), “lipid metabolism” (MSMO1), and “autophagy” (NDP52, MAP1LC3B, SQSTM1, GABARAPL2), as well as protease inhibitors (α1-antitrypsin and PCI) were degraded during DENV-2 infection and rescued by bafilomycin, providing evidence that DENV dysregulates these pathways. Western blot validation was carried out using the respective cell lysates treated with bafilomycin C1 or DMSO. While the abundances of NDP52, BNIP3, and MAP1LC3B were validated, other results were inconsistent with those of the LC-MS/MS analysis and require further investigation.

Date of Award	1 Oct 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Luca Shytaj (Supervisor) & Andrew D Davidson (Supervisor)