Establishing thermal proteomics to study cellular dynamics in alphavirus infection

Viral pathogens, such as alphaviruses, are obligatory intracellular parasites that reprogram the host cell machinery for their replication. Alphaviruses, including the human pathogen chikungunya virus (CHIKV), can take over the host cell within a few hours. This thesis aims to establish the thermal proteomics technique, Mass- Spectrometry Coupled Cellular Thermal Shift Assay (MS-CETSA), to further investigate the cellular dynamics and identify and characterize viral host factors during the early infection. MS-CETSA enables the quantification of the thermal stability (TS) of the cellular proteome by extracting and measuring the soluble proteome after heat treatment, which leads to protein precipitation, followed by detergent-free lysis. Additionally, whole-cell protein abundance (WCA) after urea lysis and soluble abundance (SA) with detergent-free lysis are quantified and characterized.

In the key project (Project 2), this setup allowed us to detect protein-level differences upon Semliki Forest virus (SFV) infection in two different cell lines and datasets. This led to novel insights into the mechanism of transcriptional arrest through modulation of the RNA polymerase II complex and the interaction of SFV nsP3 with host factors such as G3BP. We discovered that in the very early stage of infection, already at 2 hours post-infection, POLR2A (Rpb1) and the nsP3- interactor BIN1 were the only strongly destabilized proteins observed, validating the sensitivity and specificity of our approach. At a later infection stage, the infection significantly modulated the thermal stability or abundance of proteins involved in nonsense-mediated decay (NMD), glucose and nucleotide metabolism, and autophagosomes.

In preparation for this project, we optimized our MS-CETSA approach to remove any experimental design bias that had previously affected our data. As described in Project 1, a Mock infection control experiment of this original experimental design was reanalyzed to evaluate the impact of confounding factors on the thermal proteome. This led to the identification of a list of volatile proteins and the development of an updated experimental design applied in Projects 2 and 3. In the final Project 3, an MS-CETSA experiment was conducted in a BSL3 environment, comparing chikungunya virus (CHIKV) with the live-attenuated vaccine CHIKV-Δ5nsp3 (VLA1553) to potentially identify host factors involved in the virus's pathogenicity. Data analysis for this project is still pending at the time of writing.