Mass spectrometry-based proteomics of virus-host interactions: from systems to structure

Abstract

This thesis investigates viral infection across multiple levels of biological organization using mass spectrometry-based proteomics, complemented by other approaches. Viral infections are inherently complex and involve coordinated interactions between viral factors and the host, ranging from systemic responses, to intracellular signaling, antigen structure, innate and adaptive immunity. To capture this complexity, the thesis integrates plasma proteomics, phosphoproteomics, glycoproteomics, and methodological advances in de novo antibody sequencing, together with cryo-EM and live-cell microscopy. Chapter 1 of the thesis establishes the biological and analytical background, introducing key aspects of viral biology, host responses to infection, and the application of mass spectrometry-based proteomics in virology. Chapter 2 addresses the systemic host response to dengue virus infection through high-throughput plasma proteomics of a large pediatric cohort. By comparing subclinical infection, dengue fever, and severe dengue across multiple disease phases, this work demonstrates that circulating proteins capture immune activation, inflammation, endothelial dysfunction, and disease progression. These results show that plasma is a powerful source of information on host physiology during infection and may support biomarker discovery for disease severity and patient stratification. Chapter 3 focuses on intracellular signaling during Ebola virus replication using bottom-up proteomics, phosphoproteomics, and microscopy. This work reveals extensive rewiring of kinase-driven signaling pathways, particularly involving cyclin-dependent kinases, and shows that Ebola virus replication drives cells toward an S/G2-like state to support replication and transcription. Functional validation with small-molecule inhibitors demonstrates how phosphoproteomics can identify host-directed therapeutic targets. Chapter 4 investigates the structural characterization of cetacean coronavirus spike proteins through an integrative approach of cryo-EM and glycoproteomics. Cryo-EM resolved the overall architecture of the spike proteins, while glycoproteomics revealed glycan composition at site-specific resolution. In addition to canonical N-linked glycosylation, extended O-glycosylated regions were identified, showing that viral glycosylation can be more heterogeneous and less predictable than previously recognized. These findings have important implications for antigen structure, host adaptation and immune recognition. Chapter 5 introduces methodological advances in de novo antibody sequencing. By combining HTA proteases with optimized hybrid mass spectrometric acquisition strategies, this work improves sequence coverage and increases the robustness of antibody sequence reconstruction. The resulting workflows provide a basis for future studies of antibody responses to viral infection and immune profiling. Chapter 6 integrates the findings of the thesis and places them in the context of current virology and proteomics research. A central conclusion is that viral infection is best understood through integrative analytical strategies. Across all chapters, viruses were found to consistently reshape the host environment. At the same time, many viruses converge on hijacking a limited set of host pathways, including cytoskeleton organization, translation, cell cycle regulation, DNA damage response, RNA metabolism, and immune signaling. This supports the concept of pan-viral host dependencies and suggests that shared host pathways may represent promising targets for broad-spectrum antiviral strategies. Overall, this thesis demonstrates that mass spectrometry-based proteomics provides a powerful and versatile strategy to study viral infections. When integrated with structural biology and imaging, proteomics enables a multidimensional understanding of virus-host interactions, from organism-level responses to molecular mechanisms.