Proteomics-based Analysis of RSV nucleic acid/protein InteractionS

Projektinformationen

PARIS

ID Finanzhilfevereinbarung: 101107996

DOI

10.3030/101107996

Projekt abgeschlossen

EK-Unterschriftsdatum 8 Mai 2023

Startdatum 1 April 2024

Enddatum 31 März 2026

Finanziert unter

• Marie Skłodowska-Curie Actions (MSCA)

Gesamtkosten

Keine Daten

EU-Beitrag

€ 189 687,36

Koordiniert durch

TECHNISCHE UNIVERSITAET MUENCHEN
Germany

Periodic Reporting for period 1 - PARIS (Proteomics-based Analysis of RSV nucleic acid/protein InteractionS)

Berichtszeitraum: 2024-04-01 bis 2026-03-31

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

Respiratory Syncytial Virus (RSV) is a major cause of severe lower respiratory tract infections and a major cause of mortality in children under 5 years old and the elderly worldwide. While RSV impose a huge disease burden on public healthcare system, the development of specific antivirals is impeded by a lack of knowledge of virus multiplications mechanisms, virus-host interactions or pathogenicity. Here, we propose to comprehensively map RSV host interactions and characterize the cellular response to infection through an integrated multi-omics approach and using experimental systems close to natural infection. By using state-of the- art-of proteomics and transcriptomics, we characterize the protein-protein interactome, analyze the proteome of cells expressing the individual viral proteins (effectome), and assess the influence of RSV infection on cellular mRNA expression, protein abundance, and phosphorylation. We systematically map the identified viral-induced perturbations onto a network of known cellular protein-protein interactions and signalling pathways. This comprehensive network will highlight RSV-induced perturbations and pinpoint RSV host dependency-factors, whose functional importance and therapeutic potential will be assessed by conducting genetic and drug-based loss and gain of function experiments. Biochemical and structural methods, and further analyses of the cellular response in genetically or drug-perturbed cells will yield insights into the molecular function of selected proteins. The proposed multilevel approach will thus reveal yet unstudied proteins and pathways determining RSV growth, pathology or viral host defense mechanisms, which will shed light on unresolved questions of RSV biology, and reveal hotspots amenable to therapeutic intervention.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

We profiled the interactomes of both RSV A and RSV B strains using affinity purification coupled to mass spectrometry (AP-MS) and in parallel, we measured the total proteome of cells expressing the individual viral proteins to systematically connect the cellular targets of each viral protein with the downstream changes they induce on proteome levels (effectome). We assessed the influence of viral infection on the cellular mRNA expression (transcriptome) and protein abundance (proteome) in a time-resolved manner. To better understand the mechanisms underlying perturbation of cellular signalling, we also assessed protein phosphorylation. The homogeneity of the experimental systems used in each omic approach allowed the integration of all datasets by applying system biology data analysis methodologies. Using network propagation approaches, the identified viral-induced perturbations were mapped onto a network of known cellular protein-protein interactions and signalling pathways. This approach promoted the identification of functional connections between the cellular proteins that interact with the viral structures (interactome) and the cellular perturbations induced upon infection (transcriptome, proteome and phosphoproteome) or in response to the expression of individual viral proteins (effectome). We also intersected our findings with other omics databases previously generated by the host laboratory using homologous enrichment methods, employed other publicly-available or still unpublished omics databases generated by others to identify common and virus-specific host responses as well as host proteins which differentially promotes or restrict virus replication. Based on the integrative analysis, we investigated the functional importance of several high-scoring candidates. We corroborate the effect of 50 protein candidates on viral replication using a CRISPR/Cas9-based loss-of-function approach. Finally, we focused on two candidate proteins which modulate RSV infection and characterized their molecular function by performing a combination of biochemical and structural methods or in-depth analyses of the cellular response in genetically or drug-perturbed cells.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

The work in this project substantially advances our understanding of the molecular interplay between RSV and the host cell by systematically characterizing host factors and pathways involved during RSV infection through a multi-omics approach. The generated datasets provide novel insights into the cellular mechanisms regulating viral replication and antiviral responses, thereby expanding our understanding of RSV biology and host–pathogen interactions. By identifying host pathways and cellular processes associated with RSV infection, this work may support the future development of innovative host-directed antiviral strategies with broader antiviral potential and a reduced likelihood of resistance emergence. Furthermore, the knowledge generated through this project establishes conceptual and methodological frameworks that are relevant not only for RSV research, but also for the study of other RNA viruses with similar replication strategies. The project also promotes knowledge dissemination and Open Science by generating datasets and analytical frameworks that will serve as valuable resources for the scientific community. More broadly, the integration of large-scale omics approaches fosters interdisciplinary research and facilitates future studies of virus–host interactions.