Vlachová, Štěpánka (2025) Host Cell Remodelling during SARS-CoV-2 Infection: Mechanistic Insights into Autophagy and ER-Driven Membrane Rearrangements- Dissecting the Role of Autophagy-Related Factors and Atlastin-Mediated ER Dynamics in Double-Membrane Vesicle Biogenesis. [Tesi di dottorato]

	Documento PDF Stepanka_Vlachova_37_COMPLETO.pdf Visibile a [TBR] Amministratori dell'archivio Download (9MB) | Richiedi una copia
	Documento PDF Stepanka_Vlachova_37_PARZIALE.pdf Download (2MB)

Abstract

Positive-sense single-stranded RNA (+ssRNA) viruses, including SARS-CoV-2, are known to extensively remodel host cell membranes to establish viral replication organelles, which have the form of double-membrane vesicles (DMVs) in case of Coronaviruses. While viral non-structural proteins are known to drive DMV biogenesis, the contribution of host factors remains mostly elusive. Here, we identify a non-canonical autophagy pathway and ER-shaping proteins as critical host determinants of DMV formation and functional coordination. Using high-content siRNA screening targeting 48 autophagy-related genes, we uncovered 19 host dependency factors and 2 restriction factors that modulate SARS-CoV-2 infection. Among these LC3C and the ATG4D protease, able to delipidate LC3, emerged as key regulators. Functional validation across multiple cell lines and β-coronaviruses (SARS-CoV-2 and HCoV-OC43) revealed that ATG4D depletion impairs viral replication and infectivity, independent of canonical autophagy flux. Notably, ATG7-mediated LC3 lipidation is dispensable, suggesting that SARS-CoV-2 preferentially exploits unlipidated LC3C maintained by ATG4D. Reconstitution experiments with phosphomutant LC3C variants demonstrate that the non-phosphorylated, ATG4D-accessible form of LC3C supports viral replication, whereas the phosphomimetic form does not. These findings indicate that SARS-CoV-2 hijacks recycled LC3C to facilitate viral replication, bypassing the degradative autophagy pathway. Parallel investigations into ER-shaping proteins reveal that atlastin-2 (ATL2), but not ATL3, is essential for provide DMV structural integrity and coordinate viral replication and assembly. ATL2 localises to three-way ER junctions and interacts with viral proteins nsp3 and nsp4. ATL2 knockout leads to the formation of smaller, open DMVs and Spike protein retention in the ER, impairing virion assembly and subsequently infectivity. Reconstitution of the ATL2B, but not ATL2A, isoform restores viral infectivity, underscoring isoform-specific functionality. Together, these findings delineate a novel host-driven mechanism for DMV formation and organisation involving non-canonical autophagy and ER membrane dynamics proteins. This work provides mechanistic insight into host–virus interactions and identifies potential therapeutic targets for disrupting coronavirus replication.

Downloads

Actions (login required)

Modifica documento