title: Design and development of bottom-up assembled SARS-CoV-2 miniviruses to investigate the impact of lipid species on infectivity
creator: Yagüe Relimpio, Ana
subject: ddc-500
subject: 500 Natural sciences and mathematics
description: Research of highly pathogenic viruses, such as SARS-CoV-2, is often challenging due to their inherent variability and complexity, and has to be performed in a high biosafety environment. The use of bottom-up synthetic biology provides tools to investigate specific aspects of the viral life cycle in a controlled manner. To address this I designed bottom-up synthetic SARS-CoV-2 miniviruses, named MiniVs, with the aim to study the influence of lipids on the attachment process of the viral cycle. To conduct a functional screening comparing Omicron and Alpha spike glycoproteins I utilised both ACE2-functionalised and non-functionalised planar supported lipid bilayers and giant unilamellar vesicles and discovered that Omicron had a previously unreported affinity towards lipid membranes. I measured the dissociation constants of both variant spikes to a lipid membrane and demonstrated that the affinity of Omicron spike is twice as high as the one of Alpha. These findings were further validated using two cell lines, with and without ACE2 receptor expression. Moreover, modification of the plasma membrane composition proved the importance of cholesterol in the attachment step of SARS-CoV-2. Loading the cellular membrane with different amounts of cholesterol enhanced the attachment of Omicron MiniVs. The same treatment had the opposite effect on Alpha MiniVs suggesting that the effect observed on the cell membrane is not due to membrane reorganisation and receptor clustering. Measuring the interaction between Omicron spike and SLBs with increasing amounts of cholesterol indicated that there is no specific affinity between the protein and the lipid. Finally I produced spike proteoliposomes, which consisted of full-length transmembrane Alpha or Omicron spike inserted into small unilamellar vesicles. I demonstrated that the full-length spike glycoprotein can be inserted into vesicles while remaining functional and binding to the ACE2 receptor. Proteoliposomes that contained a higher content of cholesterol on their membrane showed an increased interaction with the ACE2 receptor. Altogether, the research presented here underscores the advantages of using synthetic bottom-up assembled viruses to investigate the importance of lipids on viral infectivity, specifically during viral attachment. The results obtained highlight the differences between SARS- CoV-2 variants Alpha and Omicron, and partially explain their distinct transmissibility and pathogenicity patterns. The use of MiniVs and proteoliposomes offers a novel platform for studying specific aspects of the viral life cycle that are challenging to examine with conventional biological methods.
date: 2025
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserver/36065/1/thesis.pdf
identifier: DOI:10.11588/heidok.00036065
identifier: urn:nbn:de:bsz:16-heidok-360652
identifier:   Yagüe Relimpio, Ana  (2025) Design and development of bottom-up assembled SARS-CoV-2 miniviruses to investigate the impact of lipid species on infectivity.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/36065/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng