Development of Receptor-Integrated Magnetically Labeled Liposomes for Investigating SARS-CoV-2 Fusion Interactions

The impacts of highly pathogenic enveloped viruses, such as SARS-CoV-2, have turned scientific inquiry toward the fusion mechanisms responsible for viral pathogenesis and to seek cost-effective and adaptable strategies to mitigate future outbreaks. Current approaches for studying SARS-CoV-2 fusion include computational studies, pan-coronavirus viral inhibitors, and modified peptides and lipopeptides, along with various nanotechniques. Although these methodologies have illuminated the fusion mechanisms, they possess key limitations that prevent their widespread utility in outbreaks, including high financial or instrumental costs, operational proficiency, cytotoxicity, or viral specificity. This work measures changes in spin-spin T₂ magnetic (transverse) relaxation times using a benchtop NMR instrument and introduces a bioanalytical approach to quickly quantify fusion interactions between the SARS-CoV-2 spike protein and liposome-coated iron oxide nanosensors (LIONs). Additionally, this study modifies the LION platform by appending the angiotensin-converting enzyme (ACE2) receptor, thereby creating LIONs-ACE2 that mimics the ACE2 host cell receptor targeted by SARS-CoV-2. Furthermore, SARS-CoV-2 fusion to other receptors reported to be involved is also examined. Environmental factors impacting fusion, such as calcium ion concentration, cholesterol composition, pH, neutralizing antibodies, and lower temperature, are investigated. Finally, molecular dynamics (MD) simulation studies reveal that the receptor binding domain (RBD) of the spike protein interacts more favorably with ACE2 than the lipid bilayer in the opened conformation, yet the closed conformation of RBD interacts with the bilayer with a similar energy as with ACE2. These findings reveal how the LION platform offers a customizable, fast-acting, inexpensive, and accessible mechanism for examining the fusion process of SARS-CoV-2 and other enveloped viruses.