Bottom-Up Creation of Virus-like Particles via Post-Insertion of Protein-Lipid Conjugates.

Virus-like particles (VLPs) are widely used as noninfectious platforms for vaccines, drug delivery, and synthetic biology. Here we report a bottom-up approach to generate liposomal VLPs via post-insertion of protein-lipid conjugates into preformed liposomes. Proteins were covalently coupled to a benzylguanine (BG) lipid via the bioorthogonal and irreversible SNAP-tag/BG reaction, and the resulting protein-lipid conjugate enabled detergent-free insertion into liposomal lipid bilayers. Using a SNAP-tagged GFP conjugate, we first confirmed membrane association by liposome cosedimentation and detected increased GFP fluorescence on individual liposomes by flow cytometry. We next applied the method to the SARS-CoV-2 Spike ectodomain. A Spike-SNAP recombinant protein was expressed in Expi293F cells, and its conjugation capability with BG lipid was verified by a binding inhibition assay using a fluorescent BG substrate. Incubation of virus-sized liposomes with Spike-lipid conjugates resulted in lipid-anchor-dependent recovery of Spike in the liposome fraction and increased Spike-associated fluorescence by flow cytometry without detectable changes in particle size. Negative-stain transmission electron microscopy showed a virus-like texture on Spike-modified liposomes compared with unmodified liposomes. In an ACE2 plate-binding assay, Spike-modified liposomes showed higher binding ability than unmodified liposomes, and the binding was reduced by Congo Red, a reported inhibitor of the Spike-ACE2 interaction, and a neutralizing anti-Spike antibody. By decoupling the design of the conjugate from that of the liposome, this post-insertion approach can facilitate rapid and systematic optimization of VLPs for mechanistic investigations and application-oriented studies.