Presentation Information
[P01-135]Bottom-up construction of virus-lke particles by post-insertion of Spike-lipid conjugates into the liposomal membrane.
○Iori Kobayashi1, Shogo Yoshimoto1, Takeshi Yokoyama2, Yoshikazu Tanaka2, Tomoaki Matsuura3, Shigeki Kiyonaka1, Katsutoshi Hori1 (1. Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University Hori Laboratory (Japan), 2. Department of molecular and chemical life sciences, Graduate School of life sciences, Tohoku University (Japan), 3. Earth-Life Science Institute, Institute of Future Science, Institute of Science Tokyo (Japan))
Keywords:
liposome,virus,bottom-up approach,synthetic virus-like particles,surface modification
Purpose
Virus-like particles (VLPs) are attractive noninfectious platforms for vaccines, drug delivery, and synthetic biology, but conventional top-down production often limits control over particle composition and surface functionality. Although bottom-up assembly on liposomes offers a modular alternative, post-insertion-based construction of synthetic VLPs using viral envelope protein-lipid conjugates remains underexplored. Here, we aimed to establish a detergent-free strategy to generate synthetic VLPs by post-inserting Spike-lipid conjugates into virus-sized liposomes through irreversible SNAP-tag/benzylguanine coupling.
Methods
A recombinant SARS-CoV-2 Spike ectodomain fused to a foldon trimerization motif and a SNAP-tag (Spike-SNAP) was expressed in Expi293F cells and purified by affinity chromatography. Conjugation of Spike-SNAP to a benzylguanine-functionalized lipid (BG lipid) was verified by a competitive labeling assay using a fluorescent SNAP substrate. The resulting Spike-lipid conjugates were incubated with preformed virus-sized liposomes of approximately 100 nm diameter for post-insertion. After washing, the liposomes were analyzed by liposome co-sedimentation assay, flow cytometry, dynamic light scattering (DLS), and negative-stain transmission electron microscopy (TEM). ACE2-binding activity was evaluated in a 96-well plate assay, with Congo Red used as an inhibitor control.
Results
Spike-SNAP retained reactivity toward the BG lipid. In the liposome co-sedimentation assay, more Spike-SNAP was recovered in the liposome fraction for the Spike-lipid conjugate than for unconjugated Spike-SNAP, indicating lipid-anchor-dependent membrane association. Flow cytometry also showed increased Spike-associated fluorescence on liposome particles, supporting successful surface display of Spike. DLS showed only a slight increase in particle size, whereas negative-stain TEM revealed a virus-like surface texture absent from unmodified controls. In addition, Spike-modified liposomes bound to immobilized ACE2, and this binding was significantly reduced by Congo Red without compromising liposome integrity.
Discussion
These findings demonstrate that post-insertion of protein–lipid conjugates enables detergent-free, modular construction of synthetic VLPs that mimic key features of native viruses. The SNAP-tag/benzylguanine reaction provides a selective and irreversible linkage for VLP design.
Conclusion
This study establishes a bottom-up strategy for synthetic VLP construction via post-insertion of protein–lipid conjugates and demonstrates functional display of viral proteins on virus-sized liposomes.
Virus-like particles (VLPs) are attractive noninfectious platforms for vaccines, drug delivery, and synthetic biology, but conventional top-down production often limits control over particle composition and surface functionality. Although bottom-up assembly on liposomes offers a modular alternative, post-insertion-based construction of synthetic VLPs using viral envelope protein-lipid conjugates remains underexplored. Here, we aimed to establish a detergent-free strategy to generate synthetic VLPs by post-inserting Spike-lipid conjugates into virus-sized liposomes through irreversible SNAP-tag/benzylguanine coupling.
Methods
A recombinant SARS-CoV-2 Spike ectodomain fused to a foldon trimerization motif and a SNAP-tag (Spike-SNAP) was expressed in Expi293F cells and purified by affinity chromatography. Conjugation of Spike-SNAP to a benzylguanine-functionalized lipid (BG lipid) was verified by a competitive labeling assay using a fluorescent SNAP substrate. The resulting Spike-lipid conjugates were incubated with preformed virus-sized liposomes of approximately 100 nm diameter for post-insertion. After washing, the liposomes were analyzed by liposome co-sedimentation assay, flow cytometry, dynamic light scattering (DLS), and negative-stain transmission electron microscopy (TEM). ACE2-binding activity was evaluated in a 96-well plate assay, with Congo Red used as an inhibitor control.
Results
Spike-SNAP retained reactivity toward the BG lipid. In the liposome co-sedimentation assay, more Spike-SNAP was recovered in the liposome fraction for the Spike-lipid conjugate than for unconjugated Spike-SNAP, indicating lipid-anchor-dependent membrane association. Flow cytometry also showed increased Spike-associated fluorescence on liposome particles, supporting successful surface display of Spike. DLS showed only a slight increase in particle size, whereas negative-stain TEM revealed a virus-like surface texture absent from unmodified controls. In addition, Spike-modified liposomes bound to immobilized ACE2, and this binding was significantly reduced by Congo Red without compromising liposome integrity.
Discussion
These findings demonstrate that post-insertion of protein–lipid conjugates enables detergent-free, modular construction of synthetic VLPs that mimic key features of native viruses. The SNAP-tag/benzylguanine reaction provides a selective and irreversible linkage for VLP design.
Conclusion
This study establishes a bottom-up strategy for synthetic VLP construction via post-insertion of protein–lipid conjugates and demonstrates functional display of viral proteins on virus-sized liposomes.
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