[1S01e] 10. ASPIRE - Part 3

5:58 PM - 6:13 PM JST(8:58 AM - 9:13 AM UTC)

[1ASPR-23] Artificial mimics of enveloped viruses

○KAZUNORI MATSUURA¹ (1. Tottori University (Japan))

Keywords:

Enveloped viral replica,Self-assembly,Peptide,Lipid,Membrane protein,Drug delivery system

Recently, progressive development of nano-architectures, self-assembled from rationally designed peptides, have also enabled the construction of viral capsid-like nanocapsules consisting of peptides. We previously found that a 24-residue β-annulus peptide (INHVGGTGGAIMAPVAVTRQLVGS), which is involved in the formation of the dodecahedral inner skeleton of the tomato bushy stunt virus, spontaneously self-assembles into a hollow artificial viral capsid with a size range of 30−50 nm. The artificial viral capsid can encapsulate various guest molecules such as DNA, quantum dot, His-tagged GFP, and mRNA. Since the C-terminus is directed to the outside of the capsid, nanoparticles and proteins were decorated on the surface of the capsids. The budding of human immunodeficiency virus (HIV) from an infected host cell is induced by the modification of structural proteins bearing long-chain fatty acids, followed by their anchoring to the cell membrane. Although many model budding systems using giant unilamellar vesicles (GUVs) induced by various stimuli have been developed, constructing an artificial viral budding system of GUVs using only synthesized molecules remains challenging. Recently, we report the construction of an alkyl anchor-modified artificial viral capsid budding system from a lipid bilayer of GUV. We have succeeded in constructing the enveloped viral replica (EVR) by complexing cationic lipid bilayer with anionic artificial viral capsid self-assembled from β-annulus-EE peptides (INHVGGTGGAIMAPVAVTRQLVGSEE). EVR with cationic surface can invade into HepG2 cells by membrane fusion. We also demonstrated that EVR presenting breast cancer antigens induced significant antibody production in mouse. Membrane protein connexin-43 (Cx43) is a four-fold transmembrane protein that forms gap junction structures to transport molecules between cells. Here, we constructed EVR embedded with Cx43 by using cell-free protein expression system and demonstrated molecular transport from the EVR into Cx43-expressing liposomes and HepG2 cells through gap junctions. When Cx43-embbeded EVR encapsulated with fluorescent dye TMR were added to Cx43-expressing HepG2 cells, transports of fluorescent dye into them were observed by CLSM. SARS-Cov-2, which is a kind of enveloped virus, has spike proteins (S-proteins) on its envelope surface. The S protein binds to the ACE2 receptor displayed on the host cell surface to enter the virus into the cell. We constructed EVR equipped with S-protein derived from SARS-Cov-2 using cell-free protein expression. In addition, we evaluated the binding of the S-protein-equipping EVR to the ACE2 receptor using quartz crystal microbalance (QCM) and imaging flow cytometry. CLSM imaging revealed that the S-protein-equipping EVR bound to cells expressing ACE2 receptor. Influenza virus is equipped with hemagglutinin (HA) on the envelope, which invades into the host cells by endocytosis through specific recognition to sialic acid-containing glycans. Recently, we constructed TMR/NBD-labeled EVR equipped with HA derived from influenza virus via cell-free protein expression and evaluated their intracellular invasion mechanism based on glycan recognition. We examined the effects of various endocytosis inhibitors and sialidase treatment on uptake into HepG2 cells. The results suggested that EVR equipped with HA invades into the cells by endocytosis through binding of HA to sialic acid.