A kind of enveloped virus, SARS-CoV-2, has spike proteins (S proteins) on its envelope surface, which bind to the angiotensin converting enzyme 2 (ACE2) receptor displayed on the host cell surface to enter the virus into the cell. We have previously succeeded in constructing an enveloped artificial viral capsid by complexing cationic lipid bilayer with anionic artificial viral capsid self-assembled from β-annulus-EE peptides (INHVGGTGGAIMAPVAVTRQLVGSEE) through electrostatic interaction. We also have created an enveloped viral replica equipped with a membrane protein, connexin 43, on the envelope using cell-free expression. In this study, we constructed an enveloped viral replica equipped with S protein derived from SARS-CoV-2 using cell-free protein expression, and evaluated the binding behavior of the viral replica to the ACE2 receptor.

The enveloped viral replica equipped with S protein was constructed by cell-free expression of plasmid fragment encoding the S protein in the presence of the enveloped viral capsid. TEM image showed the formation of 100-200 nm spherical structures possessing spike-like structures on the surface. The binding behavior of the S protein-equipped viral replica to AF647-labeled ACE2 as increasing the concentration, whereas enveloped capsid without S protein hardly bound. The dissociation constant and Hill coefficient were calculated to be K_d = 2.11 ± 0.11 nM, n = 2.68 ± 0.06 by Hill plot. In addition, the binding of the S protein-equipped viral replica to the ACE2-immobilized gold substrate was evaluated by quartz crystal microbalance (QCM) analysis. As a result, it was shown that the S protein-equipped viral replica strongly bound to ACE2 similar to the result of IFM (K­_d = 1.50 ± 0.12 nM calculated by Langmuir plot).