Abstract
Natural enveloped viruses, in which nucleocapsids are covered with lipid bilayers, contain membrane proteins on the outer surface that are involved in diverse functions, such as adhesion and infection of host cells. Previously, we constructed an enveloped artificial viral capsid through the complexation of cationic lipid bilayers onto an anionic artificial viral capsid self-assembled from β-annulus peptides. Here we demonstrate the embedding of the membrane protein Connexin-43 (Cx43), on the enveloped artificial viral capsid using a cell-free expression system. The expression of Cx43 in the presence of the enveloped artificial viral capsid was confirmed by western blot analysis. The embedding of Cx43 on the envelope was evaluated by detection via the anti-Cx43 antibody, using fluorescence correlation spectroscopy (FCS) and transmission electron microscopy (TEM). Interestingly, many spherical structures connected to each other were observed in TEM images of the Cx43-embedded enveloped viral replica. In addition, it was shown that fluorescent dyes could be selectively transported from Cx43-embedded enveloped viral replicas into Cx43-expressing HepG2 cells. This study provides a proof of concept for the creation of multimolecular crowding complexes, that is, an enveloped artificial viral replica embedded with membrane proteins.
Highlights
Enveloped viruses such as the influenza virus, human immunodeficiency virus, and coronavirus are nano-sized multimolecular crowding complexes consisting of nucleocapsids covered by a lipid bilayer.[1,2,3] Membrane proteins embedded in the outer surface of enveloped viruses are involved in diverse functions, including adhesion and infection of host cells
This study provides a proof of concept for the creation of multimolecular crowding complexes, that is, an enveloped artificial viral replica embedded with membrane proteins
The transmission electron microscopy (TEM) image of the post-expression solution ([pURE-Cx43] = 17.3 nM), showed spherical structures of 50–100 nm (Fig. 1B), and the size distribution obtained from TEM images and dynamic light scattering (DLS) were 103 Æ 46 nm and 58 Æ 11 nm, respectively (Fig. S2 and S3A, Electronic supplementary information (ESI)†)
Summary
Enveloped viruses such as the influenza virus, human immunodeficiency virus, and coronavirus are nano-sized multimolecular crowding complexes consisting of nucleocapsids covered by a lipid bilayer.[1,2,3] Membrane proteins embedded in the outer surface of enveloped viruses are involved in diverse functions, including adhesion and infection of host cells. Spike proteins embedded in the envelope of the coronavirus play an important role during host cell infection.[4,5] The influenza virus has two different membrane proteins, haemagglutinin and neuraminidase, on its envelope, which are involved in infection and budding, respectively.[6,7]. There have been no examples of construction of artificial enveloped viral capsids embedded with membrane proteins, which remains a challenging task
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