Embedding a membrane protein into an enveloped artificial viral replica - PubMed (original) (raw)

. 2021 Dec 21;3(2):231-241.

doi: 10.1039/d1cb00166c. eCollection 2022 Feb 9.

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Embedding a membrane protein into an enveloped artificial viral replica

Hiroto Furukawa et al. RSC Chem Biol. 2021.

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.

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Conflict of interest statement

There are no conflicts of interest to declare.

Figures

Fig. 1

Fig. 1. (A) Schematic illustration and (B) TEM images of enveloped viral capsid embedded with Cx43 ([β-annulus-EE] = 16 μM, [DOTAP] = 48 μM, [DOPC] = 480 μM). Cx43 was expressed from 17.3 to 34.6 nM pURE-Cx43 using the PURE system. The samples were stained with EM stainer.

Fig. 2

Fig. 2. (A) Western blot analysis of Cx43 expressed from 17.3 nM pURE-Cx43 in the presence of enveloped viral capsid ([β-annulus-EE] = 16 μM, [DOPC] = 480 μM (DOTAP/DOPC = 0/10); [β-annulus-EE] = 16 μM, [DOTAP] = 9.6 μM, [DOPC] = 480 μM (DOTAP/DOPC = 0.2/10); [β-annulus-EE] = 16 μM, [DOTAP] = 24 μM, [DOPC] = 480 μM (DOTAP/DOPC = 0.5/10); [β-annulus-EE] = 16 μM, [DOTAP] = 48 μM, [DOPC] = 480 μM (DOTAP/DOPC = 1/10)) in 10 mM Tris–HCl buffer (pH 7.0). (B) Western blot analysis of the concentration dependence of pURE-Cx43 (0, 17.3, 34.6, 69.1 nM) in the presence of enveloped viral capsid ([β-annulus-EE] = 16 μM, [DOTAP] = 48 μM, [DOPC] = 480 μM (DOTAP/DOPC = 1/10)) in 10 mM Tris–HCl buffer (pH 7.0).

Fig. 3

Fig. 3. (A) Normalised auto-correlation curves of 10 nM Alexa Fluor 488-labelled anti-Cx43 antibody in the presence of Cx43-embedded viral replica (red), free Cx43 (blue), and in the absence of Cx43 (antibody alone, green) in 10 mM Tris–HCl buffer (pH 7.0) at 25 °C. (B–D) Measured (solid) and fitted (dot) auto-correlation curves of 10 nM Alexa Fluor 488-labelled anti-Cx43 antibody in the presence of (B) Cx43-embedded viral replica ([β-annulus-EE] = 4 μM, [DOTAP] = 12 μM, [DOPC] = 120 μM) or (C) free Cx43, and (D) in the absence of Cx43 (the antibody alone). Cx43 was expressed from 17.3 nM pURE-Cx43 using the PURE system.

Fig. 4

Fig. 4. Concentration dependence of Alexa Fluor 488-labelled anti-Cx43 antibody on the ratio of the slow component determined by FCS curve fitting in the presence of (A) free Cx43 and (B) Cx43-embedded viral replica ([β-annulus-EE] = 4 μM, [DOTAP] = 12 μM, [DOPC] = 120 μM) at 25 °C. Cx43 was expressed from 17.3 nM pURE-Cx43 using the PURE system. Binding curve of the antibody to Cx43 is characterised by the Langmuir equation.

Fig. 5

Fig. 5. TEM images of (A) anti-Cx43 antibody (42 nM) and the gold nanoparticle-labelled secondary antibody (14.8 nM) against anti-Cx43 antibody, the mixture of the anti-Cx43 antibody and the gold nanoparticle-labelled secondary antibody with (B) the enveloped viral capsid ([β-annulus-EE] = 4 μM, [DOTAP] = 12 μM, [DOPC] = 120 μM), (C) Cx43-embedded viral replica ((DOTAP/DOPC = 1/10) [β-annulus-EE] = 4 μM, [DOTAP] = 12 μM, [DOPC] = 120 μM), and (D) Cx43-expressed liposome ([DOTAP] = 12 μM, [DOPC] = 120 μM) in 10 mM Tris–HCl buffer (pH 7.0). Cx43 was expressed from 17.3 nM pURE-Cx43 using PURE system. TEM samples were stained with EM stainer. Red arrows indicate the gold nanoparticle-labelled secondary antibody.

Fig. 6

Fig. 6. TEM images of anti-Cx43 antibody (42 nM) and the gold nanoparticle-labelled secondary antibody (14.8 nM) against anti-Cx43 antibody under the Cx43-embedded viral replica ((DOTAP/DOPC = 1/10) [β-annulus-EE] = 4 μM, [DOTAP] = 12 μM, [DOPC] = 120 μM) in 10 mM Tris–HCl buffer (pH 7.0). Cx43 was expressed from 34.6 or 69.1 nM pURE-Cx43 using PURE system. TEM samples were stained with EM stainer. Red arrows indicate the gold nanoparticle-labelled secondary antibody.

Fig. 7

Fig. 7. Transport of 5-TMR from Cx43-embedded viral replica into HepG2 cells. (A) CLSM images of HepG2 cells incubated with 5 μM Alexa Fluor 488-labelled anti-Cx43 antibody. Channels for Alexa Fluor 488 (green), Hoechst 33342 (cyan), and bright field for CLSM images. (B) CLSM images of HepG2 cells incubated with 5-TMR-encapsulated Cx43-embedded viral replica or enveloped capsid without Cx43 ([β-annulus-EE] = 10 μM, [DOTAP] = 30 μM, [DOPC] = 300 μM, [5-TMR] = 1.7 μM). Cx43 was expressed from 17.3 nM pURE-Cx43 using PURE system. Channels for 5-TMR (magenta) and Hoechst 33342 (cyan) for CLSM images. (C) Box plot of the fluorescence intensity distribution of 5-TMR in HepG2 cells normalised outside of cells (N = 60). The _P_-value was calculated by one-way analysis of variance followed by Mann–Whitney U test.

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